Summary:
Pathogenic microorganisms’ infections constitute an ongoing and significant challenge
for various sectors related to, for example, water purification systems, food packaging,
synthetic textiles, constructions, healthcare and medical care, household sanitation, etc., as
bacterial infections worldwide are known to kill more people than any other cause. Up to now,
the use of antibiotics is the main weapon in the fight against infectious diseases but, due to
their misuse and overuse, antibiotic resistance becomes as one of the leading public health
threats of the 21st century. On the other hand, various disinfectants such as hypochlorite,
triclosan, hydrogen peroxide, silver salts, etc., are used in daily life to fight against microbial
contamination, but due to their short shelf life and various health-related safety issues, their
usage is restrained. Thus, nowadays, especially after the Covid-19 pandemic, there is an
urgent need to discover new antimicrobial agents to fight microbial infections.
In this study, new antibacterial agents were developed based on halloysite nanotubes
(HNTs). Halloysite nanotubes are a type of alumino-silicate clay (Al2(OH)4Si2O5*2H2O), having
the same chemistry as kaolinite but instead of the flat sheets typical of kaolinite, the sheets
have been rolled up under favourable geological conditions to form ultra thin hollow cylinders.
Chemically, HNTs are consisting of siloxane groups on the external surface and aluminol
groups in the inner lumen. The specific morphology as well as the chemical structure makes
HNTs ideal candidates for physical or chemical modification with a variety of functional groups,
either at their inner cavity or their outer surface, affording novel materials to be used in several
fields, including biomedicine, environmental science and catalysis.
This work is focused on the development of functionalized halloysite nanotubes with
polyethylene glycol (PEG) groups having various antibacterial groups at their distal end. The
aim of this work was not only to enhance the antibacterial properties of HNTs, but also to
reduce their cytotoxicity as well as to increase their hydrophilicity, and thus their dispersibility
in various aqueous media. In this concept, the commercially available halloysite nanotubes
were initially purified and shortened, using treatment with ultrasound followed by sequential
centrifugations, as it is known that the shortened nanotubes can be considered more suitable
for biological applications. The shortened HNTs were activated in alkaline environment and
then selected functional groups (amino or carboxylate) were introduced at their external
surface. Subsequently, these functionalized HNTs were modified with three different
derivatives of poly(ethylene glycol) (PEG) in order to reduce the HNTs cytotoxicity and also to
improve their dispersibility in aqueous media. For this purpose, suitable antibacterial groups,
i.e. quaternary ammonium, sulfobetaine, and triphenylphosphonium groups, were introduced
to the distal end of PEG, having a molecular weight of 3000Da, which subsequently interacted
with the amino- or carboxyl- functionalized ΗΝΤs, affording the hybrids HNTs-PEGQ, HNTs
PEGS και HNTs-PEGTPP, respectively, at two different functionalization degrees.
The obtained hybrid materials were structurally characterized using FTIR and XRD,
revealing the successful PEGylation of HNTs, while their functionalization degrees were
calculated by TGA and by using the ninhydrin assay. The morphology of the PEGylated HNTs
were studied by SEM and TEM confirming that the tubular shape of HNTs remained intact after
the functionalization, while the presence of a uniform PEG shell on the sidewalls of HNTs was
proved by STEM imaging as evident by the corresponding energy-dispersive X-ray (EDX)
mapping images. Moreover, as concluded by visual observation over time and ζ-potential
measurements, these hybrid systems can be efficiently dispersed in aqueous media, affording
stable aqueous dispersions for at least 2 months. The enhanced dispersion properties are
attributed to the presence of PEG on the surface of HNTs that induces hydrophilicity and also to the charged antibacterial groups that provide the surface charge necessary to cause
electrostatic repulsion inhibiting HNTs agglomeration.
Finally, the antibacterial activity of the hybrid materials was assessed against the Gram
(-) bacterial strain Escherichia coli and the Gram (+) bacterial strain Staphylococcus aureus. It
was found that HNTs-PEGQ and HNTs-PEGS exhibited almost the same antibacterial activity
as that of the parent HNTs, while HNTs-PEGTPP showed enhanced antibacterial properties
against both tested organisms, especially against Gram (-) E. Coli bacteria, revealing that the
presence of triphenylphosphonium group endowed the hybrids with enhanced antibacterial
properties. SEM images revealed that probably due to the ionic character of the hybrids,
PEGylated HNTs strongly interact with charged components of the outer leaflet of the bacterial
cytoplasmic membrane and cell walls through multiple interactions, including electrostatic and
hydrogen bonding, leading to cell envelope damage, leakage of the bacterial cytoplasm to the
external medium and finally to cell lysis. It is of importance to note that, all hybrids exhibited
minimal cytotoxicity against selected mammalian cells in the concentration range that showed
antibacterial activities, indicating that these hybrid nanomaterials and especially HNTs
PEGTPP, have great potential to be used as safe and efficient antibacterial agents.
Keywords:
Halloysite, functionalized nanotubes, nanoparticles, polyethylene glycol, PEGylation, functionalized polymers, antibacterial groups, colloidal stability, antibacterial properties.
