MODIFIED CHITOSAN NANOPARTICLE BY RADIATION SYNTHESIS: AN APPROACH TO DRUG DELIVERY AND BIO-BASED ADDITIVE FOR BIOMEDICAL APPLICATIONS

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1 MDIFIED ITSAN NANPARTILE BY RADIATIN SYNTESIS: AN APPRA T DRUG DELIVERY AND BI-BASED ADDITIVE FR BIMEDIAL APPLIATINS W. Pasanphan, P. Rimdusit, T. Rattanawongwiboon, S. hoofong; TAILAND Summary Self-assembly chitosan nanoparticle (snp) has been synthesized via radiolytic methodology using gamma irradiation. The systematic condition in preparation was studied. hitosan nanoparticle was modified using hydrophobic core of deoxycholic acid (D) and stearyl methacrylate (SMA) and the hydrophilic shell of polyethylene glycol monomethacrylate (PEG). The hydrophobic/hydrophilic snp was prepared for drug carrier molecule. The SMA-sNP was also conjugated with pyperidine, hindered amine light stabilizer function, to achieve a bio-based additive for biomedical plastic. 1. INTRDUTIN Naturally occurring polymers, especially polysaccharide such as chitosan and alginate, have been in recent years extensively researched as a primary material in forming carriers. hitosan is a biodegradable polysaccharide derived by partial deacetylation of chitin, which is a copolymer of glucosamine and N-acetyl-d-glucosamine linked together by β glycosidic bonds [1]. hitosan can be degraded into N-acetyl glucosamine by general lysozymes in the body, which is subsequently excreted as carbon dioxide via the glycoprotein synthetic pathway [2]. hitosan has been widely used in pharmaceutical and medical areas, due to its favorable biological properties such as biodegradability, biocompatibility, low toxicity, hemostatic, bacteriostatic, fungistatic, anticancerogen, and anticholesteremic properties [3]. It is well known that polymer micelles have unique core shell architecture that composed of hydrophobic segments as internal core and hydrophilic segments as surrounding corona in aqueous medium. The hydrophobic core provides a loading space for water-insoluble drugs, whereas the modification of hydrophilic shell affects pharmacokinetic behavior [4]. Additionally, the nanoscaled polymer micelles exhibit many advantages for the use of drug delivery carriers, such as prolonged circulation; tumor localization by enhanced permeability and retention (EPR) effect [5]; and the controlled drug release by using stimuli-sensitive copolymers [6]. Self-assembly through chemical modification is one of the most widely used processes for chitosan nanoparticle fabrication. This aggregation process, caused by incorporation [7] of hydrophobic organic substances into micelles, can play very important roles in adsorption, transfer, and slow release of drug when treating humans. Various hydrophobilized polymers have been synthesized by modifying an organic group on a hydrophilic polymer [8]. Phthaloylchitosan mpeg as a hydrophobic/hydrophilic microsphere with the average size of 1500 nm was developed [9]. Deoxycholate-modified chitosan has widely been proposed as solid capsules for lipid drug carriers. Pang et al. [10] reported their deoxycholate-chitosan with the size of nm and it had the ability to encapsulate tocopherol acetate and stearic methyl ester. Kim et al. [11] carried out a study to control the particle size and size distribution of self-aggregates of deoxycholic acid-modified chitosan as a gene delivery carrier within the range nm. 188

2 Based on the previous reports, chemical modification is the most widely used protocol in preparing hydrophobic/hydrophilic self-assembly chitosan nanoparticles. In this way, the conjugating agent as well as the purification steps is required. In addition, the nanoscale material has been defined to be less than 100 nm [12]. Therefore, the systematic protocol in reducing and controlling the particle size of chitosan still requires further studies to achieve not only the simple and effective preparation protocol but also a superior potential product. In the present work, radiolytic methodologies to modify chitosan nanoparticle were studied. The modified chitosan nanoparticles are mainly proposed into two medical application purposes, i.e. (i) drug carrier for drug controlled release and (ii) bio-based additive for biomedical plastic. Based on this view point, the systematic examination of fabrication condition of self-assembly chitosan nanoparticle using radiolytic methodology is investigated. Gamma radiation from o-60 was used as the high energetic radiation in order to produced radiolysis products and subsequently induces radical reaction. This mild process would be expected as a novel simple and effective protocol in preparing self-assembly chitosan nanoparticle containing hydrophobic core with hydrophilic shell as a drug carrier device. The procedure would also be extended to prepare the antioxidant conjugated hydrophobic-modified chitosan nanoparticle as an antioxidant nanofiler for medical plastic, e.g. polylactic acid (PLA). 2. MDIFIED ITSAN NANPARTILE BASED SELF-ASSEMBLY AS A DRUG ARRIER FR DRUG NTRLLED RELEASE SYSTEM 2.1. Systematic preparation study on chitosan nanoparticle formulation via gamma irradiation [13] In this section, the direct effect of gamma radiation on the particle formulation due to the physical of chitosan was studied. Three route of chitosan formations, i.e.s-flake, s-colloid, and s-acid were used. Fig. 1 indicates the effect of irradiation condition due to chitosan formulation. The observation from TEM gave us information on the particle shape and the determination of particle size. The irradiations of the s-flake (Fig. 1(a)) and s-colloid (Fig. 1(b)) gave the individual spherical shape with the average size of 95 and 2 nm, respectively. n the other hand, it was difficult to identify the irradiated hi-acid as a spherical shape. hitosan particles from hi-acid were found in variable shapes, i.e. spherical, whisker, spiral, etc. 189

3 a b FIG. 1. TEM images and size distribution of chitosan nanoparticle from 10 kgy gamma irradiation to (a) s-flake and (b) s-colloid (Pasanphan et al., 2010). Fig. 2(a) and (b) shows that the higer the radiation dose, the smaller the particle size. Irradiation of s-colloid seems to bring the particle size down to lower than that of s-flake. The particle sizes are down to less than 100 nm, i.e nm, when using g-ray dose as low as 5 kgy. The variations in particle shape and size were observed in hi-acid condition. ur observation implied that the s-colloid would serve as a proper physical formulation in preparing chitosan nanoparticle via gamma irradiation [Error! Bookmark not defined.]. s-flake s-colloid s-acid FIG. 2. Relationship between particle size and gamma radiation dose of (a) s-flake and s-colloid and (b) s-acid (Pasanphan et al., 2010). 190

4 Particle size (nm) Particle size (nm) 2.2. Preparation of deoxycholate-chitosan nanoparticle (D-s): chemical conjugation of D onto gamma irradiated chitosan To formulate the hydrophobic core to irradiated chitosan, deoxycholic acid (D) was chemically conjugated onto irradiated s-flake and s-colloid. The preparation was carried out in a heterogeneous system according to our previous report [14]. hitosan nanoparticles obtained in the previous preparation were in the formulation of colloid. We suspected that the D could be conjugated on the surface of the colloidal particle. (A) (B) Dose (kgy) d a b c Dose (kgy) a b c FIG. 3. (A) Paticle size of (a) irradiated s-flake, (b) irradiated s-flake after conjugated with D, (c) irradiated s-colloid, (d) irradiated s-colloid after conjugated with D[Error! Bookmark not defined.].(b) Particle size of (a) s-colloid, (b) D-s from heterogeneous, and (c) D-s from homogeneous reactions [13, 15]. In the aqueous hydrophilic system, the self-assembly formulation occurred when the hydrophobic conjugated-d side groups turned into the interior particle to serve as hydrophobic core. Fig. 3(A) indicated that the pre-irradiation dose, physical formulation of chitosan, and D conjugation influences to the size of D-s nanoparticle. The average sizes of D-s were observed to be as small as 46 nm for s-colloid (Fig. 3d) and 72 nm for hi-flake (Fig. 3b) when D was conjugated onto 10 kgy gamma pre-irradiated chitosan in Fig. 3(c) and (a), respectively. Fig. 3(B) shows that the 10 kgy pre-irradiation could reduce the particle size of D-s when conjugating under simple heterogeneous reaction, which only ED conjugating agent was used. Pre-irradiation before Dconjugation would be alternative protocol instead of the homogenous reaction, which the ED/NS system under several steps of preparation should be carried out [15] PEG modified hydrophobic-chitosan nanoparticle gamma radiation synthesis In the previous section, the effect of pre-irradiation dose to D-s particle shape and size was observed. nly hydrophobic core was prepared in such view point. ere, the radiolytic methodology in preparing the polyethylene glycol (PEG) hydrophilic-shell is proposed. 191

5 3 2 N N N3 N 2 3 N3 N N N N 2 N2 3 N2 3 N N PEG modified deoxycholate-chitosan nanoparticle (D-s-PEG) Polyethylene glycol monomethacrylate was grafted onto the surface of D-s particle under aqueous mild system using gamma irradiation with the dose of 2, 4, 6, 8, and 10 kgy (7.7 kgy/h, o-60). TEM images in Fig.4 clearly show the PEG shell of D-s-PEG particle even 2 kgy irradiation. (a) (b) (c) (d) (e) 100 nm 100 nm FIG. 4. TEM images of PEG shell conjugated onto D-s (D-s-PEG nanoparticle) 100 nmprepared from radiation synthesis with different doses of (a) 2 kgy, (b) 4 kgy, (c) 6 kgy), (d) 8 kgy, and (e) 10 kgy). Atomic force microscope (AFM) micrograph (Fig. 5a) confirms the nanoparticle formation as expected (Fig. 5b). The image implied that the core area of D-s-PEG may provide the hollow core as the morphology seem to collapse in the middle portion after drying on the mica substrate. (a) N3 2 N3 3 N 3 N N3 N3 3 3 N N 3 3 N 2 3 N 3 N 2 3 N 2 N2 N3 3 2 N 3 N 2 3 N3 3 N N N 3 N N N N 2 N2 3 N2 N N 3 3 N 2 N N N N N N2 N N2 3 N2 N 2 N2 N2 N2 3 N N N 3 N 2 N3 2 N 3 N 3 2 N 3 N 3 N3 N 3 N N PEG (b) Deoxycholate hitosan 3 FIG. 5. (a) AFM images and (b) schematic draw of D-s-PEG nanoparticle. 192

6 Size (nm) Fig. 6 shows the effect of radiation dose on the %grafting of PEG onto D-s and subsequently affected to hydrophilic shell thickness. The consistency of shell thickness and the % grafting was observed (Fig 6A). Particle size was approximately 80 nm in the case of 2 kgy (Fig. 6A). (A) (B) FIG. 6. (A) Plot of particle sized and %grafting against radiation dose of D-s-PEG nanoparticle. (B)Particle, core, and shell size of D-s-PEG PEG modified stearyl-chitosan nanoparticle (SMA-s-PEG) The preparation of SMA-s-PEG was completely carried out via radiolysis reaction. Strearyl methacryllate was instead used as hydrophobic core of chitosan to form steary-chitosan (SMA-s). In the second step, the PEG was then be grafted onto SMA-s via radiolytic methodology as mention in the previous section. The comparative TEM images and size distribution plot (Fig. 7) suggested that SMA-s-PEG gives very small particle size approximately 40 nm with narrow size distribution (Fig. 7b) when compared to the SMA-s (Fig. 7a). Additionally, it was found that this preparation system gave the self-assembly chitosan nanoparticle with the size less than D-s- PEG (Fig. 7c). 193

7 nm (a) 200 nm 200 nm 40 6 nm (b) nm (c) 100 nm 100 nm FIG. 7. TEM images and size distribution plot of (a) SMA-s, (b) SMA-s-PEG, and (c) D-s-PEG. 194

8 3. MDIFIED ITSAN NANPARTILE AS A GREEN ADDITIVE FR MEDIAL PLASTI 3.1. SMA-grafted-chitosan nanoparticles (SMA-s) using gamma radiation synthesis Stearyl methacrylate was grafted onto non-chitosan (s0) and 40 kgy pre-irradiated chitosan (s40) via radiolytic methodology. The s:sma of 1:5 gave the highest %grafting of SMA onto s0 at 25 kgy (Fig. 8a) and that of 1:7 for s40 at 10 kgy (Fig. 8b). (a) (b) FIG. 8. SMA-grafing percentage of (a) SMA-s0 and (b) SMA-s40 with various s: SMA ratio and radiation grafing doses. The particle sizes of SMA-s0 and SMA-s40 were determined in the condition that the individual particles were formed as shown in Fig. 9. At the same radiation grafting dose the particle size of SMA-s40 was found to be smaller than SMA-s0. SMA-s0 (1:5) SMA-s40 (1:5) NA NA FIG. 9. omparative particle size between SMA-s0 and SMA-s40 at s:sma of 1:5 moles grafting under various radiation doses Pyperidine (PPD) conjugated onto SMA-s nanoparticles (SMA-s-PPD) The SMA-s0 (1:5 SMA:s, 25 kgy) and SMA-s40 (1:7 SMA:s, 10 kgy) was chemically conjugated with pyperidine (PPD) molecule to obtain SMA-s0-PPD and SMA-s40-PPD, respectively. Fig. 10 indicates the smaller particle size observed after SMA-s0 and SMA-s40 195

9 were conjugated with PPD. The particle size of SMA-s0-PPD and SMA-s40-PPD were approximately 85 and 60 nm. (A) SMA-s SMA-s-PPD (B) (a) 119 nm 84 nm SMA-s SMA-s-PPD (b) 500 nm 121 nm nm 61 nm Size (nm) 200 nm 100 nm 0 40 Dose of pre-irradiate (kgy) FIG. 10.(A) TEM images of SMA-s and SMA-s-PPD from (a) non-irradiated, s0 and (b) 40 kgy pre-irradiated chitosan, s40.(b) Particle size plot of SMA-s and SMA-s-PPD SMA-s and SMA-s-PPD nanoparticle compounding to biomedical plastic The SMA-s0-PPD and SMA-s40-PPD 200 nmnanoparticles were compounded with polylactic acid (PLA) to observe the compatibility. It was found from SEM images that SMA-s0-PPD and SMA- s40-ppd show good compatible with PLA when comparing with non-modified chitosan and with SMA-s0 of the lowest grafting percentage. PLA S phase S-control S0-SMA (1:1) 25 kgy: 11% PLA phase S0-SMA (1:5) 25 kgy-ppd S40-SMA (1:7) 10 kgy-ppd FIG. 11. SEM images of SMA-s and SMA-s-PPD nanoparticle compounding with PLA. 196

10 4. NLUSINS The present experiments proved that radiolytic methodology is a simple and effective procedure beside chemical one to fabricate hydrophobic/hydrophilic self-assembly chitosan nanoscale particle. ydrophobic core would provide a loading space for water-insoluble drugs, whereas the modification of hydrophilic shell affects pharmacokinetic behavior. To use gamma irradiation not only induce modification via radical reaction but also diminish chemical use as well as reduce the step of preparation. AKNWLEDGEMENTS The authors gratefully acknowledge IAEA s oordinated Research Project (RP), International Atomic Energy Agency (IAEA) for the financial supports. Appreciation is expressed to ffice of Atoms for Peace (AP) and Thailand Institution of Nuclear Technology (TINT), Thailand for providing some facilities. I am thankful to Assoc. Prof. Dr. Suwabun hirachanchai for providing TEM (itashi 7650) instrument. 197

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