Thesis for the Degree of
Master of Science in Pharmacy
Development of Nitric Oxide-releasing
Polymeric Nanoparticles for
Antibacterial and Wound Healing
Activity
August, 2015
The Graduate School
Pusan National University
Department of Manufacturing Pharmacy
Hasan Nurhasni
Thesis for the Degree of
Master of Science in Pharmacy
Development of Nitric Oxide-releasing
Polymeric Nanoparticles for
Antibacterial and Wound Healing
Activity
Supervisor: Yoo Jin-Wook
August 2015
The Graduate School
Pusan National University
Department of Manufacturing Pharmacy
Hasan Nurhasni
This Thesis for the
Degree of Master of Science in Pharmacy
By
Hasan Nurhasni
Has been approved
June 16, 2015
Chair Jung Yunjin
____________
Committee member Lee Joon-Hee
____________
Committee member Yoo Jin-Wook
____________
CONTENT
Page
TABLE .......................................................................................
iv
LIST OF FIGURES ..................................................................
iv
ABBREVIATIONS .................................................................
vii
I.
Introduction ........................................................................
1
II. Materials and Methods .....................................................
4
1. Materials ..............................................................................................
4
2. Synthesis of PEI/NONOate..................................................................
4
3. Characterization of PEI/NONOate ......................................................
5
4. Fabrication of nanoparticles .................................................................
6
5. Characterization of nanoparticles ........................................................
6
6. NO measurement in PEI/NONOate and NO/PPNPs ...........................
7
7. In vitro NO release .............................................................................
8
8. Antibacterial activity............................................................................
8
9. Nanoparticles adhesion to the bacteria ................................................
9
10. Anti-biofilm activity ............................................................................
10
11. In-vivo biofilm .....................................................................................
10
i
12. In vitro cytotoxicity study ....................................................................
12
13. Induction of diabetes ............................................................................
12
14. In vivo wound healing activity .............................................................
13
15. Histological processing of wound area ................................................
14
16. Statistical analysis ................................................................................
14
III. Results and Discussion ....................................................
15
1. Synthesis of PEI/NONOate..................................................................
15
2. Characterization of PEI/NONOate ......................................................
16
3. Characterization of nanoparticles ........................................................
22
4. In vitro NO release ...............................................................................
24
5. Antibacterial activity............................................................................
26
6. Nanoparticles adhesion to the bacteria ................................................
30
7. Anti-biofilm activity ............................................................................
33
8. Biofilm characterization of in vivo biofilm..........................................
35
9. In vitro cytotoxicity study ....................................................................
38
10. Blood glucose level ..............................................................................
40
11. In vivo wound healing activity .............................................................
41
IV. Conclusion .........................................................................
55
ii
V.
References ..........................................................................
56
VI. Abstract ..............................................................................
61
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TABLE
Page
Table 1. Characterizations of NPs ....................................................
21
LIST OF FIGURES
Page
Figure 1. (A) Synthesis of PEI/NONOate and (B) fabrication of NOreleasing PLGA-PEI NPs .............................................................................
15
Figure 2. Characterization of PEI/NONOate by 1H-NMR..........................
17
Figure 3. Characterization of PEI/NONOate by FT-IR...............................
18
Figure 4. Characterization of PEI/NONOate by UV-Vis spectra ................
20
Figure 5. Characterization of nanoparticles ................................................
22
Figure 6. Zeta potential distribution ............................................................
23
Figure 7. In vitro release profile of PEI/NONOate and NO/PPNPs ...........
25
Figure 8. Antibacterial activity of PPNPs and NO/PPNPs against MRSA
and P. aeruginosa .........................................................................................
iv
27
Figure 9. Confocal microscopy images of MRSA (left panel) and P.
aeruginosa (right panel) after 24 h of treatment with nanoparticles at
various concentrations .................................................................................
28
Figure 10. The percent (%) survival of MRSA (left panel) and P.
aeruginosa (right panel) after 24 h of treatment with nanoparticles at
various concentrations. ................................................................................
29
Figure 11. Adhesion of PLGA NPs, PPNPs and NO/PPNPs to gram
positive MRSA and gram negative P.aeruginosa ........................................
32
Figure 12. Anti-biofilm activity of NO/PPNPs. MRSA biofilm were
grown in coupon for up to 24 h in the presence or absence of NO/PPNPs.
34
Figure 13. Biofilm formation in cutaneous mouse wounds ........................
36
Figure 14. Construction of an EPS-rich matrix and 3D biofilm
architecture ...................................................................................................
37
Figure 15. Viability of L929 mouse fibroblast cells following 24 hexposure to nanoparticles at various concentrations ..................................
39
Figure 16. Changes in blood glucose level after a single i.p. injection of
Streptozotocin ..............................................................................................
40
Figure 17. Normal wound healing activity in Balb/c mice .........................
43
Figure 18. Histological analysis of normal wound in Balb/c mice .............
44
Figure 19. Biofilm-based wound challenged in healthy Balb/c mice .......
45
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Figure 20. Histological analysis of biofilm-based wound challenged in
healthy Balb/c mice......................................................................................
46
Figure 21. Biofilm-based wound challenged in diabetic Balb/c mice ........
47
Figure 22. Histological analysis of biofilm-based wound challenged in
diabetic Balb/c mice. ....................................................................................
48
Figure 23. Biofilm-based wound challenged in healthy ICR Mice ............
49
Figure 24. Histological analysis of biofilm-based wound challenged in
healthy ICR mice .........................................................................................
50
Figure 25. Biofilm-based wound challenged in diabetic ICR Mice ...........
51
Figure 26. Histological analysis of biofilm-based wound challenged in
diabetic ICR mice ........................................................................................
52
Figure 27. Biofilm-based wound challenged in diabetic db/db mice .........
53
Figure 28. Histological analysis of biofilm-based wound challenged in
diabetic db/db mice ......................................................................................
vi
54
ABBREVIATIONS
NO
Nitric oxide
PLGA
Poly (lactide-co-glycolide)
NPs
Nanoparticles
PEI
Polyethylenimine
PPNPs
PLGA-PEI nanoparticles
NO/PPNPs
Nitric Oxide-releasing PLGA-PEI nanoparticles
SEM
Scanning electron microscopy
1
Proton nuclear magnetic resonance
H-NMR
FT-IR
Fourier transforms infrared spectroscopy
UV-Vis
Ultraviolet-visible
DPBS
Dulbecco’s phosphate-buffered saline
NONOate
Diazeniumdiolate
MRSA
Methicillin-resistant Staphylococcus aureus
CFU
Colony forming units
PI
Propidium iodide
EPS
Extracellular polymeric substance
vii
Abstract
Nitric oxide (NO)-releasing nanoparticles (NPs) have emerged as a wound healing
enhancer and a novel antibacterial agent that can circumvent antibiotic resistance.
However, the NO release from nanoparticles over extended periods of time is still
inadequate for clinical application. In this study, we developed NO-releasing
polymeric nanoparticles (NO/PPNPs) composed of poly (lactide-co-glycolide) (PLGA)
and polyethylenimine (PEI)/NO adduct (PEI/NONOate) for prolonged NO release,
antibacterial and wound healing activity. Successful preparation of PEI/NONOate was
confirmed by proton nuclear magnetic resonance (1H-NMR), Fourier transform
infrared spectroscopy (FT-IR), and ultraviolet/visible (UV/Vis) spectrophotometry.
NO/PPNPs were characterized by particle size, surface charge and NO loading. The
NO/PPNPs showed a prolonged NO release profile over 6 days without any burst
release. The NO/PPNPs exhibited potent bactericidal activity against methicillinresistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa concentrationdependently and showed the ability to bind on the surface of the bacteria. it is found
that the NO released from the NO/PPNPs mediates bactericidal activity and is not
toxic to healthy fibroblast cells. Furthermore, NO/PPNPs acted as biofilm dispersal
and accelerated wound healing and epithelialization in normal wound, biofilm-based
wound in healthy mice and in diabetic mice. Therefore, our results suggest that the
viii
NO-releasing polymeric NPs presented in this study could be a suitable approach for
treating wounds and various skin infections.
Keywords: Nitric oxide-releasing nanoparticles, PLGA, PEI, antimicrobial, wound healing
ix