Object

Alternative title:

Biophysical analysis of the permeabilization mechanism of outer bacterial membrane with the participation of dendritic nanoparticles

Abstract:

Antibiotic resistance is an ever-growing threat to public health due to the overuse and misuse of antibiotics, including in outpatient clinics, hospitals, veterinary medicine, agriculture and many other areas of life. The World Health Organization (WHO) estimates that microbial resistance to antibiotics contributes to 1.27 million deaths worldwide each year, including 4.95 million deaths as an indirect cause. In 2019, Poland recorded 5,600 deaths directly related to antibiotic resistance and 24,100 deaths indirectly related to this phenomenon in the same year. The number of deaths due to antibiotic resistance in the world may increase to up to 10 million annually by 2050. Between 2000 and 2020, global antibiotic use increased by 65% and continues to rise. The latest WHO report only mentions 27 new antibiotics that are currently at the clinical trial stage. Of this number, only a few seem to be innovative enough to deal with the most resistant bacteria. For comparison, at the same time, over a thousand oncology drugs are in this phase of research.
In the years 2017-2021, only one new antibiotic was registered, capable of defeating superbugs from the WHO list. Specialists in this field have identified the most dangerous bacteria called ESKAPE bacteria. The name is an acronym defining a group of drug-resistant bacteria, which includes: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. These are pathogens capable of escaping the action of available antibiotics, representing a new paradigm in the field of pathogenesis, transmission and resistance.
When considering the problem of antibiotic resistance in order to design new effective methods to combat this problem, we should primarily focus on the outer bacterial membrane (OM) of Gram-negative bacteria. This membrane is a serious complication because it is an extremely problematic barrier that prevents most available antibiotics from penetrating into the bacteria. For this reason, all types of nanomaterials and antibacterial proteins are extremely attractive substances. Therefore, newly designed agents should focus primarily on the bacterial outer membrane (OM), i.e. agents that permeabilize the membrane and change its fluidity. Another extremely important aspect when designing new means is the lipid composition of individual bacterial membranes of various groups of bacteria. The composition of membranes affects the surface (membrane) charge and therefore may contribute to differences in the mechanism of action of the materials used.
The aim of the research of this doctoral thesis was a biophysical analysis of the mechanism of permeabilization of the outer bacterial membrane with the participation of dendritic nanoparticles, taking into account the lipid composition of the outer membrane of the bacterial models used. Knowledge of the mechanism associated with bacterial membrane permeabilization may contribute to the development of new alternative methods effective in the fight against resistant strains in the future.
At the beginning, two types of dendronized carbosilane silver nanoparticles were analyzed: dendronized silver nanoparticles (Dend-AgNPs) and pegylated dendronized silver nanoparticles (PEG-Dend-AgNPs), as systems alone and in the presence of lysozyme. First, the antibacterial activity of both nanoparticles and their systems with lysozyme was checked. The activity of dendronized silver nanoparticles and lysozyme was measured on an exponentially growing culture of P. aeruginosa. The obtained results show a similar antibacterial effect when both types of nanoparticles are used. When lysozyme is included in the system, the antibacterial effect becomes much better.
In order to investigate the mechanism of action of nanoparticles, an experiment was carried out examining the process of permeabilization of the bacterial outer membrane (OM). The obtained data present two different mechanisms of action. In the case of Dend-AgNPs, the permeabilization effect is much stronger, these nanoparticles damage the OM, allowing lysozyme to penetrate inside the bacteria. PEG-Dend-AgNPs, on the other hand, aggregate on the surface of bacteria, the permeabilization effect is weaker, nanoparticles cause aggregation of bacteria, inhibiting their growth. Additionally, the killing effect is supported by the generation of reactive oxygen species (Skrzyniarz et al, International Journal of Biological Macromolecules 2023).
Then, the type of interactions of the tested dendritic nanoparticles with the bacterial membrane was checked on a liposomal model (liposomal model of the outer bacterial membrane of the bacterial strain P. aeruginosa). In the case of Dend-AgNPs, the results showed a static type of interaction between the membrane and the nanoparticle, forming a stable complex. An exothermic type of reaction and spontaneity at low temperatures were demonstrated. Completely different results were obtained in the case of PEG-Dend-AgNPs. The interaction between the nanoparticle and the membrane is dynamic, creating a less stable complex. An endothermic and spontaneous reaction was demonstrated only at high temperatures.
Bacterial morphology and bacterial peptidoglycan degradation of strains treated with nanoparticles were checked by transmission electron microscopy (TEM) and fluorescence microscopy using the fluorescent marker HADA (FDAA 7-hydroxycoumarincarbonylamino- D-alanine). Photos taken using a TEM microscope showed black points, i.e. dendritic silver nanoparticles, accumulating on the bacterial surface. In the case of Dend-AgNPs, a disruption of the bacterial cell membrane and the release of internal substances is visible. When it comes to PEG-Dend-AgNPs, you can see bacterial cells aggregating. Fluorescence microscopy photos show that Dend-AgNPs damage bacterial peptidoglycan, and when using lysozyme, the effect is even stronger and partial degradation of peptidoglycan occurs. However, in photos with PEGDend- AgNPs this effect is much weaker.
The second work included in the doctoral dissertation was research on the permebilization of the bacterial membrane by two types of cationic carbosilane imidazole dendrimers: D1 (BDTRP 001) C98H200N16Si138+ and D2 (BDSTM 001) C160H268C18N24Si13. The influence of the lipid composition of bacterial membranes on the level of membrane destabilization was also investigated. For this purpose, liposomal models of three types of bacteria were made: P.aeruginosa, E.coli and A.baumanii (Skrzyniarz et al, Journal of Colloid and Interface Science, 2024).
First, the physicochemical properties of the prepared liposomes and dendrimers were examined in order to examine their size and electrokinetic potential. Dynamic light scattering (DLS) and Zeta potential experiments were performed.
In order to investigate the properties of various liposomal models (and thus investigate differences in the reaction mechanism depending on the liposomal composition) depending on the dendrimer used, an experiment was performed to investigate changes in membrane fluidity. For this purpose, two membrane markers were used: TMA-DPH (integrated into the hydrophilic layer) and DPH (incorporated into the hydrophilic layer). The experiment was performed to check which layer of the outer membrane and how the dendrimers used in the experiment affect it. The results obtained are different for both dendrimers used. Only in the case of D1 were changes in the fluidity of the outer lipid layer observed.
The next step was to carry out fluorescence quenching tests using the Cyanine 5 (Cy5) marker, which was used to label one of the lipids used to build liposomes (DOPE). Thermodynamic analysis showed that a high concentration of lipids, i.e. CL (cardiolipin) and DOPE (1,2- dioleoyl-sn-glycero-3-PE) in the membrane content of a given bacterium guarantees greater affinity of antibacterial dendrimers and causes greater permeabilization of the bacterial membrane and an increase in its permeability.
TEM microscopy was used to examine the effects of dendrimers on each liposomal model. The obtained photos presenting models treated with dendrimer D1 show damaged or disintegrating liposomes and their fragments, while in the case of D2, spherical, undamaged shapes of liposomes can be observed.
The last stage of the work was in vitro testing. The aim of this experiment was to check whether membrane destabilization can cause endolysin to penetrate the outer bacterial membrane, degrade the peptidoglycan and consequently increase the killing effect. An exponentially growing culture of P. aeruginosa bacteria and endolysin were used for this analysis. In relation to the first dendrimer (D1), an antibacterial effect occurs. The outer membrane is damaged. The addition of endolysin does not increase the killing effect. The second dendrimer (D2) itself does not destroy the outer bacterial membrane, but significantly affects its fluidity. In this way, it allows endolysin to penetrate the interior of the bacteria.
The aim of the presented work was a biophysical analysis of the mechanism of permeabilization of the outer bacterial membrane using dendritic nanoparticles. The research confirmed the significant role of dendronized nanoparticles and antibacterial proteins as future agents in the fight against antibiotic-resistant bacterial strains. These substances have many positive features, such as the great ability to modify the surface of dendrimers, the possibility of permeabilization and the impact on the fluidity of biological membranes, as well as many others. However, when designing new agents, a very important aspect should be taken into account, which is the lipid composition of the outer bacterial membrane of individual bacterial strains. In the above dissertation, it was proven that the composition of membranes influences the type of interaction of various dendritic nanoparticles with the outer bacterial membrane. Understanding the mechanism accompanying the integration between the bacterial membrane and the nanomaterials is crucial in the development of new therapeutic agents, which in the future may prove crucial in the fight against the constantly growing problem of drug resistance.

Additional notes:

Streszcz. ang.  ; Zawiera ilustracje Zawiera bibliografię

Identifier:

oai:bibliotekacyfrowa.ujk.edu.pl:13350

Computer catalogue:

click here to follow the link

Language:

pol

Degree name:

doktor

Date obtained:

4.06.2025

Degree grantor:

Uniwersytet Jana Kochanowskiego w Kielcach

Supervisor:

Ciepluch, Karol

Reviewer:

Bukowska, Bożena  ; Jańczewski, Dominik Gruszecki, Wiesław I.

Field:

Dziedzina nauk ścisłych i przyrodniczych

Scientific disciplines:

Nauki biologiczne

Faculty:

Wydział Nauk Ścisłych i Przyrodniczych

Type:

rozprawa doktorska

Access rights:

tylko w Oddziale Informacji Naukowej

Publication status:

niepublikowana