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Surface-Engineered Albumin-Phospholipid Nanotube-like Nanoconstructs for Potent Antibacterial and Interfacial Biofilm Disruption

  • 조영흔 (서강대학교 일반대학원)

초록(요약문)

Antibacterial and Anti-biofilm Strategies against Pathogenic Bacteria using Biocompatible Albumin-Phospholipid Nanoparticles Antibiotic treatment is increasingly limited not only by resistance in planktonic bacteria but also by tolerance of mature biofilms. This dissertation investigated phospholipid-based nanotube-like structures assembled onto albumin nanoparticles （pLN-NPs） as a biocompatible nanoplatform for controlling both actively growing pathogens and mature biofilms. In planktonic systems, pLN-NPs showed bactericidal activity against Gram-negative and Gram-positive pathogens and acted through physical disruption of bacterial membranes. Unlike conventional antibiotics that target easily mutable metabolic pathways, the unique structural design of pLN-NPs physically compromises bacterial membranes, providing a robust mechanism that is fundamentally less prone to the development of bacterial resistance. Using Vibrio vulnificus （V. vulnificus） as a principal model, this study further showed that pLN-NPs reduced pathogenicity in vivo and enhanced the efficacy of conventional antibiotics. The dissertation then extended the analysis to mature V. vulnificus biofilms. In this setting, pLN-NPs preferentially interacted with superficial biofilm regions and were associated with EPS-matrix destabilization and membrane perturbation of biofilm-embedded bacteria. Their anti-biofilm activity was retained across multiple matrix-related mutant backgrounds, suggesting that the response was not governed by any single tested surface determinant. In addition, pretreatment with pLN-NPs improved the anti-biofilm efficacy of kanamycin. Together, these findings indicate that pLN-NPs function as an active interfacial nanoplatform with dual bactericidal and anti-biofilm activity, and support their potential use as antibiotic-sensitizing nanomaterials for clinically difficult-to-treat bacterial infections. This work links nanoscale surface architecture to antibacterial function across two biologically distinct bacterial states.

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목차

Chapter 1. Literature review
Strategies for overcoming antibiotic resistance and bacterial biofilms using phospholipid-based nanotube-assembled protein nanoparticles (pLN-NPs)
1. Antibiotic resistance and unmet clinical need .............................................................................. 13
1.1 Global burden of antimicrobial resistance .................................................................................. 13
1.2 Major resistant pathogens and the ESKAPE problem .............................................................. 14
1.3 Molecular mechanisms of antibiotic resistance ......................................................................... 15
1.4 Limitations of conventional antibiotic discovery ...................................................................... 16
1.5 Why membrane-active alternatives are needed ......................................................................... 17
2. Bacterial biofilms as a major therapeutic barrier ....................................................................... 20
2.1 Stages of biofilm formation and maturation .............................................................................. 20
2.2 Composition and functions of the biofilm matrix ...................................................................... 20
2.3 Diffusion limitation and spatial heterogeneity in biofilms ........................................................ 21
2.4 Persister cells and biofilm-associated antibiotic tolerance ........................................................ 21
2.5 Clinical importance of mature biofilms ..................................................................................... 22
3. Target pathogens ............................................................................................................................ 25
3.1 Vibrio vulnificus: clinical severity and pathogenesis ............................................................... 25
3.2 Surface polysaccharides and biofilm determinants of V. vulnificus ........................................ 25
3.3 Staphylococcus aureus as a Gram-positive comparator ........................................................... 27
3.4 Why these two organisms were selected for this dissertation .................................................. 27
4. Nanotechnology in antimicrobial therapy ................................................................................... 29
4.1 General rationale for antibacterial nanotechnology .................................................................. 29
4.2 Inorganic and organic nanomaterial platforms ........................................................................ 29
4.3 Membrane-disruptive nano-antibiotics ..................................................................................... 30
4.4 Nanoparticle strategies against bacterial biofilms .................................................................... 31
4.5 Nanoparticle-enabled antibiotic potentiation ........................................................................... 32
4.6 Translational considerations ...................................................................................................... 33
5. Albumin nanoparticles as a biocompatible engineering scaffold .............................................. 35
5.1 Albumin as a carrier material .................................................................................................... 35
5.2 Fabrication and surface properties of albumin nanoparticles .................................................. 35
5.3 Translational rationale for the use of albumin nanoparticles .................................................. 36
6. Self-assembly of phospholipid nanostructures and pLN-NPs design rationale ...................... 36
6.1 Amphiphile packing and liposome formation .......................................................................... 36
6.2 Curvature elasticity and non-spherical lipid morphologies ..................................................... 37
6.3 Lipid nanotubes and tubular transitions ................................................................................... 37
6.4 Literature-based interpretation of pLN formation on albumin nanoparticles ........................ 38
6.5 Engineering significance and boundary of the claim ................................................................ 39
7. Preliminary formulation screening and process rationale for pLN-NPs fabrication ............... 39
7.1 Effect of albumin nanoparticle core size on pLN-NPs morphology and antibacterial activity ................................................................................................................................................ 40
7.2 Effect of DSPC/DSPE-PEG-NHS ratio on pLN-NPs antibacterial activity ............................ 44
7.3 Simplification of the fabrication route from microbubble-assisted preparation to direct liposome-mediated assembly ............................................................................................................. 46
7.4 Effect of liposome preparation temperature on pLN-NPs antibacterial activity .................... 46
7.5 Effect of 100 kDa Amicon tube processing on final pLN-NPs activity .................................... 47
7.6 Selection of the final pLN-NPs formulation ............................................................................... 48
8. Goals of this study ......................................................................................................................... 50
9. References ....................................................................................................................................... 53


Chapter 2.
Bactericidal effect of pLN-NPs against actively growing pathogens via physical membrane disruption
1. Abstract .......................................................................................................................................... 61
2. Introduction .................................................................................................................................... 62
3. Materials and methods .................................................................................................................. 64
3.1 Synthesis of protein-based nanoparticles ................................................................................... 64
3.2 Preparation of phospholipid liposomes and fabrication of pLN-NPs ..................................... 64
3.3 Determination of protein concentration and estimation of the molar concentration of pLN-NPs ...................................................................................................................................................... 65
3.4 Culture conditions ........................................................................................................................ 66
3.5 Bacterial susceptibility assay ....................................................................................................... 66
3.6 Electron microscopic analysis of pLN-NPs-treated bacteria ................................................... 67
3.7 Propidium iodide uptake assay and fluorescence microscopy ................................................. 69
3.8 Fluorescent labeling and confocal imaging of pLN-NPs association with bacterial cells ...... 69
3.9 Cytotoxicity assay in human-derived cell lines ......................................................................... 70
3.10 In vivo biodistribution, histological analysis, and serum cytokine assay in mice ................. 70
3.11 Mouse mortality assay ............................................................................................................... 72
3.12 Bacterial colonization of mouse organs ................................................................................... 72
3.13 Animal model ............................................................................................................................. 73
3.14 Statistical analysis ...................................................................................................................... 73
4. Results ............................................................................................................................................ 73
4.1 Physicochemical characterization and colloidal stability of pLN-NPs .................................... 73
4.2 Approximate molar concentration of pLN-NPs based on protein concentration and DLS analysis ............................................................................................................................................... 77
4.3 Dose-dependent bactericidal activity of pLN-NPs against actively growing pathogens ......... 79
4.4 The precursor components of pLN-NPs do not reproduce the bactericidal effect .................. 81
4.5 Limited cytotoxicity of pLN-NPs toward human-derived cell lines ........................................ 83
4.6 pLN-NPs induce structural damage in bacterial cells .............................................................. 85
4.7 pLN-NPs associate with the surface of Vibrio vulnificus cells ................................................. 88
4.8 pLN-NPs increase membrane permeability in wild-type Vibrio vulnificus but not in the CPS-deficient mutant ................................................................................................................................ 90
4.9 Reduced susceptibility of the CPS-deficient wbpP mutant to pLN-NPs treatment ................ 93
4.10 Biodistribution and acute histological safety of intraperitoneally administered pLN-NPs ................................................................................................................................................... 95
4.11 Limited serum cytokine response after intraperitoneal injection of pLN-NPs ..................... 99
4.12 pLN-NPs reduce the pathogenicity of Vibrio vulnificus in mice ........................................... 101
4.13 pLN-NPs enhance bacterial susceptibility to conventional antibiotics ................................ 104
5. Discussion ..................................................................................................................................... 107
6. References ..................................................................................................................................... 113


Chapter 3.
Eradication of mature biofilms by pLN-NPs and synergistic sensitization to conventional antibiotics
1. Abstract ......................................................................................................................................... 119
2. Introduction ................................................................................................................................... 120
3. Materials and methods ................................................................................................................. 122
3.1 Preparation of thiolated HSA nanoparticles ............................................................................ 122
3.2 Preparation of liposomes and fabrication of pLN-NPs ........................................................... 123
3.3 Bacterial strains, mature biofilm formation, and nanoparticle treatment ............................. 124
3.4 Quantification of biofilm biomass by crystal violet staining .................................................. 124
3.5 Fluorescent labeling of pLN-NPs, albumin nanoparticles, and liposomes and confocal laser scanning microscopy ....................................................................................................................... 124
3.6 Quantification of fluorescence intensity and depth distribution ............................................ 125
3.7 Phenomenological analysis of surface capture and erosion-like decay .................................. 125
3.8 Membrane permeability assay in biofilm-embedded bacteria ................................................ 127
3.9 Scanning electron microscopy .................................................................................................. 127
3.10 Kanamycin dose selection and sequential treatment assay .................................................. 127
3.11 Statistical analysis .................................................................................................................... 128
4. Results .......................................................................................................................................... 128
4.1 Dose- and time-dependent biofilm reduction by pLN-NPs .................................................... 128
4.2 Selection of 12 h as the standard treatment window .............................................................. 131
4.3 Superficial localization and structural perturbation by pLN-NPs ........................................ 134
4.4 Distinct interfacial responses of liposomes and albumin nanoparticles ................................ 138
4.5 Membrane permeability of biofilm-embedded bacteria ......................................................... 141
4.6 SYTO9/PI staining of mutant biofilms .................................................................................... 144
4.7 PI/SYTO9 ratios in mutant biofilms ....................................................................................... 147
4.8 Biofilm reduction in mutant backgrounds ............................................................................... 149
4.9 Kanamycin dose selection ......................................................................................................... 151
4.10 Kanamycin sensitization by pLN-NPs ................................................................................... 153
5. Discussion ..................................................................................................................................... 156
6. References ..................................................................................................................................... 163


Chapter 4.
Development of melittin polymersomes inducing lysosomal rupture
1. Abstract ......................................................................................................................................... 167
2. Introduction ................................................................................................................................... 168
3. Materials and methods ................................................................................................................. 170
3.1 Materials ..................................................................................................................................... 171
3.2 Preparation of PM NPs and PMH NPs ................................................................................... 171
3.3 Characterizations of PM NPs and PMH NPs .......................................................................... 172
3.4 Hemolysis assay of PM NPs and PMH NPs ........................................................................... 174
3.5 In vitro fluorescence imaging of cellular uptake ..................................................................... 174
3.6 In vitro toxicity study of PMH NPs ........................................................................................ 176
3.7 In vivo distribution of PMH NPs ............................................................................................ 177
3.8 In vivo therapy and histological analysis of PMH NPs ......................................................... 177
3.9 Statistical analysis ...................................................................................................................... 178
4. Results and discussion ................................................................................................................. 178
4.1 Characterization of PM NPs and PMH NPs ........................................................................... 178
4.2 Cellular viability ........................................................................................................................ 189
4.3 Hemolytic activity of PMH NPs .............................................................................................. 196
4.4 Cellular uptake of PMH NPs .................................................................................................... 196
4.5 In vivo biodistribution of intravenously administered PMH NPs ......................................... 203
4.6 In vivo anti-cancer therapeutic efficacy of PMH NPs ........................................................... 207
5. Conclusion .................................................................................................................................... 211
6. References ..................................................................................................................................... 212


Chapter 5.
Hyaluronan-coated polymersomes for macrophage-directed inflammatory modulation
1. Abstract ......................................................................................................................................... 217
2. Introduction ................................................................................................................................... 218
3. Materials and methods ................................................................................................................. 220
3.1 Materials ..................................................................................................................................... 220
3.2 Preparation of PM and PMHA ................................................................................................. 220
3.3 Characterization of PM and PMHA ........................................................................................ 221
3.4 In vitro drug release studies ..................................................................................................... 222
3.5 Cell culture ................................................................................................................................. 223
3.6 In vitro cytotoxicity assay ......................................................................................................... 223
3.7 Hemolysis assay of MEL and PMHA ....................................................................................... 224
3.8 In vitro flow cytometry and cellular uptake imaging ............................................................ 225
3.9 In vitro gene expression ............................................................................................................ 225
3.10 Quantification of TNF-α and COX-2 by ELISA .................................................................. 226
3.11 Combination index analysis .................................................................................................... 227
3.12 Statistical analysis .................................................................................................................... 228
4. Results and discussion ................................................................................................................. 228
4.1 Design and synthesis of HA-coated nanoparticles for inflammation-targeted therapy ......... 229
4.2 Drug loading and in vitro release profile ................................................................................. 233
4.3 Cell viability and hemolysis assay of PM and PMHA ........................................................... 234
4.4 In vitro cellular uptake studies ................................................................................................. 238
4.5 Elucidation of cellular uptake route: role of TLR4 ................................................................. 241
4.6 Validation of pro-inflammatory cytokine expression .............................................................. 242
5. Conclusion .................................................................................................................................... 246
6. References ..................................................................................................................................... 247


Chapter 6.
Overall conclusion ......................................................................................................................... 252

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