Preparation of bio-polymeric nanoparticles by electrostatic field system

Yu-Hsien Kao1,2, Shwu-Jen Chang1, Chin Wen Chuang2, Shyh-Ming Kuo1, Shu-Ying Chen1, Ru-Ting Lu1

1Department of Biomedical Engineering, I-Shou University, No. 8, Yida Road, Yanchao District, Kaohsiung City 82445,Taiwan2Department of Electric Engineering, I-Shou University, No. 8, Yida Road, Yanchao District, Kaohsiung City 82445, TaiwanE-mail: smkuo@isu.edu.tw

Published in Micro & Nano Letters; Received on 1st August 2012; Revised on 13th September 2012

This Letter describes a new method to prepare collagen, gelatin, chitosan and hyaluronic acid nanoparticles by using an electrostatic fieldsystem (EFS) in aqueous phase. Observations from transmission electron microscopy shows that good spherical shape and well dispersed col-lagen, gelatin, chitosan and hyaluronan nanoparticles ranging from about 4–200 nm in diameter could be produced under certain experimentalsettings, including the usage of various cross-linking reagents. In short, the lower temperature setting about 48C is needed when preparingcollagen I nanoparticles; however, a higher temperature setting about 178C and a higher concentration of collagen II at 0.2 mg/ml isneeded to prepare collagen II nanoparticles. Gelatin nanoparticles could be produced under a longer treatment period at 5 h; however, thegelatin nanoparticles aggregates together yielding a larger cluster at a longer 5 h treatment period. Chitosan and hyaluronan nanoparticlescould only be produced under low concentrations of cross-linking reagents treatment. Working ranges of the important parameters for the pro-duction of bio-polymeric nanoparticles beyond the traditional technique have been defined. EFS technology provides a simple yet powerfultechnique for fabricating bio-polymeric nanoparticles. In fact, these nanoparticles are used as ingredients, scaffolds or carriers in the chondro-cytes, hepatocytes and stem cells differentiation applications all show promising and encouraging results.

1. Introduction: As known, nanoparticles show unique physicaland chemical properties that are different from those of the conven-tional materials, owing to their ultra-fine size and at a sub-cellularlevel. These nanoparticles materials offer extraordinary potentialfor development of new materials: for example, the ability to dis-perse in water to form a clear colloid solution that promotes intimatecontact at the interfaces for improving poor water solubility of drugsuptake and thereafter reducing the use of drug dosage [1, 2].Generally speaking, an increased surface area, high adsorptivecapacity and the size reduction in the nanometre size rangeprovide a critical yet comfortable dimension that is conducive tophysiophysiological phenomena and functions at a compatiblelevel. The idea to match the domain sizes of the interacting sub-stances to a manageable level is nothing new, but only with theadvances in nanotechnology in recent years we can begin to putthis aspiration into practice in many fields and especially in biome-dical engineering [3–5].

Until now, many efforts or methods have been devoted to thepreparation of nanoparticles including emulsification-solvent evap-oration and precipitation methods [6, 7]. Dry and wet milling pro-cesses are also widely used to reduce the particle size to preparenanoparticles. However, these techniques result in broad particlesize distribution and non-uniformity in particle surface character-istics [8]. Long milling processing times are often required toachieve a particle size of nanometre scale and decrease its practical-ity in tissue engineering field. Also, organic solvent and de-oilingprocesses in the emulsification-solvent evaporation would impedethe use in live cells and tissues carrier applications. This studydescribes a new method for the production of bio-polymeric nano-particles by using an EFS method in aqueous phase environment.By controlling and setting the processing conditions, the appliedelectrostatic field strength and waveform, the temperature, the bio-polymeric solution concentration and reaction period, a series ofbio-polymeric nanoparticles were prepared.

Bio-polymeric materials or biomaterials, offer the advantage ofbeing similar to macromolecular substances of the biologicalenvironment, which demonstrates biocompatibility. Furthermore,an interesting characteristic of bio-polymeric materials is theirability to be degraded by naturally occurring enzymes, which guar-antee that the implant will be eventually metabolised by physiologi-cal mechanisms. Collagen is a protein, which forms the majority

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part of extracellular matrix. Many research efforts have focusedon how to produce an ordered collagen matrix and demonstratethe cells cultured in this matrix having better growth close to thatof in vivo environment [9]. However, the prepared collagen matriceswere usually the fibrillar structural forms. It appears that interest inproviding and studying particular structures of collagen matriceswould probably lead to new vision and applications in the biomater-ial fields, especially in the nanometre size of particles. This Letterdescribes a new method for the production of bio-polymeric nano-particles by an electrostatic field system (EFS). The optimal prepar-ing parameters on the nanoparticles were characterised and analysedby transmission electron microscopy (TEM). The preliminaryresults demonstrated that, if we use certain experimental settings,under an appropriate electrostatic field environment, we canproduce nanoparticles with good spherical shape and in a sizerange of about from 4 to 200 nm in diameter.

2. Experimental2.1. Materials: Chitosan was purchased from TCI (Tokyo, Japan)with a molecular weight of 3 × 105 Da and deacetylation degreeof 83%. Sodium tripolyphosphate (Na5P3O10, 5%) and sodiumhydroxide (NaOH, 1N) were purchased from SHOWA (Tokyo,Japan). Gelatin, alginate and acetic acid were purchased fromSigma (St. Louis, MO). Hyaluronan, with a molecular weight of9 × 105 Da (from Streptococcus zooepidemicus) was purchasedfrom Fluka (Switzerland). Type I and Type II collagens wereextracted and purified from calf skin and calf articular cartilage,respectively, in our laboratory. All chemicals used in this studywere of reagent grade. High-voltage power supply was suppliedby Bertan (series 230, USA). Function waveform generator wassupplied by Hewlett-Packard (series 33120A, USA).

2.2. Construction of EFS and production of bio-polymeric nanopar-ticles: An EFS was constructed by using two parallel plates (Cooperplate, 18 × 6 cm) with a gap distance of 2 cm. The electrostaticfield strength between two plates were supplied by a DC powersupply and controlled by a function generator (fixed as 50 mHzsquare waveform in this study). The whole system was placedinside a thermal-control chamber for safety purposes and set to adesignated temperature. 1 ml of bio-polymeric solution (0.2 mg/ml) was poured into a plastic Petri dish and then placed at the

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centre position of the two plate electrodes. The optimal experimen-tal settings, including temperature, applied electrostatic fieldstrength, concentration of bio-polymeric solution, the reactiontime and cross-linking reagents were varied and examined toprepare a good spherical shape of nanoparticles. The schematicpresentation of experimental EFS and the procedures in productionof bio-polymeric nanoparticles are shown in Fig. 1.

2.3. Electron microscopy observation: To evaluate the prepared bio-polymeric nanoparticles under the influences of the above-men-tioned experimental settings, samples of each experimental runwere taken at the predetermined periods of time and immediatelyplaced on Formvar-coated Cooper grids. These grids were thennegatively stained with 1.5% phosphotungstic acid. After that,these dry grids were examined and analysed under transmissionelectron microscope (Philips, Model CM 200, the Netherlands)observation.

3. Results and discussion: Table 1 shows the optimal experimentalsettings of bio-polymeric solution for preparing spherical and well-dispersed nanoparticles by the EFS method. Alginate could notproduce spherical nanoparticles but only flask-shaped particles,because of the high viscosity and instant cross-linking reactionwith CaCl2 reagent. However, we could produce collagen I, col-lagen II, gelatin, chitosan and hyaluronan nanoparticles of goodspherical shape with a diameter in the range of 4–200 nm by con-trolling certain experimental settings, including the usage of variouscross-linking reagents. In the case of collagen I and collagen IInanoparticles preparation, because of an entropy-driven processthat is termed fibrillogenesis, a larger force causing the collagenmolecules to self-assemble and then form collagen fibrils, the

Figure 1 Schematic presentation of EFS and preparation procedures forbio-polymeric nanoparticles

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temperature settings require crucial controlling. The nanoparticlescould be produced under a relative lower temperature setting(Figs. 2a and b). A fibrous structure of collagen was formedinstead of a nanoparticle shape at the temperature setting of 378C(Fig. 2c). On the contrary, gelatin, a product of partially hydrolysedcollagen, could be produced into gelatin nanoparticles about 20 nmin diameter under a less strict temperature setting (Fig. 3a). Thegelatin nanoparticles aggregated together yielding a larger clusterat a longer 5 h treatment period (Fig. 3b).

Perhaps because of the compositional component of chitosan,polysaccharide structure [poly(1,4)-b-D-glucopyranosamine], itneeds a longer treatment period of (5 h) and relative low concen-tration of cross-linking reagent to produce good spherical shapeof chitosan nanoparticles (Fig. 4b). Parts of the chitosan moleculesbegun to coil together and formed less spherical particles, on thecontrary, more irregular shape of chitosan sheets formed under1 h treatment period (Fig. 4a). Furthermore, we could produce chit-osan nanoparticles of the size about 5 nm in diameter by usingnegative-charged alginate as cross-linking reagent with similarexperimental settings (Fig. 4c).

Fig. 5 shows the TEM observation of hyaluronan nanoparticlesprepared by EFS method under the experimental settings set at elec-trostatic strength 2.5 kV/cm, treatment period of 1 h and 0.001 NFeCl3 as cross-linking reagent (Fig. 5). It was noted that it

Figure 2 Transmission electron micrographs of nanoparticles froma Collagen Ib Collagen IIc Collagen I, temperature set at 378CExperimental settings were listed at Table 1

Table 1 Characteristics of bio-polymeric nanoparticles

Electrostatic field strength, kV/cm Reaction period, h Reaction temperature, 8C Cross-linking reagent Diameter, nm

Collagen I 2.5 1 4 NU �10Collagen II 2.7–4.5 3 17 NU �4Gelatin 2.5 3 25 NU �20Chitosan 2.5 1 25 0.01N NaOH IS

2.5 3 25 0.01N NaOH IS2.5 5 25 0.001N NaOH 100–2002.5 5 17 alginatea �5

Hyaluronan 2.5 1 25 0.001N FeCl3 �52.5 3 27 0.001N FeCl3 IS

NU: not used; IS: irregular shape.a0.0134 mg alginate added.

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Figure 3 Transmission electron micrographs of gelatin nanoparticlesa Experimental setting: 228C, electrostatic strength set at 2.5 kV/cm,treatment period set at 3 hb Same with (a), except that the treatment period was set at 5 h

Figure 4 Transmission electron micrographs of chitosan nanoparticlesa Experimental settings: electrostatic strength set at 2.5 kV/cm, treatmentperiod set at 1 h, cross-linking reagent 0.01 N NaOHb Treatment period set at 5 h, cross-linking reagent 0.001 N NaOHc Same with (b) except for the cross-linking reagent: alginate

Figure 5 Transmission electron micrographs of hyaluronan nanoparticlesprepared by EFS methodExperimental settings: electrostatic strength set at 2.5 kV/cm, treatmentperiod set at 1 h, gelation reagent 0.001 N FeCl3

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needed a strict experimental setting at treatment period and the con-centration of gelation reagent to produce hyaluronan nanoparticleswith good sphericity. It was probably contributed to low mechanicalstrength of hyaluronan, the hyaluronan molecules formed debrisinstead of spherical particles, under a treatment period longer than1 h. The preliminary results indicate that spherical hyaluronan nano-particles could only be produced after a mild electrostatic treatmentand gelation reaction in aqueous environment.

4. Conclusions: This Letter shows that we can prepare collagen,gelatin, chitosan and hyaluronan nanoparticles under certain set-tings. We have investigated the critical conditions for temperaturerange, electrostatic field strength applied and reaction reagentsthat can produce nanosized particles. For collagen materials, astrict lower temperature setting is necessary to produce collagennanoparticles. When the temperature is increased to 378C, the spon-taneous reconstitution of collagen I and collagen II would take overand form the normal fibrillar structure. However, gelatin material isinsensitive to the temperature variation and it is easier to preparenanosized particles under the same electrostatic system and exper-imental settings. For chitosan and hyaluronan materials, we canproduce nano-sized particles by selection of mild cross-linking reac-tion of low concentrations of NaOH and FeCl3, respectively. Fromthe preliminary results obtained, we may expect that the waveformof the applied voltage (50 mHz square waveform) used in this studythat produce alternating electrical polarity of the EFS (shown inFig. 1), also, the high voltage applied to a dilute bio-polymeric sol-ution may have dissociated the bio-polymer into separated stringand then these individual strings have coiled into nanoparticlesunder this flip-flop electrostatic field environment. In fact, thedetailed formation mechanisms, especially the applied waveform,which influence the size of nanoparticles of these results, stillneed to be explored.

However, it is interesting to note that this is the first attempt toproduce bio-polymeric nanoparticles directly under an EFSmethod in aqueous environment. We have coated collagen I nano-particles onto an asymmetric chitosan membrane [Chitosan mem-brane cross-linked with genipin, (CG membrane)] to prepareChitosan membrane cross-linked with genipin/collagen nanoparti-cles (CGC membrane). Collagen I nanoparticles were uniformlydistributed throughout the CGC membrane and had good sphericalshapes with diameters of 20–30 nm as shown in Fig. 6. This CGCmembrane exhibited better cell growth. Moreover, an in vivo histo-logical assessment indicated that covering the wound with the CGCmembrane resulted in quicker epithelialisation and reconstruction ofthe wound than CG membrane or commercial wound dressing [10].The obtained results indicated that addition of collagen I nanoparti-cles may enhance or improve the cellular behaviours and yieldanother potential of traditional materials applications either inbasic material engineering or tissue engineering. In fact, chitosannanoparticles are being used in DNA vaccine carriers and are

Figure 6 Field emission scanning electron micrographs of the CGC mem-brane containing collagen I nanoparticles [9]

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under evaluation now. Collagen I nanoparticles are used in the cul-tivation of mesenchymal stem cells to differentiate into cardiomyo-cytes by a demethylating agent, 5-azacytidine. Addition of collagenII nanoparticles into the culturing medium, could improve/enhancethe secreting functions of glycosaminoglycan of chondrocytes. Thetemporary results obtained using these bio-polymeric nanoparticleseither used as ingredients, scaffolds or carriers in the chondrocytes,hepatocytes and stem cells differentiation all show promising andencouraging results.

5. Acknowledgment: This study was financially supported by agrant from the National Science Council, Taiwan (grants NSC99-2632-E-214-001-MY3 and DOH100-TD-N-111-011).

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Micro & Nano Letters, 2012, Vol. 7, Iss. 10, pp. 997–1000

doi: 10.1049/mnl.2012.0588