Thermo-responsive triple-function nanotransporter for efficient chemo-

photothermal therapy of multidrug-resistant bacterial infection

Multidrug-resistant organisms are bacteria and other microorganisms that have developed resistance to
antimicrobial drugs. It develops when antibiotics are taken often longer than necessary or when they
are not needed. So scientists come with new strategies with high antimicrobial efficacy against
multidrug-resistant bacteria. In this article, the writers describe a smart triple-functional nanostructure,
namely TRIDENT (Thermo-Responsive-Inspired Drug-Delivery Nano-Transporter) for dependable
bacterial extermination. Fluorescence monitoring and synergistic chemo-photothermal killing are more
effective and vigorous. From this paper, they can also learn that NIR (near-infrared radiation) generates
temperature rise which did not only melt the nanotransporter but also irreversibly damaged bacterial
membranes which can lead to rapid bacterial death. Low doses of imipenem-encapsulated TRIDENT
could exterminate clinical methicillin-resistant Staphylococcus aureus both in vitro and in vivo but in
that term imipenem alone had limited effect.

Introduction:
Sepsis is a potentially life-threatening condition that occurs when the body's response to an infection
damages its tissues. When the infection-fighting processes turn on the body, they cause organs to
function poorly and abnormally. To preventing local infection using various antibiotics which lowering
the risk of retrogression but prophylactic antibiotic medication suddenly outbreak of multidrug-
resistant (MDR) or extremely drug-resistant(XDR)pathogenic bacteria. Now to prevent MDR or XDR
nanotechnology-based antibacterial strategies are being used. Near-infrared (NIR) light is an exogenous
stimulus that could be used to deliver sustained drug release. Photothermal therapy(PTT) refers to
efforts to use electromagnetic radiation for the treatment of various medical conditions. PTT activated
by NIR can penetrate tissues deeply that can cause a little damage to the surrounding area and also fight
against pathogenic bacteria by disrupting cell membrane, denaturing proteins, and bacterial death. In
this process, they use nanoparticles like iron oxide, graphene, black phosphorus, and other polymer
nanoparticles which are capable of delivering antibiotics to the infected sites for chem-photothermal
therapy under NIR irradiation. IR-780 iodide (IR780) is a NIR fluorescence dye with high and stable
fluorescence intensity and has been utilized in photothermal therapy. There are many stimulus
nanostructures, from them thermoresponsive nanostructure (TRN) can work with a large latent heat of
fusion and reversible solid-liquid transition over a narrow temperature range which is more convenient
in the term of controlled drug release. In particular, TRN has the following features:
1.High biocompatibility and no leakage of encapsulated antibiotics.
2.Convenient incorporation of diverse functional molecules into the nanotransporter
3.Prompt release of encapsulated antibiotics upon exposure to NIR
4.Synergistic antibacterial activity involving PTT and antibiotic killing.
In this experiment, they developed a smart biocompatible TRIDENT for synergistic elimination of
MDR bacteria through combined chemo-photothermal therapy. For this purpose, a TRN formulated
from natural fatty acids with a tunable melting point around 43 °C is selected as the hydrophobic
vehicle for encapsulation of both imipenem (IMP, a broad-spectrum antibiotic) and IR780 (a
photosensitizermolecule).
Results:
Synthesis and characterization of TRIDENT: For synthesis and characterization they utilized lauric acid
and stearic acid to prepare the TRIDENT for biocompatibility and biodegradability. Here they
characterized spherical morphology of the TRIDENT with a homogeneous size that could be observed
in transmission electron microscopy (TEM) image, UV-vis absorbance spectra confirmed the
successful encapsulation of both IMP and IR780 in the TRN and encapsulation efficiency (EE) and
loading efficiency (LE). Afterward, they tasted the size and morphology of the TRIDENT in water,
phosphate-buffered solution (PBS), Luria-Bertani broth (LB) medium.

Figure 1: Preparation and characterization of TRIDENT

Thermo-responsive properties of the TRIDENT system: A series of experiments were carried out to
analyze thermo-responsive properties such as differential scanning calorimetry (DSC) where TRN
prepared with weight ratio LA/SA of 3:5:1 had the suitable melting point (43°c) thermo-responsive
therapy in a physiological setting. And in the temperature increment of TRIDENT suspension, it was
concluded that a 12°C increase in temperature is suitable to create serious damage to the bacterial cell
when the temperature starts at 37°C in vitro and in vivo. Besides, through the investigation of the
morphology of the TRIDENT before and after irradiation, it was observed that laser irradiation led to a
significant change in particle size but not the morphology of TRIDENT.
In vitro antibacterial activities: To test the toxicity on cells the in vitro cytocompatibility of TRIDENT
was appraised from normal mammalian tissues including mouse embryo cells (3T3 cells) and human
umbilical vein endothelial cells (HUVECs). From this, they suggested that TRIDENT had no cytotoxic
effect on mammalian cells because the viability of both 3T3 cells and HUVECs was above 80% even at
a TRIDENT concentration up to 80 μg mL−1. Again to test the synergistic antibacterial behaviors of
the TRIDENT against antibiotic-sensitive Escherichia coli (E.coli) and Staphylococcus aureus
(S.aureus) under NIR irradiation. After that investigation, standard plate counting showed that NIR
irradiated TRIDENT was highly efficacious against both species. To clarify the connection between
bacterial viability and temperature they also investigated the thermal response of these bacteria to
various temperatures. Also, log10 (CFU mL−1) (CFU: colony-forming unit) was used to calculate the
viability of bacteria treated with different conditions. Around 2log10CFU reduction could be monitored
when the bacteria were subjected to 49 °C for 5 min. These in vitro antibacterial activities could also be
studied using Live/Dead dual-color fluorescent staining. These experiments was conducted by
replacing the standard antibiotic sensitive strains with clinically isolated multidrug-resistant E. coli
(MDREC) and methicillin-resistant Staphylococcus aureus (MRSA). Synergistic antibacterial
behaviors of TRIDENT to treat clinical MDR or XDR bacteria which can’t be dealt with using
conventional antibiotics.

Figure 3: In vitro antibacterial activities of TRIDENT against clinical multidrug-resistant (MDR)

bacteria (MDREC and MRSA).

In vivo eradication of skin infections: A mouse model with MDREC and MRSA skin infection was
considered to investigate the antibacterial activity in vivo. The in vivo enrichment of the nanomaterials
at the infection site was monitored in real-time, providing visual guidance for the selection of the
optimal treatment time which demonstrated the NIR-irradiated TRIDENT has the promising
bactericidal potential against MDREC and MRSA infection. After that to examine H&E staining the
recovery of the infected skin tissues. Severely disrupted dermis structures and infected skin lesions
were observed in the samples from groups treated with PBS, IMP@TRN, IR780@TRN,
IMP/IR780@TRN, T-IMP, and NIR alone (Fig. 6k and Supplementary Figure 12d). In contrast, the
skin tissues obtained from TRIDENT + NIR showed relatively intact histological characteristics,
followed by the groups treated with IR780@TRN + NIR and 3× IMP. Finally, these result shows that
the superior therapeutic efficacy of NIR-irradiated TRIDENT. Similar healing processes and
therapeutic results were also observed when the infection models were induced with antibiotic-sensitive
E. coli and S. aureus (Supplementary Figs. 13–15). Thus, the proposed strategy had in vivo
antibacterial activities against both antibiotic-sensitive bacteria and clinically relevant MDR bacteria at
a low antibiotic dosage. Furthermore, to better validate the capability of the nanotransporter to curb
localized infection,
the change of two major sepsis-associated biochemical markers (procalcitonin (PCT), c-reactive protein
(CRP)) in the blood of the MRSA-infected mice after treatment with PBS, NIR-irradiated
IMP/IR780@TRN, and 3× IMP was monitored which showed that the occurrence of severe systemic
infection like sepsis from local bacterial infection can be prevented by the TRIDENT system.

Figure 4: Antibacterial activities of TRIDENT in vivo and The sterilization mechanism of TRIDENT.

Biosafety of TRIDENT: For evaluating potential acute biohazards associated with TRIDENT they
performed blood biochemical analysis and histological analysis of mouse sample. The mice samples
are treated with free IMP had increased levels of blood urea nitrogen and creatinine and hyaline casts.
Overall, these results clearly demonstrated that TRIDENT possesses superior biosafety and can greatly
reduce the toxicity of IMP to the kidney.
Methods:

1. Materials: Stearic acid (95%) and lauric acid (97%), Imipenem (IMP,98%), IR780 iodide (IR780,
98%) soybean lecithin (Mw-750), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy
(polyethylene glycol)-2000] (Mw = 2000, DSPE-PEG2000).

2. Preparation of the IMP/IR780 thermo-responsive nanostructure (TRIDENT): For the preparation of

TRN solution need lauric acid and stearic acid at a mass ratio of 3.5:1, and the final concentration was
4 mg mL-1 , then added drop by drop of TRN stock solution containing IMP and IR780 and also added
4% aqueous solution of phospholipids formed by lecithin and DSPE-PEG2000 at 55 °C. The
solification process of the nanoparticles done with ice water.

3. Characterization of TRIDENT: The phase change temperatures of TRN, IMP@TRN, IR780@TRN

and IMP/IR780@TRN were measured by differential scanning calorimeter. The size and morphology
of IMP@TRN, IR780@TRN and IMP/IR780@TRN were characterized by dynamic light scattering
and transmission electron microscopy (HITACHI, Ht-7700), respectively.

4. Photothermal properties of TRIDENT: IMP/IR780@TRN was dispersed in PBS at five

concentrations. After that the prepared solution was added to cuvette and irradiated. For accurate
morphology of TRIDENT the irradiation needed to be stopped and the TRIDENT solution was
immediately immerged in liquid nitrogen to be rapidly solidified.

5. Light-triggered IMP release of TRIDENT: For this 1mL TRIDENT solution were separately added to
four dialysis tube and shaken. NIR added to these four tubes and shaken and vibrated and the
absorbance was measured by high performance liquid chromatography.

6. Cytotoxicity of TRIDENT: The cytotoxicity of IMP/IR780@TRN was investigated using a cell

counting kit-8 (CCK-8) assay. 3T3 cells and HUVECs were seeded in 96-well plates and starting
incubation.

7. Bacterial culture: E. coli, S. aureus and clinically isolated MDREC or MRSA were cultured in
Luria–Bertani (LB) broth medium in a shaking incubator at 37°C and harvested at the logarithmic
growth. The concentration bacteria is monitored by measuring the optical density.

8. In vitro antibacterial experiments: In this method, bacteria were diluted and treated with different
formulations. Then the solution of bacterial suspension was spread on LB plates and CFU was counted.

9. Morphology of bacteria after treatment: In this process different treatments were added to the
bacterial suspension and the suspension treated with IR780@TRN + NIR, IMP/IR780@TRN + NIR
and NIR alone were irradiated with an 808 nm NIR laser at a power density. The bacterial suspension
were then centrifuged and then washed three times with PBS.
10. Live/dead staining assay: The viability of bacteria in the test samples was qualitatively assessed
using a live/dead Baclight bacterial viability kit and all experiments were carried out according to the
manufacturer’s instructions. In this bacteria are treated with different seven formulations and after
washing with NaCl, the bacteria was stained with green-fluorescent nucleic acid stain and after red-
fluorescent nucleic acid stain.

11. Mouse infection model: For this method they used 6-8 week old female mice. The infection model
was established by subcutaneously inoculating clinically isolated MDREC or MRSA. The temperatures
of infected skin were recorded by an IR camera.

12. In vivo antibacterial experiments: In these experiment they were working with mice. Mice were
randomly divided into seven groups 24 h after bacterial inoculation and 100 μL PBS IMP@TRN,
IR780@TRN, IMP/IR780@TRN, IR780@TRN + NIR, IMP/IR780@TRN + NIR were
subcutaneously injected twice, on days 1 and 7, NIR laser was applied to group of IR780@TRN + NIR,
IMP/IR780@TRN + NIR after the two subcutaneous injections. After observation the healing process
of the infected areas through macroscopic photographs and damaged areas and body weights of the
infected mice were measured throughout the whole treatment period. To determine the number of
bacteria at the infection site, the infected tissues were homogenized in PBS (1 mL) and the suitably
diluted tissue solutions were plated on LB agar plates. And the bacterial colony were counted after the
incubation for 24 h at 37 °C.

13. Analysis of sepsis biomarkers: For estimation of the levels of biomarkers associated with sepsis,
experimental mice were divided into four groups 24h after inoculation. Before treatment, from one
group of the infected mice and healthy mice they collected blood sample for further analysis. After that
the three groups were separately treated with PBS, free IMP and IMP/IR780@TRN + NIR by two
subcutaneous injections on days 1 and 7. In this process healthy mice were used as negative controls.
At the end of the treatment period blood sample collected from infected mice and the levels of
procalcitonin and c-reactive protein were measured by enzyme-linked immunosorbent assay kits.

14. In vivo biosafety assessment: To verify the biosafety of IMP/IR780@TRN, mice were randomly
divided into three groups and treated with PBS, IMP/IR780@TRN + NIR and free IMP by two
subcutaneous injections on days 1 and 7. The blood serum samples were analyzed for two renal
function markers, blood urea nitrogen.

From this article, we can learn that

• To control drug resistance nanoparticle-based targeted delivery strategies have been developed.
• Thermo-responsive gating materials used for antibiotics to construct an NIR triggered
nanomaterial complex(TRIDENT).
• TRIDENT has three effective bacterial killing functions.
• NIR-irradiated TRIDENT killed MRSA.
• The TRIDENT also used for treating potential MDR bacterial infections.
• TRIDENT decreased the possibility of sepsis and promoted the rehabilitation of infected site.