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. 2015 Apr 22:10:3065-80.
doi: 10.2147/IJN.S82199. eCollection 2015.

Nitric oxide-releasing poly(lactic-co-glycolic acid)-polyethylenimine nanoparticles for prolonged nitric oxide release, antibacterial efficacy, and in vivo wound healing activity

Hasan Nurhasni  1 , Jiafu Cao  1 , Moonjeong Choi  1 , Il Kim  2 , Bok Luel Lee  1 , Yunjin Jung  1 , Jin-Wook Yoo  1
Affiliations

Nitric oxide-releasing poly(lactic-co-glycolic acid)-polyethylenimine nanoparticles for prolonged nitric oxide release, antibacterial efficacy, and in vivo wound healing activity

Hasan Nurhasni et al. Int J Nanomedicine. .

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 NPs over extended periods of time is still inadequate for clinical application. In this study, we developed NO-releasing poly(lactic-co-glycolic acid)-polyethylenimine (PEI) NPs (NO/PPNPs) composed of poly(lactic-co-glycolic acid) and PEI/diazeniumdiolate (PEI/NONOate) for prolonged NO release, antibacterial efficacy, and wound healing activity. Successful preparation of PEI/NONOate was confirmed by proton nuclear magnetic resonance, Fourier transform infrared spectroscopy, and ultraviolet/visible 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 efficacy against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa concentration-dependently and showed the ability to bind on the surface of the bacteria. We also found that the NO released from the NO/PPNPs mediates bactericidal efficacy and is not toxic to healthy fibroblast cells. Furthermore, NO/PPNPs accelerated wound healing and epithelialization in a mouse model of a MRSA-infected wound. Therefore, our results suggest that the NO/PPNPs presented in this study could be a suitable approach for treating wounds and various skin infections.

Keywords: PEI; PLGA; antimicrobial; nitric oxide-releasing nanoparticles; wound healing.

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Figures

Figure 1
Figure 1
Synthesis of PEI/NONOate, followed by NO/PPNPs fabrication. Notes: (A) Synthesis of PEI/NONOate and (B) fabrication of NO/PNNPs. Abbreviations: NONOate, diazeniumdiolate; NO, nitric oxide; PLGA, poly(lactic-co-glycolic acid); PEI, polyethylenimine; NPs, nanoparticles; THF, tetrahydrofuran; NO/PPNPs, NO-releasing PLGA-PEI nanoparticles; PVA, poly(vinyl alcohol); DCM, dichloromethane; NaOCH3, sodium methoxide; MeOH, methanol.
Figure 2
Figure 2
Characterization of PEI/NONOate by 1H NMR and FTIR. Notes: (A) 1H NMR spectra of PEI and PEI/NONOate. (B) FTIR spectra of PEI/NONOate, PLGA, PPNPs, and NO/PPNPs. The red circles in A and B refer to proton signals of methylene groups and diazeniumdiolates group. Abbreviations: 1H NMR, proton nuclear magnetic resonance; PEI, polyethylenimine; PLGA, poly(lactic-co-glycolic acid); FTIR, Fourier transform infrared spectroscopy; PPNPs, PLGA-PEI nanoparticles; NO/PPNPs, NO-releasing PLGA-PEI nanoparticles; NO, nitric oxide; NONOate, diazeniumdiolate.
Figure 3
Figure 3
Characterization of PEI/NONOate by UV-Vis spectra. Notes: (A) UV-Vis absorption of PEI/NONOate (violet line), PEI/NONOate with Griess reagent (green line). (B) UV-Vis spectra of PEI/NONOate in different concentrations. (C) UV-Vis spectra of PEI/NONOate in DPBS at 37°C at different times. Abbreviations: NED, N-1-naphthylethylenediamine; UV-Vis, ultraviolet-visible; PEI, polyethylenimine; DPBS, Dulbecco’s phosphate-buffered saline; NONOate, diazeniumdiolate.
Figure 4
Figure 4
Characterization of nanoparticles. Notes: (A) SEM images of PPNPs and NO/PPNPs, bars represent 500 nm. (B) Size distribution of PPNPs and NO/PPNPs. Abbreviations: SEM, scanning electron microscopy; PLGA, poly(lactic-co-glycolic acid); PEI, polyethylenimine; PPNPs, PLGA-PEI nanoparticles; NO/PPNPs, NO-releasing PLGA-PEI nanoparticles; NONOate, diazeniumdiolate; NO, nitric oxide.
Figure 5
Figure 5
In vitro release profile of PEI/NONOate and NO/PPNPs. Notes: All samples were placed in DPBS at 37°C; data presented are mean ± standard deviation; n=3. Abbreviations: PEI, polyethylenimine; PPNPs, PLGA-PEI nanoparticles; NO/PPNPs, NO-releasing PLGA-PEI nanoparticles; NONOate, diazeniumdiolate; NO, nitric oxide; PLGA, poly(lactic-co-glycolic acid); DPBS, Dulbecco’s phosphate-buffered saline; h, hour(s).
Figure 6
Figure 6
Antibacterial activity of PPNPs and NO/PPNPs against MRSA and Pseudomonas aeruginosa. Notes: The number of CFU (A) of MRSA and (B) of P. aeruginosa. Data shown are mean ± standard deviation; n=3. Abbreviations: PLGA, poly(lactic-co-glycolic acid); PEI, polyethylenimine; PPNPs, PLGA-PEI nanoparticles; NO/PPNPs, NO-releasing PLGA-PEI nanoparticles; MRSA, methicillin-resistant Staphylococcus aureus; CFU, colony forming units; NO, nitric oxide.
Figure 7
Figure 7
Confocal microscopy images and the percent (%) survival of MRSA (left panel) and Pseudomonas aeruginosa (right panel) after 24 hours of treatment with nanoparticles at different concentrations. Notes: (A) PPNPs, (B) NO/PPNPs, (C) percent (%) survival against MRSA and (D) percent (%) survival against P. aeruginosa. Syto-9 fluorescence (green) indicates intact membrane of healthy bacteria, PI fluorescence (red) indicates membrane destruction and cell death. Bacterial survival at each point is presented as a percentage relative to the control group (buffer alone). Data shown are mean ± standard deviation; n=3. (A and B) Bars represent 20 μm. Abbreviations: PLGA, poly(lactic-co-glycolic acid); PEI, polyethylenimine; PPNPs, PLGA-PEI nanoparticles; NO/PPNPs, NO-releasing PLGA-PEI nanoparticles; MRSA, methicillin-resistant Staphylococcus aureus; PI, propidium iodide; NO, nitric oxide.
Figure 7
Figure 7
Confocal microscopy images and the percent (%) survival of MRSA (left panel) and Pseudomonas aeruginosa (right panel) after 24 hours of treatment with nanoparticles at different concentrations. Notes: (A) PPNPs, (B) NO/PPNPs, (C) percent (%) survival against MRSA and (D) percent (%) survival against P. aeruginosa. Syto-9 fluorescence (green) indicates intact membrane of healthy bacteria, PI fluorescence (red) indicates membrane destruction and cell death. Bacterial survival at each point is presented as a percentage relative to the control group (buffer alone). Data shown are mean ± standard deviation; n=3. (A and B) Bars represent 20 μm. Abbreviations: PLGA, poly(lactic-co-glycolic acid); PEI, polyethylenimine; PPNPs, PLGA-PEI nanoparticles; NO/PPNPs, NO-releasing PLGA-PEI nanoparticles; MRSA, methicillin-resistant Staphylococcus aureus; PI, propidium iodide; NO, nitric oxide.
Figure 8
Figure 8
Adhesion of PLGA NPs, PPNPs, and NO/PPNPs to bacteria. Notes: (A) MRSA and (B) Pseudomonas aeruginosa. Nanoparticles were incubated with bacteria for 2 hours and images were obtained using a confocal microscope. Bacterial membrane (green) stained with Syto-9 and nanoparticles (red) labeled with nile red for visualization. Abbreviations: PLGA, poly(lactic-co-glycolic acid); PEI, polyethylenimine; PPNPs, PLGA-PEI nanoparticles; NO/PPNPs, NO-releasing PLGA-PEI nanoparticles; MRSA, methicillin-resistant Staphylococcus aureus; NO, nitric oxide; NPs, nanoparticles.
Figure 9
Figure 9
Viability (%) of L929 mouse fibroblast cells following 24-hour exposure to nanoparticles at different concentrations (n=8). Abbreviations: PLGA, poly(lactic-co-glycolic acid); PEI, polyethylenimine; NPs, nanoparticles; PPNPs, PLGA-PEI nanoparticles; NO/PPNPs, NO-releasing PLGA-PEI nanoparticles; NO, nitric oxide.
Figure 10
Figure 10
Wound healing assay in mice. Notes: (A) Representative photographs of MRSA-infected wounds of BALB/c mice treated with or without NO/PPNPs. (B) Area reduction (%) profiles of the wounds. Values are mean ± standard deviation, n=4, *P<0.05 compared with untreated group. Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; NO/PPNPs, NO-releasing PLGA-PEI nanoparticles; PLGA, poly(lactic-co-glycolic acid); PEI, polyethylenimine; NO, nitric oxide.
Figure 11
Figure 11
Histological sections of normal skin, untreated, and NO/PPNPs stained with hematoxylin and eosin. Notes: Histological analysis of BALB/c mice at day 7, scale bar =50 μm. The arrows colored with black, blue, yellow, white, and red indicate edema, ulceration, early epithelialization, mononuclear inflammatory cell, and fibroblast cell, respectively. Abbreviations: NO/PPNPs, NO-releasing PLGA-PEI nanoparticles; PLGA, poly(lactic-co-glycolic acid); PEI, polyethylenimine; NO, nitric oxide; HF, hair follicles; Epi, epidermis; Seb, sebaceous glands.
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