Formation mechanism of monodisperse, low ...

Colloids and Surfaces B: Biointerfaces 90 (2012) 21–27

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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique Wen Fan a,b,1 , Wei Yan b,1 , Zushun Xu b , Hong Ni a,∗ a b

School of Life Science, Hubei University, Wuhan 430062, China Ministry-of-Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei University, Wuhan 430062, China

a r t i c l e

i n f o

Article history: Received 1 September 2011 Received in revised form 16 September 2011 Accepted 19 September 2011 Available online 2 October 2011 Keywords: Low molecular weight chitosan Nanoparticles Monodisperse Ionic cross-linking Drug carrier

a b s t r a c t Chitosan nanoparticles have been extensively studied for drug and gene delivery. In this paper, monodisperse, low molecular weight (LMW) chitosan nanoparticles were prepared by a novel method based on ionic gelation using sodium tripolyphosphate (TPP) as cross-linking agent. The objective of this study was to solve the problem of preparation of chitosan/TPP nanoparticles with high degree of monodispersity and stability, and investigate the effect of various parameters on the formation of LMW chitosan/TPP nanoparticles. It was found that the particle size distribution of the nanoparticles could be significantly narrowed by a combination of decreasing the concentration of acetic acid and reducing the ambient temperature during cross-linking process. The optimized nanoparticles exhibited a mean hydrodynamic diameter of 138 nm with a polydispersity index (PDI) of 0.026 and a zeta potential of +35 mV, the nanoparticles had good storage stability at room temperature up to at least 20 days. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Chitosan is a linear polysaccharide composed of randomly distributed ␤-(1-4)-linked d-glucosamine and N-acetyl-dglucosamine. Due to the advantageous biological properties of chitosan, such as relative non-toxicity, biocompatibility, biodegradability, cationic properties, bioadhesive characteristics and permeability-enhancing properties, chitosan-based particles have been extensively studied for delivery of anti-cancer agents, therapeutic proteins, genes, antigens, and so on [1–3]. In recent years, low molecular weight (LMW) chitosan nanoparticles have shown great potential in the applications of drug delivery and non-viral vector for gene delivery [4–6]. This is because, compared with high molecular weight (HMW) chitosan, LMW chitosan shows better solubility, biocompatibility, bioactivity, biodegradability and even less toxicity [7–9]. Furthermore, many studies have emphasized the significance of size and revealed the advantages of nanoparticles over the microspheres [10]. Among variety of methods developed to prepare chitosan nanoparticles, ionic gelation technique has attracted considerable attention due to this process is non-toxic, organic solvent free, convenient and controllable [11]. Ionic gelation technique is based

on the ionic interactions between the positively charged primary amino groups of chitosan and the negatively charged groups of polyanion, such as sodium tripolyphosphate (TPP), which is the most extensively used ion cross-linking agent due to its non-toxic and multivalent properties [12]. This physical cross-linking process not only avoids the use of chemical cross-linking agents and emulsifying agents which are often toxic to organisms, but also prevents the possibility of damage to drugs, particularly biological agents [13]. However, chitosan/TPP nanoparticles prepared by conventional methods usually have a wide particle size distribution and poor stability, which limit their usefulness in certain applications. Up to now, it is still a challenge to prepare chitosan/TPP nanoparticles with high degree of monodispersity and stability by a convenient and effective method. Thus, in this study, we choose to use LMW chitosan with high degree of deacetylation, and focus on the reproducible preparation of chitosan/TPP nanoparticles with those desirable characteristics, which wished to promote the development of chitosan/TPP nanoparticles in the applications of drug and gene delivery. 2. Materials and methods 2.1. Materials

∗ Corresponding author. Tel.: +86 27 88661237; fax: +86 27 88661571. E-mail address: [email protected] (H. Ni). 1 These two authors contributed equally to this work. 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.09.042

Low molecular weight (LMW) water-soluble chitosan (viscosity 35 cps, deacetylation degree 91.5%) derived from crab shell, was

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W. Fan et al. / Colloids and Surfaces B: Biointerfaces 90 (2012) 21–27

purchased from Jinhu Crust Product Co., Ltd., China. Sodium tripolyphosphate (TPP), glacial acetic acid, sodium hydroxide and all other chemicals were analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd., China. Ultrapure water was used throughout this study.

2.2. Preparation of LMW chitosan/TPP nanoparticles LMW chitosan nanoparticles were prepared according to a modified method of Calvo et al. [14], based on the ionic gelation of chitosan with TPP anions. Based on an optimization procedure designed by us, a number of parameters were investigated by changing one parameter while keeping the others constant. These varying parameters included concentration of chitosan solution, concentration of TPP solution, temperature of chitosan solution, pH of chitosan solution, mass ratio of chitosan to TPP, concentration of acetic acid solution, ambient temperature during cross-linking process and stirring speed. The optimization procedure was as follows: LMW chitosan was dissolved in an aqueous solution of acetic acid to form a 0.5 mg/mL chitosan solution. The concentration of acetic acid was 0.4 times (0.2 mg/mL) that of chitosan. The chitosan solution was stirred overnight at room temperature using a magnet stirrer. The pH of the resulting solution was around 3.6 and this was adjusted to 4.7–4.8 using 20 wt% aqueous sodium hydroxide solution. The chitosan solution was then passed through a syringe filter (pore size 0.45 ␮m, Millipore, USA) to remove residues of insoluble particles. TPP was dissolved in ultrapure water at a concentration of 0.5 mg/mL and also passed through a syringe filter (pore size 0.22 ␮m, Millipore, USA). To prepare chitosan nanoparticles, a magnetic stirrer was placed in a chest freezer, in which the ambient temperature was controlled at 2–4 ◦ C, temperature fluctuations and flow of cold air should be avoided as much as possible. Ten milliliters of chitosan solution in a 25 mL round-bottom flask was preheated in a water bath at 60 ◦ C for 10 min, the flask was then placed on the magnetic stirrer stirring at 700 rpm, 3.0 mL of 2–4 ◦ C TPP solution was quickly added to the chitosan solution with a plastic Pasteur pipette. The reaction was carried out for 10 min and the resulting suspension was subjected to further analysis.

2.3. Characterization and morphology of LMW chitosan/TPP nanoparticles The z-average particle size, particle size distribution and polydispersity index (PDI) of the chitosan/TPP nanoparticles were measured at 25 ◦ C by dynamic light scattering (DLS) on a high performance particle sizer (HPPS-5001, Malvern, UK). The zeta potential values of the nanoparticles were performed using a Zetasizer 3000 HAS (Malvern, UK). The samples were left standing overnight at room temperature for stabilization and were then inverted several times before the measurements. Mean values were obtained from the analysis of three different batches, each of them measured three times. The morphological characteristics of the nanoparticles were examined using a high resolution transmission electron microscope (TEM, Tecnai G20, FEI, Netherlands). A droplet of suspension was placed on a carbon film-covered copper grid (200 mesh) without being stained. Five minutes later, the excess liquid was removed by touching the edge of the copper grid with a piece of filter paper. The sample was then air-dried before observation by TEM.

3. Results and discussion 3.1. Effect of the concentration of chitosan solution or TPP solution The characteristics of the chitosan/TPP particles prepared with different concentrations of chitosan or TPP were studied. The results indicated that the particle size increased with increasing the concentration of either chitosan or TPP. Calvo et al. [14] found that the formation of chitosan/TPP nanoparticles was only possible for some specific concentrations of chitosan and TPP. This fact was also verified in our study that in order to avoid the formation of any micro-particles, the concentration of chitosan or TPP needed to be below 1.5 mg/mL and 1.0 mg/mL, respectively. In these concentration ranges, it seemed that the concentration of chitosan or TPP had little effect on the monodispersity of the nanoparticles, since their PDI values were all below 0.05. It is known that under acidic conditions, there is electrostatic repulsion between chitosan molecules due to the protonated amino groups of chitosan, meanwhile, there also exist interchain hydrogen bonding interactions between chitosan molecules. Below a certain concentration of LMW chitosan (2.0 mg/mL as reported), the intermolecular hydrogen bonding attraction and the intermolecular electrostatic repulsion are in equilibrium [15]. Therefore, in this concentration range, as chitosan concentration increases, chitosan molecules approach each other with a limit, leading to a limited increase in intermolecular cross-linking, thus larger but still nanoscale particles are formed. Above this concentration, microparticles are easily formed probably due to the stronger hydrogen bonding interactions leading to plenty of chitosan molecules involved in the cross-linking of a single particle. The formation of micro-particles usually leads to a flocculent precipitate due to the electrostatic repulsion between particles is not sufficient to maintain the stability of these large particles. It was also found that chitosan at low concentration could form stable nanoparticles even at a low mass ratio of chitosan to TPP, while chitosan at higher concentration could only form stable nanoparticles at a higher mass ratio of chitosan to TPP. For example, when the concentration of TPP was fixed at 0.5 mg/mL, a chitosan concentration of 0.5 mg/mL could form stable nanoparticles at a mass ratio of 3.3:1, while a chitosan concentration of 1.0 mg/mL would form aggregates at this mass ratio. To explain this phenomenon, it is inferred that as chitosan concentration decreases, the intermolecular distance increases, thus leading to a decrease in intermolecular cross-linking between chitosan molecules while an increase in cross-linking density between chitosan and TPP, namely an increase in the ratio of moles of TPP to the moles of chitosan repeating units [13]. This can be utilized to prepare chitosan/TPP nanoparticles with smaller size, since an appropriate increase in the mass ratio is conducive to reduce the particle size, which will be discussed in detail in Section 3.2. 3.2. Effect of the mass ratio of chitosan to TPP Fig. 1 shows the effect of the mass ratio of chitosan to TPP on the particle size and zeta potential by adding different volumes of TPP to 10.0 mL of chitosan solution. With increasing TPP volume from 2.5 mL to 3.5 mL (mass ratio from 4.0:1 to 2.9:1), the particle size first gradually decreased from 172 nm to 133 nm and then increased dramatically to 237 nm. The zeta potential decreased almost linearly from +39 mV to +26 mV due to the neutralization of protonated amino groups by TPP anions. Within this mass ratio range, it seemed that the mass ratio was not the crucial parameter for monodispersity since the resulting PDI values were all below 0.06. However, a TPP volume of 3.6 mL would lead to a flocculent precipitate.


Fig. 1. Effect of the mass ratio of chitosan to TPP on particle size and zeta potential by adding different volumes of TPP to 10.0 mL of chitosan solution.

The large positive charge density due to the high degree of deacetylation and protonation makes chitosan molecules have a large number of potential cross-linking sites. When TPP volume was below 2.5 mL, the reaction solution would be a clear solution without visible opalescence, indicating that the TPP volume was inadequate to lead to the formation of a cross-linked structure of chitosan. As TPP volume increased from 2.5 mL to 3.3 mL, the particle size decreased due to increased cross-linking density between chitosan and TPP. As TPP volume continued to increase from 3.3 mL to 3.5 mL, it can be inferred that chitosan molecules were almost fully cross-linked and the excess TPP would lead to more chitosan molecules involved in the formation of a single nanoparticle, resulting in larger particle size. When TPP volume was 3.6 mL, the low surface charge density of the particles was no longer able to maintain the stability of these large particles during stirring, resulting in the precipitation of particles.

3.3. Effect of the pH of chitosan solution The effect of the pH of chitosan solution on the formation of chitosan/TPP particles was investigated by adjusting the pH from 3.6 to 5.5 (3.6, 4.0, 4.2, 4.5, 4.7, 5.0, 5.2, 5.5). The results indicated that the critical mass ratio of chitosan to TPP for the formation of an opalescent suspension decreased with the pH decreasing. When the pH was below 4.5, it was not easy to produce nanoparticles with unimodal particle size distribution, while when the pH was above 5.2, micro-particles were unavoidable present in the suspension. Chitosan is a weak polyelectrolyte with a pKa around 6.5, the protonation degree of chitosan is mainly controlled by solution pH. Shu and Zhu [12] have studied the relationship between the pH of chitosan solution and the protonation degree of chitosan (the deacetylation degree and viscosity average molecular weight of the reported chitosan were 86% and 460,000, respectively). Their results show that as the pH of chitosan solution increased from 4.7 to 8, the protonation degree of chitosan decreased rapidly from 100% to 0%, indicating that there exists a critical pH above which chitosan starts to be deprotonated. Meanwhile, the charge number and ionic species of TPP are affected by solution pH. In original TPP solution (pH 9.7), the concentration of tripolyphosphoric ions (P3 O10 5− and HP3 O10 4− ) is high but the concentration of hydroxide ions is also present. The hydroxide ions or tripolyphosphoric ions in the medium can competitively react ionically with the protonated amino groups of chitosan by deprotonation or ionic cross-linking, respectively [12,16]. Moreover, the hydroxide ions are supposed

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Fig. 2. Effect of the temperature of chitosan solution on particle size.

to preferentially bind to the protonated amino groups due to their higher mobility [17]. Thus, it is inferred that as TPP solution is mixed with chitosan solution, when the pH of chitosan solution is below the critical pH, the hydroxide ions in TPP solution are partially neutralized by the hydrogen ions in chitosan solution. The remaining hydroxide ions (if any) have little effect on the protonation degree of chitosan and the chitosan maintains its fully protonated state. Then the protonated amino groups are linked via tripolyphosphoric ions in spite of the strong intramolecular electrostatic repulsion. When the pH of chitosan solution is around the critical pH, the remaining hydroxide ions may have an apparent effect on the protonation degree of chitosan and the chitosan becomes less extended, which favors the formation of a particle structure. When the pH of chitosan solution is above the critical pH, the chitosan is already less protonated and the remaining hydroxide ions will cause a significant decrease in the protonation degree of chitosan and the potential capacity of chitosan to form cross-linking with tripolyphosphoric ions is supposed to decrease.

3.4. Effect of the temperature of chitosan solution The effect of the temperature of chitosan solution on the particle size was studied. As shown in Fig. 2, the particle size displayed a clear tendency to diminish when the temperature was increased from 10 ◦ C to 60 ◦ C, while when the temperature was above 60 ◦ C, the particle size was slightly decreased. When the temperature was 10 ◦ C, the PDI value of the resulting particles was 0.112, while when the temperature was between 25 ◦ C and 70 ◦ C, the resulting PDI values were all below 0.05. It was reported that the intrinsic viscosity of chitosan solution decreases linearly with increasing solution temperature. This is because as solution temperature increases, the ratio of radius of gyration of chitosan is decreased and the hydrogen-bonded hydration water of chitosan is reduced, resulting in an increase in chitosan chain flexibility and a decrease in specific volume of chitosan molecule, respectively [18]. Both these effects will probably facilitate the approaching of chitosan molecules and promote the formation of a compact structure during crosslinking process, resulting in a decrease in particle size. However, when the temperature is above 60 ◦ C, above effects probably tend to a limit, and thus the decrease in particle size is negligible.

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Table 1 The characteristics of chitosan/TPP particles prepared at different ambient temperatures. Ambient temperature (◦ C) 16–25 10–15 0–4 a

Mean particle size range (nm)

Particle size distributiona

PDI

150–160 135–150 130–140

Bimodal Broad unimodal Narrow unimodal

>0.2 0.1–0.15 4.0:1). For example, as the mass ratio of chitosan to TPP was 5.0:1, the mean particle size, PDI and peak width of the particles were 315.6 nm, 0.081 and 71.1 nm, respectively. At an acetic acid concentration of 0.1 mg/mL, the particles exhibited a bimodal particle size distribution, centered around 164 nm and 850 nm, respectively. However, an acetic acid concentration of 0.2 mg/mL would lead to a unimodal and narrow particle size distribution, indicating that an appropriate increase in the concentration of acetic acid is conducive to enhance the degree of protonation of chitosan, which increases the potential capacity of chitosan to form cross-linking with TPP. When the acetic acid concentration continued to increase to 0.5 mg/mL and 0.8 mg/mL, comparisons of their particle size distributions (by intensity, by number and by volume, respectively) are shown in Fig. 4. The results indicate that as the acetic acid concentration is increased from 0.2 mg/mL to 0.8 mg/mL, there are not only more smaller particles formed, but also an increase in the number of larger particles, resulting in a decrease in the monodispersity of the particles. As the concentration of acetic acid is increased from 0.2 mg/mL to 0.8 mg/mL, an increased amount of NaOH should be consumed to neutralize the excess acid and adjust the chitosan solution to pH 4.7–4.8 since this pH is conducive to the formation of chitosan/TPP nanoparticles with a unimodal size distribution, causing an increase in the ionic strength of the chitosan solution. In dilute solution, the intermolecular interaction forces are weak because of the long distance between chitosan molecules, thus the conformation of the molecules is the most important parameter that determines the physical properties [19]. In low ionic strength solution, the intramolecular electrostatic repulsion effect, also called the third electroviscous effect dominates, resulting in the chitosan molecules exist in an extended conformation. However, as the ionic strength increases, the concentration of counter-ions is raised which screens the protonated amine groups, and thus

Table 2 Effect of the concentration of acetic acid on the characteristics of chitosan/TPP particles.a

Fig. 3. Cooling curves of chitosan/TPP nanoparticles suspensions reaction at different ambient temperatures: (A) 25 ◦ C, (B) 15 ◦ C, (C) 4 ◦ C and (D) 4 ◦ C with a flow of cold air. (The data were measured by a temperature probe inserted into the suspension, sampling rate 1 s, resolution 0.06 ◦ C.)

Acetic acid (mg/mL)

Size (nm)

Peak widthb (nm)

PDI

0 0.1 0.2 0.5 0.8

Precipitation 196.0 138.1 138.4 137.6

– Bimodal 25.4 51.3 67.2

– 0.204 0.026 0.083 0.124

a The mass ratio of chitosan to TPP was 3.3:1 and the pH of chitosan solution was adjusted to 4.7–4.8, except when the acetic acid concentration was 0 mg/mL, of which pH remained unchanged. b The values were obtained from the particle size distribution by intensity and were given by the HPPS.


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aggregation of particles into larger, resulting in a broader particle size distribution. So it can be concluded that a high acetic acid concentration (or in other words, a high ionic strength) is not conducive to a narrow particle size distribution. 3.7. Effect of the stirring speed We had also investigated the effect of stirring speed on particle size distribution. The results showed that the particle size distribution was significantly narrowed by increasing stirring speed from 200 rpm to 800 rpm. However, although a continued increase in stirring speed would lead to an even narrower main peak, there was also an aggregate formed at around 1000 nm. This probably because adequate stirring can accelerate the dispersion of TPP in chitosan solution and the increased shear force is help to improve the monodispersity, while intense stirring may destroy the repulsive force between particles and lead to aggregation of particles. 3.8. Morphology of LMW chitosan/TPP nanoparticles

Fig. 4. Particle size distributions (A) by intensity, (B) by number and (C) by volume of chitosan/TPP nanoparticles prepared with different acetic acid concentrations.

the intramolecular electrostatic repulsion force decreases, which makes the molecules contracted [20]. Therefore, the increased acetic acid concentration indirectly causes an increase in the ionic strength, the less extended chitosan molecules and the increased shielding effect of counter-ions (CH3 COO− ) make chitosan molecules have less cross-linking points to be accessed by TPP. Meanwhile, chitosan molecules with a contracted worm like chain conformation entangle harder than those with an extended conformation [15]. For these reasons, fewer chitosan molecules involved in the formation of a single nanoparticle and thus a greater number of smaller nanoparticles are formed (Fig. 5). On the other hand, the increased shielding effect will decrease the electrostatic repulsion between particles, and moreover, the increased electrolyte ions will reduce the thickness of the surface hydration layer of the particles [21], which facilitate the

The typical morphology of the chitosan/TPP nanoparticles prepared at optimal conditions is shown in Fig. 6. The nanoparticles exhibited a spherical shape and had a narrow particle size distribution with size in the range of 30–50 nm. The discrepancy in the size of chitosan nanoparticles between DLS and TEM can be that chitosan nanoparticles swell in aqueous media and DLS gives a hydrodynamic diameter of nanoparticles, while TEM gives an actual diameter of nanoparticles in dry state. The part of aggregation of the chitosan/TPP nanoparticles is probably because that the hydrogen bonding interactions between chitosan nanoparticles gradually become dominant in the drying process. It also can be noticed that these nanoparticles have a deeper color in the core and surface, indicating that these regions have higher electron density distribution. As TPP contains phosphorus element, which has a higher electron density than those elements of chitosan, so it can be inferred that chitosan has higher degree of cross-linking density with TPP in these regions. 3.9. Colloid stability The colloidal stability of the chitosan/TPP nanoparticles prepared at optimal conditions was evaluated by storage at room temperature (10–20 ◦ C) for different periods of time. The results showed that after stored at room temperature for 20 days, the liquid

Fig. 5. Schematic representation of ionic crosslinking reaction between chitosan and TPP in (A) low ionic strength solution and (B) high ionic strength solution.

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of chitosan/TPP particles. These effects probably also can serve to enhance the mechanical strength of the particles, thus favor the encapsulation and slow release of drug. At last, the formation mechanisms presented in this study may also be applied to prepare monodisperse nanoparticles based on chitosan derivatives, such as N-trimethyl chitosan and carboxymethyl chitosan, which also have promising applications in biomedical fields [33,34].

Acknowledgment The authors appreciate Jinzhao Wang and Qiwei Yang for their assistance in this work.

References

Fig. 6. TEM image of chitosan/TPP nanoparticles without being stained.

was still a clear suspension with light blue opalescence and the nanoparticles did not show any statistically significant change in particle size and its distribution during the investigation (data not shown). It was suggested that chitosan/TPP nanogels behaved as a metastable system, since they underwent spontaneous aggregation and disintegration under very mild conditions in a short period of time [22]. Our results indicate that these monodisperse chitosan/TPP nanoparticles have excellent storage stability, which probably due to the low ionic strength environment, appropriate pH conditions (the final pH of the suspension was around 5.4), together with the high cross-linking density, small size, narrow particle size distribution and high surface potential of the particles [13,16,23]. Reducing the storage temperature to 4 ◦ C or using freeze-drying with a cryoprotective agent is expected to further improve the long-term storage stability of the chitosan/TPP nanoparticles [22,24,25]. 4. Conclusions Although the drug delivery system based on chitosan/TPP nanoparticles has its own advantages, the polydispersity and poor stability of the colloids seriously limit the efficiency of nanoparticle-mediated drug delivery. In a polydisperse system, larger nanoparticles usually have higher drug loading capacity, while smaller nanoparticles are expected to have higher efficiency of delivering drug to tissues or cells [26]. This contradiction means that even if the drug carrier system has a high encapsulation efficiency, the delivery efficiency may be poor. Thus, through the novel method developed by this study, which allows a highly repeatability of producing monodisperse, LMW chitosan/TPP nanoparticles, it is expected to further improve the efficiency of drug utilization, especially for highly size-dependent applications, such as gene transfer or delivery of drug via mucosal routes [27,28]. In addition, several attempts have been made to increase the encapsulation and release period of drug, and the major problem encountered is the initial burst release of drug [29–31]. This is partly associated with the low mechanical strength of the chitosan/TPP particles, and the initial burst release can be reduced by enhancing the mechanical strength of the particles [32]. It has been found in this study that an increase in the temperature of chitosan solution and a decrease in the ionic strength of chitosan solution are expected to promote the formation of a compact and sufficient cross-linking structure

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