Ã ¥ µ µª°¤¦¦´ r ´ µ¦ ª» ·· Î ª· ¥µ ·¡ r ¸ÊÁ È nª ... · 2014-10-22 · ACID) NANOFIBER FOR POTENTIAL APPLICATION AS WOUND DRESSING ÿ Ü By Miss Amornrat

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PREPARATION AND PROPERTIES OF ELECTROSPUN CHITOSAN/ POLY (LACTIC

ACID) NANOFIBER FOR POTENTIAL APPLICATION AS WOUND DRESSING

By

Miss Amornrat Tantithumrongwut

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree

Master of Engineering Program in Polymer Science and Engineering

Department of Materials Science and Engineering

Graduate School, Silpakorn University

Academic Year 2012

Copyright of Graduate School, Silpakorn University



“

”

…….…...........................................................

(

(

( (

( (



52402240 :

100

(Unhydrolyzed chitosan Hydrolyzed chitosan)

H

20%

20 19

10, 20 40

2555



52402240 : MAJOR : POLYMER SCIENCE AND ENGINEERINGKEY WORD : POLY(LACTIC ACID) CHITOSAN ELECTROSPINNING NANOFIBER

AMORNRAT TANTITHUMRONGWUT : PREPARATION AND PROPERTIES OF ELECTROSPUN CHITOSAN/ POLY (LACTIC ACID) NANOFIBER FOR POTENTIAL APPLICATION AS WOUND DRESSING. THESIS ADVISORS : NUTTAWUT CHAIYUT, Ph.D., ASSOC. PROF. KALYANEE JIRASRIPONGPUN, Ph.D. 100 pp.

Chitosan/poly(lactic acid) (PLA) nanofibers were successfully prepared by electrospinning process. Electrospinnability and morphology of the electrospun fibers were investigated by adjusting electrospinning parameters, such as spinning electric field, spinning distance, types of chitosan (unhydrolyzed and hydrolyzed), concentration of PLA solution and solution blend ratios of chitosan and PLA. Morphological study of the obtained fibers was carried out by scanning electron microscope (SEM). The suitable condition for producing chitosan/PLA fibers as follows: applied voltage is 19 kV and collection distance is 20 cm. Hydrolyzed chitosan and chitosan/PLA at ratio of 40/60 were suitable to electrospinning uniform chitosan/PLA nanofibers.

Hydrolyzed chitosan/PLA and hydrolyzed chitosan/PLA by adding gentamicin sulfate at 10, 20 and 40 mg/ml nanofibers was prepared by electrospinning process which tested and observed for physical and mechanical properties changes. This study found that the more gentamicin sulfate added to hydrolyzed chitosan/PLA solution, the less water absorbed, resulting in low vapor transmission rate of the electrospuns. Hydrolyzed chitosan/PLA and hydrolyzed chitosan/PLA by adding gentamicin sulfate showed no cytotoxicity toward growth of Vero cell.

Department of Materials Science and Engineering Graduate School, Silpakorn University Student's signature ........................................ Academic Year 2012Thesis Advisor's signature 1. ........................... 2. ...........................



Scanning Electron Microscope (SEM)



1 1

1

2

3

4

2 5

5

5

7

Low-adherent Dressing 8

8

Hydrogel 8

Hydrocolloid 8

Alginate dressing 9

Non-adhesive-semi-permeable Polyurethand Foam Dressing 9

9

9

10

10



10

10

11

11

12

12

12

13

13

13

14

14

14

14

15

16

16

16

16

17

Degree of deacetylation 17

17

Coagulation ability 17

Molecular conformation 17

18

19

19

19



20

20

21

21

24

24

24

25

25

26

26

26

2

29

29

30

33

34

35

3 36

36

37

38

39

hydrolyzed chitosan 39

39

40



41

41

43

44

44

44

45

45

46

46

47

49

4 51

51

51

53

55

Hydrolyzed chitosan PLA

56

58

59

60



61

62

63

64

5 66

66

67

69

73

74

76

82

85

93

95

(Drug release)

97

100



1 LPL

–

21

2 23

3 25

4 30

5 (Permeability)

(PET) (OPS)

(OPLA)

33

6 Unhydrolyzed chitosan Hydrolyzed chitosan 55

7

56

8

58

9 60

10 61

11 S. aureus /

64

12 /

24

65

13 (Intrinsic viscosity)

75

14

77

15

78

16

Unhydrolyzed



chitosan hydrolyzed chitosan hydrolyzed

79

17

80

18

81

19 83

20

10

83

21

20

84

22

30

84

23 (Water vapor

transmission rate: WVTR)

86

24 (Water vapor


10

87

25 (Water vapor


20

88

26 (Water vapor


40

89

27 (Water vapor rate: WVTR)

92



28 (Water adsorption)

94

29 (Cytotoxicity)

96

30 max = 400

24

98

31 max = 400

98



1 (a) neutrophils

(b) (Epithelial

cells)(c) (d)

6

2 15

3 16

4 27 5 28 6 (T

g) (Tm)

31 7 (Tg) -

31

8 (Tm) -

33

9 (LDPE)

(PS) (PLA) L:D

96:4 (PET)

34

10 43

11 44

12 46

13 UV-VIS spectrophotometer max = 400

50

14 Hydrolyzed chitosan/PLA

20

0.01

a) 13 b) 15 c) 17 d) 19

52

15 Hydrolyzed chitosan/PLA



19

0.01

a) 8 b) 12 c) 16 d) 20

54

16

a) Unhydrolyzed chitosan b) Hydrolyzed

chitosan

55

17

Hydrolyzed chitosan/PLAa) PLA

15%wtb) PLA 18%wt c) PLA 20%wt

57

18

Hydrolyzed chitosan/PLA a) 40/60 b)

50/50

59

19 /

/

62

20

24

63

21 Clear zone

Staphylococcus aureus (S. aureus)

64

22

65

23

Hydrolyzed chitosan

75

24

90

25

10%

90



26

20%

91

27

30%

91



1

1

1.1

(Wound dressing)

30

[1]



2

[2,3]

(Poly(lactic acid) ;PLA) (Polylactide)

Aliphatic polyester (Lactic acid)

PLA

PLA

[4]

(Gentamicin sulphate)

(Aminoglycosides)

(Gram

negative aerobic bacteria) Staphylococcus aureus [5,6]

Electrospinning

process)

1.2

1.2.1

1.2.2



3

1.2.3

1.2.4

1.3


1.4

1.4.1 1.4.2 1.4.3

1.4.3.1


1.4.3.2

(Water vapor permeability)



4

(Water absorption) (Cytotoxicity)

(Antibacterial test) (Drug releasability)

1.4.4

1.4.5

1.4.6

1.5

1.5.1

1.5.2

1.5.3



5

2

2.1 [7-9]

2.1.1 rocess)

(Platelets) (Fibrin)

“Haemostatic phase”

“Inflammatory phase”

(Epidermal cell)

(Epithelialization) (Fibrobrast)

“Proliferative phase”

“Maturation phase” “Remodeling phase”



6

1 (a) Neutrophils

(b) (Epithelial cells) (c)

(d)

[8]

Inflammatory

phase

(Infection)

Senescent cell

1.

7

2.

(Epithelialization) 2

3.

4.

Exudates

5.

6.

7.

8.

9.

10.

11.

12.

2.1.2

2.1.2.1 s)

8

Debride Pack

Polyester Rayon

Hypertonic Saline Sponge

2.1.2.2 -

Exudates

2.1.2.3 (Semi-

(Adhesives)

Autolytic

debridement

2.1.2.4

2.1.2.5

(Granule)

9

2.1.2.6

(Ion

exchange) (Ca+)

2.1.2.7 Non- -semi-

2.2 ) [10-13]

2

(Reinforcing fibers)

2.2.1

“Taylor

cone”

(Electrostatic repulsion)

10

Bending instability)

(Unbalance force)

2.2.2

3

2.2.2.1

2.2.2.1.1 (Concentration)

(Beads)

(Bending instability)

Power law

2.2.2.1.2

Dielectric strength

11

(Chain entanglement)

(Beads)

2.2.2.1.3 ( iscosity)

1-215

poise [11]

Stress relaxation

2.2.2.1.4

12

2.2.2.1.5 (

(Bending instability)

2.2.2.2

2.2.2.2.1

(Electrostatic force)

13

2.2.2.2.2

2.2.2.2.3

(Dimple)

2.2.2.2.4distance) (Flight time)

(Electric field strength)

(Merge) (Intra

and inter layer bonding) Scaffold

14

2.2.2.3

2.2.2.3.1 (Humidity)

Atomic force

2.2.2.3.2

Freon ®-12 2

2.2.2.3.3 (Pressure)

15

2.2.3

(

2

2 [13]

16

2.3 [14-19]

2.3.1

(Deacetylation)

(-NHCOCH3) (-NH2) 2

D-glucosamine(2-amino-2-deoxy-D-glucose)

Poly( (1 4)-2-amino-2-deoxy-D-glucose) 3

3 [14]

2.3.2

2.3.2.1

(Organic solvent)

pH 6

-

(Non-Newtonian)

(pKa) pKa

6.2-6.8

17

2.3.2.2 1×105 - 2×106

2.3.2.3 n -

- N-acetyl-D-glucosamine

D-glucosamine Degree of

deacetylation

Degree of deacetylation

2.3.2.4

Degree of deacetylation Ionic strength

- (pH)

pH

pH

pH

2.3.2.5

(Flocculating and coagulating agent)

Cheese whey

(Coordinate)


2.3.2.6

(Cationic Polyelectrolyte)

–NH+3 Conformation Mark-

Houwink Exponent ( a) a 0, 0.5-0.8 1.8

18

(Sphere) Random coil (Rod) Conformation

Ionic Strength pH


2.3.2.7

(Oligomer) (Oligosaccharide)

(Monomer) (Monosaccharide)

/ N-Acetyl-

Chitooligosaccharide Chitooligosaccharides /

N-Acetyl-D-Glucosamine D-Glucosamine

2.3.2.7.1

Random

2.3.2.7.2

Peeling Reaction

2.3.2.7.3sonication)

2.3.2.7.4

- Chitinase (EC 3.2.1.14)

(Random) (-1,4-Linkage) N-acetyl-chitooligosaccharide

Chitosanase (EC 3.2.1.132) (Random)

(-1, 4-Linkage) Chitooligosaccharide Lysozyme (EC 3.2.1.17)

Chitinase N-acetylglucosaminidase (EC3.2.1.30) N-

acetylglucosaminidase (EC3.2.1.50) N-acetylchitooligo saccharides N-

acetyl-glucosamine (Non-reducing end)

19

2.3.2.7.5

(Dry heat) 80

Glass transition temperature (Tg)

120

160 2

(0.2 M)/ (0.1M)

(Saturated steam)

115 2

120 1

120

2.3.2.8 3

(-NH2) 2 (C-2) Primary

alcohol (-CH2OH) 6 (C-6) Secondary alcohol (-CHOH)

3 (C-3) (Chemical modification)

2.2.1 [15-16]

(Biomaterials) (Biocompatibility)

(Biodegradable)

(-NH2) (-OH)

(Derivatives)

-

2.2.1.1 -

-

20

-

-

2.2.1.1.1 -

(Non–woven fabrics)

(Hydrogel) -

-

Chitosan–collagen [17] (Contact lenses) Partially

depolymerisation Spin casting

(Blood dialysis membrane)

Antibacterial, Antivirus Antitumor

-

Periodontal tissue

(Foreign body reaction)

2.2.1.1.2 , Anti- Heparin

(N-sulphate

group) Heparin - -

Lipoprotein lipase (LPL)

1 Chitin-3, 6-sulphate

Heparin 2 LPL 0.1

O-Sulphated Chitin (Toxicity, LD50 = 1.25–3.25 g/kg)

Haparin (LD50 = 1.59–2.00 g/kg) [17] -

N-Hexanoyl N-Octanoychitosan

[16]

21

1 LPL

– [16]

MW

(x103) -

Chitin 26 2.0 355 (2.0) 100 (0.1)

Chitosan 12-22 1.7 239 (3.4) 3200 (3.4)

N-Desulphated chitosan 22 0.7 n.d.c Inactive

N-Hexanoy chitosan 27 1.8 n.d.c Inactive

CM-chitosan 245-540 0.6 26 (0.10) 700 (0.7)

Heparin 21 - 174 (1.0) 950 (1.0) a The dosage was 0.1 mgkg-1 of body weight, and maximum LPL activity is shown in mole

equivalents of free acids per litre of plasma after incubation at 37 C for 30 min, and the activity

relative to heparin is shown in parentheses b With respect to the activated thromboplastin time (APTT); the activity relative to heparin is

shown in parentreses. c Not determined.

2.2.1.2

2.2.1.2.1 [18]2.2.1.2.1.1

2

(Cholesterol)

22

20

1-2 25–50 %

–

2.2.1.2.1.2 -

2

2 Glucosamine ( )

pH 6 -

Interaction

Chelating agent

(Toxin)

23

2 [18]

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

2.2.1.2.1.3

2.2.1.2.1.4

Gelatin, Bentonite, Silica sol,

Tannins, Potassium caseinate Polyvinyl pyrrolidone

Grapefruit Fining

agent

24

2.2.1.2.2 (Drug delivery and controlled release systems)

Glutaraldehyde cross–linked chitosan

2.2.1.2.3

Hyaluronic acid

(Water binding capacity)

(Wella Inc.)

2.2.1.3 [15,17] -

1981 -

- 3

-

( )

25

3 [16]

Chitosana

N-Acetylchitosanb

N-Propionlychitosanb

3.74-9.02

3.18

4.71

0.78–1.24

0.79

0.52

13.0–28.1

27.1

25.9 aA 5% chitosan solution in aqueous 2% CH2COOH was span into an aqueous 10% NaOH

containing 30%CH3COONa. b Each solution (5–8%) of sodium N-acetylchitosan salt and sodium N-propionylchitosan salt in

aqueous 14% NaOH as span into aqueous 10% H2SO4 containing 25-33% Na2SO4 and 1.3%

ZnCl2

(Anionic inks) – Hydroxymethyl chitin

[17]

2.2.1.4 [15,19

2.2.1.5 [15]

-

(Tree-protecting agents) (Animal feed)

26

(Fertilizers/Soil strabilizers)

2.2.1.6 [15]

Support

Crystalline cellulose (Polyamide) Cross–

linked

Gas chromatography ( Stationary phase) High performance liquid chromatography

Bio-reactor, Bio-sensor Ligand carrier Affinity chromatography

Carrier ( Glucose isomerase)

2.3 [20-23]

(Poly(lactic acid), PLA)

(Aliphatic polyester) (Lactic acid)

2.3.1

( -Hydroxy acids)

2-

(2-Hydroxy propionic acid)

( 4)

(Enantiomer) (Optical active)

- - -

(Racemic mixture, = 1:1 DL)

(meso-compound) (Optically inactive)

(Optical

purity)

27

4 [21]

(Azeotropic dehydrative condensation) (Direct

condensation polymerization) / (Lactide for-mation)

( 5) ( 100,000 )

(Ring-opening polymerization)

- ( -

(PLLA)) ( -

(PDLLA)) -

- (PDLA)

( ) ( )

- ( ) ,

185

175

28

5 [21]

2.3.2

(Optical purity)

(Barrier pro-

perties) - 90

(Semicrystalline polymer) -

(Amorphous)

-

(PET) (PS)

29

2.3.2.1

(Degree of crystallinity)

(Hexane) (Heptane)

(Poly(L-lactic acid), PLLA)

(Chlorinatedor fluorinated organic solvents) (Dioxane) (Dioxolane)

(Furane)

(Poly(rac-lactic acid)

(Acetone) (Pyridine) (Ethyl lactate) (Ethyl

acetate) (Tetrahydrofuran) (Xylene)

(Dimethylsul-foxide) , - (N,N- dimethylformamide)

(Methyl ethyl ketone)

2.3.2.2

1.25 (Unoriented PLA)

(Stiffness) (Strength) (Oriented)

(Polyethylene terepthalate, PET)

(Oriented PS) (Tensile and flexural modulus)

(HDPE) (PP) (PS)

(Izod impact strength) (Elongation at break)

4

30

[20]

(Ultimate tensile strength, psi)6,900-7,700 6,900-24,000

(Tensile yield strength, psi)6,600-8,900 N/A

(Tensile modulus, psi) 500,000-580,000 564,000-600,000

(Notched Izod impact, ftlb in-1)0.3-0.4 N/A

Elongation at break (%) 3.1-5.8 15-160

(Rockwell hardness) 82-88 82-88

* (Degree of orientation)

2.3.2.3 (Tg)

(Tm) ( 6)

(L:D ratio, 7)

-

50:50

31

6 (Tg) (Tm)

[20]

7 (Tg)

- [20]

32

Flory-Fox 1 =

= Tg

PLLA PDLA 57-58

K = (Excess free volume)

(End groups) PLLA PDLA

(5.5-7.3) x 104

=

(Thermal history)

(Tm)

( PLLA PDLA) 180 40-50

-

50 8

(meso-lactide)

130-

160

33

8 (Tm)

[20]

2.3.2.4

PLA

(PS) (PET) ( 9)

( )

-

(D-linonene)

(Permeability) (PET)

(OPS) (OPLA) [20]

( .)

( 2 )) PET 18 2.82 x 10-15 ± 1.50x10-17

OPS 18 4.18 x 10-15 ± 2.07x10-17

OPLA 20 1.34 x 10-14 ± 3.61x10-17

34

9 (LDPE)

(PS) (PLA) L:D 96:4

(PET) [20]

2.3.3 [22]

(Oligomer)

60

60 2

-

35

(Autocatalyzed) (Carboxylic acid)

2.3.4 [23]

-

(Biocompatible) (Bioresorbable)

2 (Sutures) (Stables)

(Wound dressing) (Surgical implants)

(Orthopedic fixation devices)

-

-

- Non-woven

- (Bumpers) (Floor mats)

-

-

36

3

3.1

1. Poly(lactic acid) (PLA) 2002D NatureWorks®

2. Chitosan (CS) Seafresh Chitosan (LAB) CO.,Ltd

150,000 Degree of deacetylation (%) 90

3. Gentamicin sulfate Vesco Pharmaceutical CO.,Ltd Gentamicin

40 mg/ml

4. Acetic acid Glacial 100% suitable for biopharmaceutical production

EMPROVE® bio Merck

5. Acetone Analysis EMSURE® ACS, ISO, Reag. PhEur Merck

6. Chloroform Commercial Labscan

7. Deionized water

8. Dimethylsulfoxide (DMSO) Analysis EMSURE® ACS Merck

9. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT)

Invitrogen

10. Distilled water

11. Ethanol

12. Ethylen diamine tetra acetic acid (EDTA: C10H16N2O8) Ameresco

13. Fetal Bovine Serum (FBS) HyClone

14. Modified Eagle’s Medium (MEM) Gibco

15. Ninhydrin Ajax

16. Nutrient agar (NA) Lab008 Lab M

17. Nutrient broth (NB) Lab014 Lab M

18. Potassium chloride (KCl) Plant cell culture test Sigma

19. Potassium phosphate (KH2PO4) Plant cell culture test Sigma

37

20. Sodium chloride (NaCl) Cell culture Reagent Sigma

21. Sodium hydroxide (NaOH) American Chemical Society certified (ACS)

Carlo Erba

22. Sodium phosphate (Na2HPO4) Plant cell culture test Sigma

23. Trypan blue Sigma

24. Trypsin Fluka

3.2

1. (Aluminium foil)

2. (Autoclave) Tommy SS-325

3. Autopipette 20-200 ml

4. Autopipette 100-1000 ml

5. Breaker)

6. Buchner funnel)

7. Centrifuge) Hettrich

8. (Centrifuge tubes) 15 ml

9. (Clamp/Clamp holder)

10. (Condenser)

11. Conical flask with joint)

12. Cotton)

13. –80 (Deep freezer) NuAire

14. Dropper)

15. (Drum collector)

16. Electrospinning machine)

17. Filter paper)

18. Forceps)

19. Graduated cylinder)

20. Hemocytometer

21. Hot plate with stirrer)

22. CO2 Incubator) NuAire

38

23. (Incubator) Memmert

24. Laminar air flow class II) NuAire

25. (Light microscope) Olympus

26. (Magnetic bar)

27. 0.2 μm (Membrane filter)

28. Metal plate collector)

29. 3 3-Necked round bottom flask)

30. (Oil bath)

31. (Petri dish)

32. pH indicator paper)

33. pH-Meter) Schott

34. (Pipette) 5 10 ml

35. Shaker) GFL 3017

36. Spatula)

37. 26 (Spinneret)

38. (Stop watch)

39. (Suction flask)

40. (Syringe)

41. (Temperature controller)

42. T-flask 25 cm2

43. Thermometer)

44. Vacuum oven with pump)

45. (Vial tube)

46. (Viscometer) Brookfield DV-I+ prime

47. Volumetric flask)

48. 96-well plate

3.3

1. (Microplate reader TECAN

ELISA

39

2. Scanning electron microscope : SEM)

CamScan MX-2000

3. Ubbelohde viscometer

4. Universal testing machine Lloyd

5. UV-VIS spectroscopy (UV-VIS) PG Instruments

T80+UV/VIS Spectrometer

3.4

3.4.1 H -CS)

Hydrolyzed chitosan (h-CS) 1

NaOH 50% 25 95 24

Deionized water

100% Deionized water Hydrolyzed chitosan

60 16

3.4.2 [35]

Hydrolyzed chitosan

(Intrinsic viscosity) 90%

Hydrolyzed chitosan 5

0.5-5.0 g/l

Mark-Houwink equation 2

[ ] = (2) K a Mark-Houwink parameter

1.8×10-3 0.93 [ ]

(Intrinsic viscosity) Huggins equation 3)

Kramer Equation 4)

Huggins equation : = [ ] + [ ] (3)

Kramer equation : = [ ] [ ] (4)

40

c = (g/l)

= relative viscosity

= ( ) ( )

= 1 = Specific viscosity

Hydrolyzed chitosan

plot

(C) (g/l) y

Huggins Kramer equation [ ] [ ]

2

3.4.3

Hydrolyzed chitosan (h-CS) Hydrolyzed chitosan

3.4.1 90% 5%

(PLA solution)

(CHCl3) ((CH3)2CO)

70:30 15, 18 20%

48

(chitosan : poly(lactic acid) blend ratio) 50/50 40/60

Hydrolyzed chitosan

Unhydrolyzed chitosan (CS)

5% 90%

(PLA solution) 20%

(chitosan : poly(lactic acid)) 50/50

41

3.4.4

3.4.3

Brookfield viscometer (Model DV-

I+prime) Spindle 2 Switch power

Spindle 2 Select spindle

Spindle 3.4.3

0.05-200

%Torque

Option Time to stop 1 Enter Set speed

Enter 2

3.4.5

2

1. hydrolyzed

chitosan/PLA Hydrolyzed chitosan/PLA

2.

Unhydrolyzed chitosan Hydrolyzed chitosan

hydrolyzed chitosan/PLA

5%wt/v

Hydrolyzed chitosan/20%wt/v

50/50 10 26

10

(Feed rate) 0.01

42

20 20 13, 15, 17

19

5%wt/v Hydrolyzed chitosan/20%wt/v f

50/50 10

26 10

(Feed rate) 0.01 19

20

8, 12, 16 20

5%wt/v Hydrolyzed chitosan/20%wt/v

5%wt/v Unhydrolyzed chitosan/20%wt/v

50/50 10

26

10 (Feed rate) 0.01

19 20

20

5%wt/v Hydrolyzed chitosan/ 15, 18

20%wt/v 50/50

10 26

10 (Feed rate)

0.01 19

20 20

5%wt/v Hydrolyzed chitosan/20%wt/v

50/50 40/60 10

26

10 (Feed rate) 0.01

43

19 20

20

10

3.4.6

5%wt/v h-CS/20%wt/v PLA

3.4.4

10 26

11 0.01

(Drum collector)

20

19

15

10 6

60 C 12

44

11

3.4.7

3.4.6

400

3.4.8

3.4.8.1

3.4.4 60

12

(Scanning electron microscope: MX-2000, CamScan)

Stub

Sputtering

45

2,000

ImageTool

3.4.8.2

3.4.5 3.4.6

5×65 Tensile test)

ASTM D882-83 Universal testing machine (Lloyd)

Load cell 1 Gage length 20 10

3.4.8.3

) -37]

3.4.6 3.4.7

3×3

(Parafilm)

(%RH)

6

12 WVTR ( ) = ( ) × 24

w t = 1 (g/hr)

A = (1.51×10-4 m2)

46

12

3.4.8.4 [29]

24 3.4.6 3.4.7

3×3 (W0)

24 We)

%

(%) = × 100

We

W0

3.4.8.5 )

3.4.7.5.1

Vero cell

African green monkey kidney cell

w t

47

3.4.7.5.2

Vero cell

Minimum essential medium (MEM) Fetal bovine serum (FBS) 10%

37 5%

log phase (Conference) 80-90%

3.4.7.5.3

3.4.5 3.4.6 1

100 20 deionized

water

200 rpm 3 0.2

3.4.7.5.4

MTT assay ISO

10993-5 2×105

96 well plate 100 μl 24 37

5% 3.4.7.5.3

0.25, 0.5, 1, 2 4 mg/ml 100 μl

24

Phosphate-buffered saline (PBS) MTT 1.0 mg/ml 50 μl

4 MTT

DMSO 50 μl 570

3.4.8.6 (

Agar diffusion

48

3.4.7.6.1


3.4.7.6.2 Nutrient agar (NA) Nutrient broth (NB)

Growth media

(Sterile)

3.4.7.6.2.1 Nutrient broth (NB)

Nutrient Broth 1.25 50 ml

pH NaOH 1 M pH 6.8 - 7.2

10 5 ml

(Sterilize) Autoclave 121

15

3.4.7.6.2.2 Nutrient agar (NA)

Nutrient Agar 4.2 150 ml

pH NaOH 1 M pH 6.8 - 7.2

Autoclave 121 15

Nutrient agar (Plate) Nutrient agar

3.4.7.6.3

3.4.5 3.4.6 Aseptic technique Laminar air

flow class II

3.4.7.6.4

(NB) 5 ml 1 loop 37 24

Nutrient agar plate (NA plate)

3.4.7.6.3 NA plate 37

24 Clear zone

Caliper

49

3.4.8.7

(Drug release) [38-39]

3.4.6 1

100 Phosphate-buffered saline (PBS) pH 7.4

10 100 37

1, 2, 3, 6, 12, 24, 48 72

5 Ninhydrin

1.25% 1.5 95 15

UV-VIS

400 Phosphate-buffered saline (PBS) pH 7.4

Phosphate-buffered saline (PBS)

pH 7.4 Ninhydrin 5:1

(Standard curve)

13

50

13

UV-VIS = 400

y = 0.0163x + 0.219 R² = 0.9844

0

0.2

0.4

0.6

0.8

1

0 10 20 30 40 50

51

4

4.1




4.1.1 H PLA

Hydrolyzed

chitosan PLA

Hydrolyzed chitosan (h-CS) 5%

(PLA solution) 20%

(chitosan : poly(lactic acid) blend ratio)

20

0.01 13,

15, 17 19 14

52

14 Hydrolyzed chitosan PLA

20

0.01

a) 13 b) 15 c) 17 d) 19

14


237.39±59.45, 210.92±45.27, 181.74±49.02

137.07±25.53 13, 15, 17 19




b

d

SEI 15 kV 10 μm

SEI 15 kV 10 μm SEI 15 kV 10 μm

SEI 15 kV 10 μm

a

c

53

[10-12]

Hydrolyzed chitosan PLA 19

4.1.2

Hydrolyzed chitosan

(h-CS) 5% (PLA solution)

20%


19 0.01

8, 12, 16 20

15

54

15 Hydrolyzed chitosan PLA

19 0.01

a) 8 b) 12 c) 16 d) 20

15

256.58±38.88, 233.14±21.92, 205.48±24.04

137.07±25.53 8, 12, 16 20

(Beads)

a b

d

SEI 15kV 10 μm SEI 15kV 10 μm


c

55

[10-12]

Hydrolyzed chitosan PLA 20

4.1.3

Unhydrolyzed

chitosan Hydrolyzed chitosan ( 6)


19 20

0.01

16

6 Unhydrolyzed chitosan Hydrolyzed chitosan

( )

Unhydrolyzed chitosan 150,000

Hydrolyzed chitosan 50,318

16

a) Unhydrolyzed chitosan b) Hydrolyzed chitosan

a b


56

16

Unhydrolyzed chitosan

Hydrolyzed chitosan

Unhydrolyzed chitosan

[10-12] Hydrolyzed chitosan

4.1.4

PLA

H


19

20 0.01

17

7

( (

15 220.33

18 573.20

20 985.76

57

17


a) PLA 15%wt b) PLA 18%wt c) PLA 20%wt

17

(Beads)

133.37±15.21, 134.87±12.11 137.07±25.53

15, 18 20 %

( 7)

(Entanglement)

b c


a

SEI 15kV 10 μm

58

[10-12]

20%

4.1.5

Hydrolyzed chitosan (h-CS)

5% (PLA solution)

20%

(chitosan : poly(lactic acid) blend ratio) 0 5 50

19 20

0.01

18

8

(

1,243.48

985.76

59

18

Hydrolyzed chitosan PLA a) 4 60 b) 0

18

137.07±25.53

369.10±45.39 50 60 %

60

50

4.2

(Tensile test)

ASTM D882 Load cell 1 Gage

length 20 10

a b


60

9

)

33.18 ± 0.64 8.92 ± 0.36 3.59 ± 0.09

10 30.36 ± 0.90 9.70 ± 0.48 3.64 ± 0.21

29.68 ± 1.16 9.73 ± 0.67 3.82 ± 0.17

40 27.70 ± 0.55 9.87 ± 0.21 4.33 ± 0.14

9

(Tensile strength)

(%Elongation)

4.3

WVTR)

10

61

10

2

1,796.03

10 1,764.24

20 1,732.45

40 1,494.04

10

4.4

19

62

19

4.5

90-100 %

10-

10-

297.89 291.33 262.87

239.42

0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

63

20

24

4.6 (

Agar diffusion test

(Clear zone)

Staphylococcus aureus (S. aureus

21 S. aureus

Clear zone

S. aureus 10-40

Clear zone 11)

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

No GS Add GS Add GS Add GS

()

64

21 Clear zone


11 S. aureus

9.50

18.25

20 22.50

40 25.00

4.7

release)

C+

C-

C+

C-

4

4

4

2

2

+2

65

12

10 7.12

20 18.04

40 37.42

3 (

33

[40]

0.00

2.00

4.00

6.00

8.00

10.00

0 10 20 30 40 50 60 70 80

( )

66

5

5.1

/

2


Hydrolyzed chitosan/PLA Hydrolyzed

chitosan/PLA PLA 20%

40/60

20 19

0.01

369.10±45.39

10 20 40

24 Universal testing machine

67

/

(Tensile strength)

(%Elongation)

(0-40 mg/ml)


S. aureus 10-40 mg/ml

3

5.2

1. /

68

2.

3.

69

[1]

cited 1 2553]; Available from:

[2] Novel chitin and chitosan

nanofibers in biomedical applications -

[3]

cited 1 2553]; Available from:

C K Preparation and characterization of biodegradable

poly(l- -

[5] cited 1 2553]; Available

-topical-

[6] Gentamicin Cream

- A review

on wound dressings with an emphasis on electrospunnanofibrous polymeric bandages

-95

[9]

-

C An Introduction to

Electrospinning and Nanofibers

[11] Electrospinning: A fascinating fiber fabrication technique

-

[12] Z A review on polymer nanofibers by

electrospinning and their applications in nanocomposites

-

[13] Bending instability of electrically

charged liquid jets of polymer solutions in electrospinning

-

-

- -

[15] -

- -

[16] Chitin and chitosan as novel biotechnological materials

-

Chitin Biotechnology Applications -

-

F Kamil Food applications of chitin and chitosans

-

[19] - cited 1 2553]; Available from:

:

-

[21] Poly(lactic acid) fiber: An overview

-

[22] Large-scale production, properties and commercial applications of polylactic

acid polymers – -

[23] Polylactides “NatureWorks® PLA -

C C C Stretching-induced crystallinity and orientation of

polylactic acid nanofibers with improved mechanical properties using an electrically

charged rotating viscoelastic jet -

[25] Chitosan bicomponentnanofibers and nanoporous fibers

-

[26] Influence of acetic acid concentration on the

solubilization of chitosan -

X Electrospinning of chitosan dissolved in concentrated

acetic acid solution -

Che K

as wound dressing

– -

[29] Electrospinning of gelatin an -

lactide) blend and its characteristics for wound dressing

-

Preparation and

characterization of electrospun

system -

[31]

-

[32] K Preparation of fish gelatin and fish

-lactide) nanofibers by electrospinning

-

[33] A Electrospinning of chitosan

nanofibers: Processing optimization -

- In vitro gentamicin

sustained and controlled release from chitosan cross-linked films

-

[35] Calculation of Mark–Houwink–Sakurada (MHS) equation viscometric

constants for chitosan in any solvent–temperature system using experimental reported

viscometric constants data -

[36] In vitro assesment of water vapour

transmission of synthesic wound dressing -

Application of benzyl

hyaluronate membranes as potential wound dressing: evaluation of water vapour and

gas permeabilities -

ez- A validated quantitative colorimetric

assay for gentamicin -

[39]

In vitro elution and dissolution of tobramycin and gentamicin from

calcium phosphate -

Amoxycillin release from a floating dosage form

based on alginates - -

74

75

13 (Intrinsic viscosity)

Concentration (g/l) 1 2 3 4 5

Hydrolyzed

chitosan

(ln rel)/c : Kraemer 0.0600 0.0592 0.0588 0.0587 0.0582

sp/c : Huggins 0.0618 0.0628 0.0643 0.0661 0.0676

23

Hydrolyzed chitosan

y 2 Kraemer Huggins [ ] [ ] Mark-Houwink parameter

(1) (2)

K = 1.8×10-3 a = 0.93 (2)

[ ] = (1.8 × 10 / ) . (1)

= [ ] /. × / × . (2)

y = 0.0015x + 0.0601

R² = 0.9914

y = -0.0004x + 0.0602

R² = 0.9281 0.0550

0.0570

0.0590

0.0610

0.0630

0.0650

0.0670

0.0690

0 2 4 6

intr

insic

visc

osity

concentration (g/l)

Huggin eq

Kraemer eq

(Huggin eq)

(Kraemer eq)

76

77

14

(nm)

13 kV 20 cm 15 kV 20 cm 17 kV 20 cm 19 kV 20 cm

1 209.52 171.43 206.03 78.54

2 188.56 209.52 202.48 133.33

3 209.52 171.43 296.31 133.33

4 205.15 231.72 202.48 120.47

5 285.71 310.07 170.37 157.07

6 286.35 247.62 134.69 133.33

7 217.18 190.48 161.62 152.38

8 198.86 210.39 171.43 137.35

9 198.86 153.57 138.67 152.38

10 374.23 212.96 133.33 172.48

Mean 237.39 210.92 181.74 137.07

Std. Dev. 59.45 45.27 49.02 25.53

78

15

(nm)

8 cm 19 kV 12 cm 19 kV 16 cm 19 kV 20 cm 19 kV

1 342.86 231.72 175.61 78.54

2 232.51 206.03 170.37 133.33

3 247.62 259.08 232.51 133.33

4 285.71 229.36 215.50 120.47

5 217.18 250.53 217.18 157.07

6 217.18 266.67 243.93 133.33

7 283.16 229.36 191.43 152.38

8 264.62 205.15 188.56 137.35

9 242.44 209.52 202.48 152.38

10 232.51 243.93 217.18 172.48

Mean 256.58 233.14 205.48 137.07

Std. Dev. 38.88 21.92 24.04 25.53

79

16


(nm)

PLA/h-CS PLA/CS

1 78.54 170.37

2 133.33 247.62

3 133.33 250.53

4 120.47 230.15

5 157.07 230.15

6 133.33 222.13

7 152.38 235.61

8 137.35 222.13

9 152.38 222.13

10 172.48 247.62

Mean 137.07 227.84

Std. Dev. 25.53 23.02

80

17

Hydrolyzed chitosan/PLA

(nm)

PLA 15%wt/h-CS PLA 18%wt/h-CS PLA 20%wt/h-CS

1 111.07 148.77 78.54

2 121.96 127.78 133.33

3 145.06 127.78 133.33

4 153.57 145.06 120.47

5 133.33 127.78 157.07

6 111.07 121.96 133.33

7 153.57 134.69 152.38

8 134.69 120.47 137.35

9 134.69 137.35 152.38

10 134.69 157.07 172.48

Mean 133.37 134.87 137.07

Std. Dev. 15.21 12.11 25.53

81

18

Hydrolyzed chitosan/PLA

(nm)

PLA:h-CS (50:50) PLA:h-CS (60:40)

1 78.54 383.33

2 133.33 396.35

3 133.33 325.49

4 120.47 419.48

5 157.07 307.13

6 133.33 355.84

7 152.38 361.40

8 137.35 434.35

9 152.38 400.45

10 172.48 307.13

Mean 137.07 369.10

Std. Dev. 25.53 45.39

82

83

19

Sample

No.

Tensile Strength

(MPa)

% Elongation

at Break (%)

Tensile Strength

at Yield (MPa)

% Elongation

at Yield (%)

Young’s

modulus (MPa)

1 33.886 3.5323 33.886 3.8364 8.833

2 33.162 3.6537 32.162 3.7350 8.611

3 33.259 3.6989 32.259 3.3876 9.523

4 32.159 3.598 32.159 3.5982 8.938

5 33.447 3.4822 33.447 3.8582 8.669

Mean 33.183 3.59302 32.783 3.6831 8.915

S.D. 0.636 0.087871 0.823 0.1945 0.364

20

10

Sample

No.

Tensile Strength

(MPa)

% Elongation

at Break (%)

Tensile Strength

at Yield (MPa)

% Elongation

at Yield (%)

Young’s

modulus (MPa)

1 30.352 3.8615 30.831 3.1998 9.635

2 31.858 3.4113 31.776 3.4450 9.224

3 29.885 3.6503 29.445 2.8580 10.303

4 30.193 3.4447 30.193 3.2526 9.283

5 29.492 3.8490 30.492 3.0286 10.068

Mean 30.356 3.6434 30.547 3.1568 9.702

S.D. 0.902 0.2140 0.856 0.2235 0.475

84

21

20

Sample

No.

Tensile Strength

(MPa)

% Elongation

at Break (%)

Tensile Strength

at Yield (MPa)

% Elongation

at Yield (%)

Young’s

modulus (MPa)

1 30.975 3.6317 30.752 3.3452 9.193

2 30.905 3.6553 29.395 3.3223 8.848

3 28.845 3.8869 29.346 2.8342 10.354

4 28.997 4.0078 28.997 2.8864 10.046

5 28.674 3.9090 29.174 2.8612 10.196

Mean 29.679 3.8181 29.533 3.0499 9.727

S.D. 1.157 0.1660 0.699 0.2599 0.666

22

30

Sample

No.

Tensile Strength

(MPa)

% Elongation

at Break (%)

Tensile Strength

at Yield (MPa)

% Elongation

at Yield (%)

Young’s

modulus (MPa)

1 26.750 4.5520 26.030 2.6454 9.840

2 27.808 4.2270 30.808 3.0495 10.103

3 27.866 4.2983 28.866 3.0032 9.612

4 27.867 4.3793 28.200 2.8010 10.068

5 28.201 4.2115 29.139 2.9954 9.728

Mean 27.698 4.3336 28.609 2.8989 9.870

S.D. 0.552 0.1390 1.732 0.1708 0.213

85

(Water vapor transmission rate: WVTR)

86

23 (Water vapor transmission rate:

WVTR)

time

(h) g) g) g)

0

1 23.5262 23.5262 0.0000

0.0000 2 23.5261 23.5261 0.0000

3 23.5262 23.5262 0.0000

1

1 23.5262 23.5150 0.0112

0.0113 2 23.5261 23.5147 0.0114

3 23.5262 23.5150 0.0112

2

1 23.1829 23.1601 0.0228

0.0224 2 23.1824 23.1601 0.0223

3 23.1826 23.1604 0.0222

3

1 23.4129 23.3813 0.0316

0.0323 2 23.4129 23.3805 0.0324

3 23.4128 23.3800 0.0328

4

1 23.5112 23.4666 0.0446

0.0447 2 23.5105 23.4662 0.0443

3 23.5112 23.4660 0.0452

5

1 23.5161 23.4600 0.0561

0.0562 2 23.5163 23.4600 0.0563

3 23.5162 23.4599 0.0563

6

1 22.8973 22.8289 0.0684

0.0685 2 22.8970 22.8286 0.0684

3 22.8972 22.8285 0.0687

87


WVTR) 10

time

(h) g) g) g)

0

1 23.3740 23.3740 0.0000

0.0000 2 23.3738 23.3738 0.0000

3 23.3738 23.3738 0.0000

1

1 23.3740 23.3623 0.0117

0.0116 2 23.3738 23.3621 0.0117

3 23.3738 23.3623 0.0115

2

1 23.4635 23.4410 0.0225

0.0225 2 23.4635 23.4410 0.0225

3 23.4633 23.4408 0.0225

3

1 23.6099 23.5766 0.0333

0.0336 2 23.6104 23.5766 0.0338

3 23.6103 23.5765 0.0338

4

1 23.4530 23.4098 0.0432

0.0435 2 23.4535 23.4097 0.0438

3 23.4533 23.4098 0.0435

5

1 23.4788 23.4245 0.0543

0.0541 2 23.4789 23.4247 0.0542

3 23.4785 23.4248 0.0537

6

1 22.8966 22.8292 0.0674

0.0678 2 22.8966 22.8290 0.0676

3 22.8975 22.8290 0.0685

88


WVTR) 20

time

(h) g) g) g)

0

1 23.6374 23.6374 0.0000

0.0000 2 23.6372 23.6372 0.0000

3 23.6378 23.6378 0.0000

1

1 23.6374 23.6253 0.0121

0.0123 2 23.6372 23.6250 0.0122

3 23.6378 23.6253 0.0125

2

1 23.5549 23.5315 0.0234

0.0234 2 23.5547 23.5313 0.0234

3 23.5547 23.5313 0.0234

3

1 23.4577 23.4228 0.0349

0.0348 2 23.4575 23.4227 0.0348

3 23.4573 23.4225 0.0348

4

1 23.3639 23.3197 0.0442

0.0440 2 23.3638 23.3198 0.0440

3 23.3638 23.3199 0.0439

5

1 23.4010 23.3472 0.0538

0.0536 2 23.4012 23.3477 0.0535

3 23.4008 23.3473 0.0535

6

1 23.5773 23.5102 0.0671

0.0670 2 23.5772 23.5102 0.0670

3 23.5772 23.5103 0.0669

89


WVTR) 40

time

(h) g) g) g)

0

1 23.4589 23.4589 0.0000

0.0000 2 23.4593 23.4593 0.0000

3 23.4588 23.4588 0.0000

1

1 23.4589 23.4464 0.0125

0.0125 2 23.4593 23.4465 0.0128

3 23.4588 23.4465 0.0123

2

1 23.5168 23.4991 0.0177

0.0178 2 23.5172 23.4992 0.0180

3 23.5169 23.4991 0.0178

3

1 23.5638 23.5348 0.0290

0.0289 2 23.5637 23.5349 0.0288

3 23.5636 23.5348 0.0288

4

1 23.3977 23.3599 0.0378

0.0376 2 23.3975 23.3599 0.0376

3 23.3973 23.3598 0.0375

5

1 23.3839 23.3342 0.0497

0.0496 2 23.3838 23.3343 0.0495

3 23.3837 23.3342 0.0495

6

1 23.4952 23.4390 0.0562

0.0562 2 23.4953 23.4391 0.0562

3 23.4953 23.4391 0.0562

90

24

25

10

y = 0.0113x - 0.0004

R² = 0.9992

-0.0200

0.0000

0.0200

0.0400

0.0600

0.0800

0 2 4 6 8

()

( )

y = 0.011x + 0.0002

R² = 0.9986

0.0000

0.0200

0.0400

0.0600

0.0800

0 2 4 6 8

()

( )

91

26

20

y = 0.0109x + 0.001

R² = 0.9979

0.0000

0.0200

0.0400

0.0600

0.0800

0 2 4 6 8

()

( )

y = 0.0094x + 0.0008

R² = 0.9944

0.0000

0.0100

0.0200

0.0300

0.0400

0.0500

0.0600

0 1 2 3 4 5 6 7

()

( )

92

27

40

27 (Water vapor transmission rate: WVTR)

Sample w/ t

(g/h)

time

(h/day) (m2)

WVTR

(g/m2 day)

PLA/h-CS 0.0113 24 0.000151 1796.03

PLA/h-CS

Add gentamicin 10 mg/ml 0.0111 24 0.000151 1764.24

PLA/h-CS


PLA/h-CS


93

(Water adsorption)

94

28 (Water adsorption)

Sample info Sample

No. g) g)

%water

absorption SD

PLA/h-CS

1 0.0031 0.0122 293.55

297.89 4.99 2 0.0031 0.0123 296.77

3 0.003 0.0121 303.33

PLA/h-CS

Add GS 10 mg/ml

1 0.003 0.0119 296.67

291.33 9.25 2 0.003 0.0119 296.67

3 0.0031 0.0118 280.65

PLA/h-CS

Add GS 20 mg/ml

1 0.0031 0.0114 267.74

262.87 8.44 2 0.0032 0.0113 253.13

3 0.0031 0.0114 267.74

PLA/h-CS

Add GS 40 mg/ml

1 0.0032 0.0107 234.38

239.42 4.37 2 0.0031 0.0106 241.94

3 0.0031 0.0106 241.94

95

(Cytotoxicity)

96

29 (Cytotoxicity)

(mg/ml)

OD %

1 2 3 4

GS 0

0 0.287 0.336 0.271 0.323 0.304 100.00

0.25 0.312 0.331 0.296 0.296 0.309 101.48

0.5 0.376 0.348 0.300 0.316 0.335 110.11

1 0.345 0.339 0.335 0.308 0.332 109.04

2 0.281 0.303 0.254 0.259 0.274 90.14

4 0.310 0.337 0.299 0.300 0.312 102.38

GS 10 mg/ml

0 0.277 0.345 0.318 0.318 0.315 100.00

0.25 0.357 0.377 0.334 0.334 0.351 111.45

0.5 0.313 0.330 0.271 0.319 0.308 98.01

1 0.311 0.345 0.307 0.325 0.322 102.38

2 0.310 0.317 0.301 0.317 0.311 98.97

4 0.300 0.315 0.306 0.341 0.316 100.32

GS 20 mg/ml

0 0.302 0.312 0.337 0.329 0.320 100.00

0.25 0.340 0.362 0.387 0.381 0.368 114.84

0.5 0.325 0.311 0.284 0.271 0.298 93.05

1 0.313 0.322 0.368 0.346 0.337 105.39

2 0.282 0.315 0.339 0.322 0.315 98.28

4 0.292 0.319 0.337 0.362 0.328 102.34

GS 40 mg/ml

0 0.318 0.317 0.322 0.368 0.331 100.00

0.25 0.366 0.318 0.354 0.339 0.344 103.92

0.5 0.296 0.345 0.394 0.334 0.342 103.32

1 0.300 0.307 0.374 0.352 0.333 100.60

2 0.313 0.324 0.359 0.389 0.346 104.53

4 0.270 0.316 0.320 0.317 0.306 92.30

97

(Drug release)

98

30 max = 400

24

GS load (mg/ml) Absorbance Actual GS (mg/m)

10 0.335 7.12

20 0.513 18.04

40 0.829 37.42

31 max = 400

GS-load (mg/ml) Time (hr) Absorbance Drug release (mg/ml)

10

1 0.262 2.64

2 0.251 1.96

3 0.237 1.10

6 0.231 0.74

12 0.222 0.18

24 0.221 0.12

72 0.22 0.06

20

1 0.284 3.99

2 0.277 3.56

3 0.267 2.94

6 0.251 1.96

12 0.242 1.41

24 0.235 0.98

72 0.223 0.25

40

1 0.357 8.47

2 0.323 6.38

3 0.286 4.11

99

31 ( ) max = 400

GS-load (mg/ml) Time (hr) Absorbance Drug release (mg/ml)

40

6 0.277 3.56

12 0.269 3.07

24 0.256 2.27

72 0.242 1.41

Absorbance

UV-VIS spectrophotometer max 400 y =

0.0618x + 0.219

y Absorbance x

100

-

20/3

92000

(080) 430-1458

E-mail address [email protected]

2548

2552

2552

2552-2554

1. Amornrat Tantithumrongwut and Nattawut Chaiyut, “Preparation of electrospun

chitosan/poly(lactic acid) nanofibers”, The 3rd National Research Symposium on Petroleum,

Petrochemicals, and Advanced Materials and The 18th PPC Symposium on Petroleum,

Petrochemicals, and Polymer, April 24, 2012, Bangkok, Thailand.

Documents

Ã ¥ µ µª°¤¦¦´ r ´ µ¦ ª» ·· Î ª· ¥µ ·¡ r ¸ÊÁ È nª ... · 2014-10-22 · ACID) NANOFIBER FOR POTENTIAL APPLICATION AS WOUND DRESSING ÿ Ü By Miss Amornrat