Nanotechnology facilitated photothermal and photodynamic therapy for enhanced antibacterial treatment

Abstract

Pathogens have acquired drug resistance to the existing antibiotics, and this has burdened public health and the economy. Over the past decade, a substantial amount of research has been carried out in the field of nanotechnology to address this issue. Among these, phototherapy with nanomaterials including photothermal (PTT) and photodynamic therapy (PDT) is gaining tremendous interest due to their advantages in fighting with drug resistant bacteria. In this thesis, techniques to enhance the current PTT and PDT have been developed.

Firstly, we employed Copper selenide (Cu2-xSe) NPs to carry out PTT on two types of bacteria E.Coli and Methicillin-resistant staphylococcus aureus (MRSA). Cu2-xSe NPs are photothermoresponsive, when NIR laser 1064 nm is exposed to these NPs, loosely bound electrons excite and undergo the localised surface plasmon resonance (LSPR) and produce heat as an output on de-excitation. This heat can kill pathogens by heating up the cytoplasm and essential proteins in the bacterial cell. PTT is a type of therapy which brings out bactericidal effects when the surrounding temperature rises, and significant amount of heat is produced. We utilized NIR bio window II 1064 nm laser with a power of 0.5-2.75W to illuminate the pathogens in the presence of Cu2-xSe NPs in the cuvette setup. The laser irradiated with beam size of approximately 1 cm diameter which was obtained after expanding the beam. As a result, these NPs attain a temperature of 57°C (0.5 W power). Photothermal properties of Cu2-xSe NPs were characterised under 1064nm laser exposure. These NPs showed efficient photothermal properties and they were observed to be stable for 20 laser on/off cycles. To evaluate their photothermal performance on pathogens, various in vitro antibacterial assays were performed, including colony count method, live dead assay, and bacterial growth kinetics. Our results showed that Cu2-xSe NPs could efficiently kill the pathogens in a concentration-dependent manner. Moreover, the increased power of the laser leads to an increased temperature, which eradicates pathogens rapidly.

Secondly, we synergistically applied chlorin e6 loaded mesoporous silica nanoparticles (Ce6-MSNs) and cyanobacteria beads for improved PDT. PDT involves a non-toxic photosensitiser, light, and oxygen. When light of a specific wavelength is exposed to a photosensitiser, it undergoes excitation and reacts with oxygen to produce reactive oxygen species (ROS). These ROS are proven to be cytotoxic for pathogens by acting on multiple targets within the cell and hence damage the cell by disturbing its equilibrium. PDT involves use of cytotoxic radicals that are highly energetic and make bonds or knock out electrons when react with the biomacromolecules present within the cell. However, hypoxia in bacterial infections is a known concern in PDT. To rectify this problem, we have developed a synergistic platform comprising of two components. One is cyanobacteria microalgae hydrogel beads and the other Ce6-MSNs. Sodium alginate hydrogel beads were synthesized to immobilize living microalgae Synechococcus elongatus (PCC 7942). Ce6-MSNs were demonstrated to be efficient PDT agents and hydrogel cyanobacteria beads were the natural and economic source of oxygen because of photosynthesis. LED light possessing wavelength of 660 nm was used as a single source and in the form of a wide fan beam which covered the whole plate containing bacterial sample. 660 nm light exposed Ce6-MSNs and cyanobacteria beads to excite and photosynthesise, respectively. Algae beads aided Ce6-MSNs to produce boosted ROS when combined with oxygen produced in situ and hence enhance the efficacy of PDT. Ce6-MSNs and algae beads were characterised with UV/Vis Spectra, dynamic light scattering (DLS) zetasizer, transmission electron microscopy (TEM), scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), and fluorescence microscope. The PDT capacity of Ce6-MSNs and Ce6-MSNs + algae beads, was tested with 1,3-diphenylisobenzofuran (DPBF) assay and 2',7'-Dichlorofluorescin diacetate (DCFH-DA) assay. To demonstrate the antibacterial efficacy of this synergistic platform, several in vitro antibacterial assays were performed in normoxia and hypoxia atmosphere for planktonic and biofilm MRSA. These methods include colony count method, ROS detection, ROS quantification, live/dead imaging, and biofilm hypoxia alleviation. Our results illustrate that oxygen from algae beads amplifies the production of ROS. This synergistic platform can mitigate the hypoxia in PDT setup.

Thirdly, two types of hydrogel beads were synthesised. One was cyano@SA bead and the other was Ce6-MSNs@SA bead. Ce6-MSNs@SA beads were proved to be an efficient PDT agent, while the cyano@SA beads were able to produce oxygen to enhance the efficacy of PDT. To evaluate the controlled drug release from Ce6-MSNs@SA beads, a model drug methylene blue was incorporated into sodium alginate hydrogel beads and the drug release profile for methylene blue for up to 10 days was monitored. Photosensitiser Ce6 is repeatedly referred as ‘drug’ due to its ability to provide therapeutic effects, kill pathogens and relieving symptoms. To demonstrate the enhanced PDT, these two types of beads were used to carry out antibacterial PDT. Our results demonstrate that Ce6-MSNs@SA hydrogel beads are potent controlled drug release depot and successfully demonstrate controlled drug release for up to 4 cycles of PDT against MRSA. In conclusion, the PTT with Cu2-xSe NPs and the synergistic PDT techniques of using cyanobacteria and MSN-Ce6 were demonstrated to be an efficient PTT and PDT agent. Implementation of these techniques can strengthen existing antibiotic treatment by which they can efficiently ablate drug-resistant pathogens.

Moreover, the oxygenated platform composed of living cyanobacteria could provide an economic and long-term supply of oxygen to replenish the oxygen in existing PDT, hence boosting the PDT efficacy by alleviating hypoxia in bacterial infections. The synergistic platform can be used in the future to heal the wound by providing a copious amount of oxygen and simultaneously curbing the bacterial growth in the wound. Additionally, PTT can be a promising alternative to existing antibiotics and provide a solution to eliminate drug-resistant bacteria inhabit the wound. Being light dependent techniques, it will be a great avenue to consider the specific and targeted area of laser illumination so that damage occurs to the pathogenic bacteria while sparing surrounding healthy cells.