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การประชุมวิชาการ มหาวิทยาลัยเกษตรศาสตร วิทยาเขตกําแพงแสน ครั้งที ่7

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สารเชิงซอนของคารบอนนาโนทูบหลายชั้นฟงชั่นนัลไลซกับดีเอ็นเอสําหรับนําสงยนี Functionalized Multi-Walled Carbon Nanotube-Dna Complex for Gene Delivery

อรพรรณ ใจอุตม1 และกาญจนพิมล ฤทธิเดช1

Orrapan Jai-ut1 and Garnpimol C.Ritthidej1

บทคัดยอ

งานวิจัยนี้ ทําการปรับปรุงผิวคารบอนนาโนทูบชนิดหลายชั้นดวยกรดเขมขน พบวาเมื่อทําการปรับปรุงผิวคารบอนนาโนทูบชนิดหลายชั้นทําใหมีความสามารถในการละลายในน้ํา จากภาพถายจากกลองจุลทรรศนแบบสองกราดพบวา คารบอนนาโนทูบชนิดหลายชั้นมีผิวที่สะอาดมากขึ้น และขนาดสั้นลง เมื่อนําไปวัดขนาดอนุภาค มีขนาดเฉลี่ยเทากับ 166.83 นาโนเมตร มีประจุเฉลี่ยเทากับ -23.2 มิลลิโวลต ทําการศึกษาการจับตัวกันของ ไคโตซานนาโนพาทิเคิลและคารบอนนาโนทูบชนิดหลายชั้นที่ปรับปรุงพื้นผิว ศึกษา FTIR spectra พบวา คารบอนนาโนทูบชนิดหลายชั้นที่ปรับปรุงพื้นผิวมีหมูคารบอกซิลิก และไคโตซานนาโนพาทิเคิล-คารบอนนาโนทูบชนิดหลายชั้นที่ปรับปรุงพื้นผิวมีหมูคารบอกซิลิกและหมูคารบอนิล เมื่อเพิ่มปริมาณคารบอกซิลิกพบวามีขนาดอนุภาคและประจุเพิ่มขึ้น โดยแตละอัตราสวนมีความแตกตางกันอยางมีนัยสําคัญทางสถิติ โดยมีขนาดอนุภาคอยูในชวง 19.18 ถึง 103.81 นาโนเมตร ประจุบนพื้นผิวอยูในชวง +14.3 ถึง +43.7mV จากนั้นนําสารประกอบเชิงซอนของไคโตซานนาโนพาทิเคิลและคารบอนนาโนทูบชนิดหลายชั้นที่ปรับปรุงพื้นผิวมาจับกับพลาสมิดดีเอ็นเอ pEPGF-C2

คําสําคัญ : คารบอนนาโนทูบ คารบอนนาโนทูบชนิดหลายช้ัน ดีเอ็นเอ ไคโตซาน

Abstract Multiwalled carbonnanotube (MWCNTs) were functionalized by treating with concentrated

sulfuric acid and concentrated nitric acid to improve solubilization and to obtain anionic particles. SEM images f-MWCNTs revealed clean surface and shortened tubes. The average particles size of f-MWCNTs and average zeta potential was 166.83 nm. and was -23.2mV. The f-MWCNTs were then attached with cationic charged chitosan (CS). FTIR spectra showed of carboxylic group on f-MWCNTs. Carboxylic group and carbonyl group were on CS/f-MWCNTs. When decreasing the CS/f-MWCNTs ratio, the particles size and zeta potential of nanoparticle complexation increased significantly. The size and zeta potential was 19.18 to 103.81 nm and +14.3 to +43.7 mV respectively. The CS/f-MWCNTs were then formed complex with pEGFP-C2 and confirmed by the electrophoretic mobility analysis.

Keywords : Carbon nanotubes, Multiwalled carbon nanotubes (MWNTs), DNA, Chitosan E-mail : [email protected]

1 ภาควิชาวิทยาการเภสัชกรรมและเภสัชอุตสาหกรรม คณะเภสัชศาสตร จุฬาลงกรณมหาวิทยาลัย กรุงเทพมหานคร 10330 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmaceutical Sciences, Chulalongkorn University Bangkok 10330


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INTRODUCTION Carbon nanotubes (CNTs) are cylindrical molecules, hexagonal arrangement of sp2 hybridized

carbon atoms. The hollow cylinders formed by rolling single or multiple layers of graphene sheet. Functionalized singlewalled carbon nanotubes (SWCNTs) has been investigated for protein and DNA delivery. They could transfect cultured cells. Many researches showed SWCNTs were nontoxic. Recently, multiwalled carbon nanotubes (MWCNTs) has been easily synthesized and the production cost is less than that of SWCNTs. MWCNTs can be functionalized at the surface to have positive charge for DNA transports. The purposes of this study were to develop f-MWCNTs for plasmid DNA delivery.

METERIALS AND METHODS

1 METERIALS Multiwalled carbon nanotubes (Bayer,Geramany), plasmid DNA purification (Geneaid,Taiwan),

pEGFP-C2 (Clontech, USA), low molegular weight chitosan 448869 (sigma-aldrich, Singapore), 1 kb DNA Ladder (Vivantis, Malaysia), Ethidium bromide (Vivantis, Malaysia), Glacial acetic acid, nitric acid,sulfuric acid,Kanamycin sulfate (Meiji,Japan), LB Broth Miller Luria-Bertani (Becton Dickinson,Germany), 6x Loading Dye (Vivantis,Malaysia), Tris/Acetic acid/EDTA (TAE) buffer (Bio-rad, USA). 2 METHODS 2.1 Synthesis of functionalize multiwalled carbonnanotube (f-MWCNTs). MWCNTs were added to the mixture of 65% nitric acid and 95% sulfuric acid (1:3 ratio) by sonication for 6 hours. F-MWCNTs were washed with ultrapurification water until neutral then dried at 80˚C over night. Dried f-MWCNTs (1% w/v) were added with ultrapurification water. 2.2 Preparation of chitasan nanoparticles(CS) 0.25%w/v low molecular weight chitasan was dissolved in the solution of 2%v/v acetic acid containing 1%TWEEN 80. CS solution was stirred and sonicated for 1 hour. 2.3 Preparation of CS/f-MWCNTs nanoparticles CS/f-MWCNTs were prepared by weight ratio 1:0.5, 1:1, 1:2 and 1:8. The mixtures were stirred overnight at room temperature. 2.4 Preparation of plasmid DNA (pEGFP-C2). Plasmid Enhanced Green Fluorescence Protein-C2 (pEGFP-C2) was amplified in E. coli and prepared by alkaline lysis technique. A single colony of E. coli was inoculated with LB broth containing Kanamycin. Bacterial cells harvested from the culture by centrifugation. Supernatant was then discarded. SDS in 0.2N NaOH was added. Potassium acetate was then added. White precipitate consist of chromosomal DNA, protien and cell walls were discarded. RNase A was added to digest RNA. Phenol/chloroform/isoamyl alcohol was added to removed the remaining protein.


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2.5 Preparation of CS/f-MWCNTs/pDNA nanoparticles CS nanoparticle, f-MWCNTs and pDNA in water were prepare by weight ratio. CS/f-

MWCNTs/pDNA ratio was 0.5:1:1, 1:1:1, 1.5:1:1, 2:1:1, 4:1:1, 6:1:1, 8:1:1 and 10:1:1. Ultrapurify water was added to equal volume. All solutions were heated to 55˚C separately for 10 min. Then, quickly mixed together and vortexed for 1 min. 2.6 Investigation of Size and surface charge of f-MWCNTs, CS nanoparticles, CS/f-MWCNTs, CS/f-MWCNTs/pDNA The zetasizer was worked to triple measure of the size distribution and surface zeta potential of f-MWCNTs, CS nanoparticles, CS/f-MWCNTs, CS/f-MWCNTs/pDNA. Solution was diluted up to 1 ml. 2.7 FTIR spectra of MWCNTs, f-MWCNTs, CS, CS/f-MWCNTs f-MWCNTs and CS/f-MWCNTs were dried overnight. Pristine MWCNTs, f-MWCNTs, CS and CS/f-MWCNTs were prepare with KBr for FTIR investigation. 2.8 SEM observations of MWCNTs, f-MWCNTs. The pristine MWCNTs and f-MWCNTs suspension were prepared for SEM samples.

RESULTS AND DISCUSSION

Table 1 shows size distribution and surface zeta potential of f-MWCNTs. The average particle size of functionalize-multiwalled carbon nanotubes was 166.83 n. and the average zeta potential was -23.2mV. Negative charges were resulted from the carboxylic acid group from the used acid functionalization. This could improve the solubility ( Fig. 1B). The sedimentation of insoluble pristine MWCNTs particles at the bottom of the bottle (Fig. 1A). SEM images f-MWCNTs revealed clean surface and shortened tubes (Fig. 2). Separation of f-MWCNT could be seen (Fig. 2B).

Table 1 shows size distribution and surface zeta potential of f-MWCNTs

f-MWCNTs Size(nm.) Zeta(mV) 1 168.5 -23 2 172.6 -22.7 3 159.4 -23.9 Aver. 166.8333 -23.2 SD 6.755985 0.6245


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Fig. 1 Pristine MWCNTs (A) and 1% w/v f-MWCNTs solutions (B) were shaked.

Fig. 2 SEM images of pristine MWCNTs (A) and f-MWCNTs (B)

When decreasing the CS/f-MWCNTs ratio, the particles size and zeta potential of nanoparticle complexation increased significantly. The size and zeta potential were 19.18 to 103.81 nm. and +14.3 to +43.7 mV respectively (Fig. 3). However, increasing the CS/f-MWCNTs ratio, decreased the particles size and zeta potential of nanoparticle complexation. The size and zeta potential were 22.46 to 199.13 nm. and +23.4 to +49.87 mV respectively (Fig. 4A). The particles size and zeta potential of CS/f-MWCNTs/pDNA were compared with CS/f-MWCNTs every ratio (Fig. 4). The particle sizes of CS/f-MWCNTs/pDNA were larger than CS/f-MWCNTs, but zeta potential of CS/f-MWCNTs/pDNA were less than CS/f-MWCNTs because the attachment of negative charge pDNA particles.

Fig. 3 The particle size and zeta potential of nanoparticle complexation when decreasing the

CS/f-MWCNTs ratio.

A B

A B


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Fig. 4 The particles size and zeta potential of CS/f-MWCNTs (A) and CS/f-MWCNTs/pDNA (B) when

increasing the CS/f-MWCNTs/pDNA ratio.

The infrared spectra of CS and CS/f-MWCNTs showed the region 2879,2871 cm-1 that were assigned to CH3-N,CH3-N;amines. The region 1631, 1649, 1635 cm-1 of f-MWCNTs, CS and CS/f-MWCNTs of indicated the carbonyl group.

Fig. 5 The infrared spectra of MWCNTs, f-MWCNTs, CS and CS/f-MWCNTs.

CONCLUSION AND SUGGESTION

f-MWCNTs have good solubility. SEM images of f-MWCNTs revealed clean surface and shortened tubes. The particle sizes and zeta potential of CS/f-MWCNTs/pDNA were showed the attachment of negative charge pDNA particles. FTIR spectra showed of carboxylic group on f-MWCNTs. These modified MWCNTs were provided benefit properties, such as drug carrier, diagnostic or therapeutic devices.

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