ProtocolsComparative study of an antimicrobial peptide and a neuropeptide conjugated with gold nanorods for the targeted photothermal killing of bacteria
Graphical Abstract
Introduction
Antibiotic resistance and impaired wound healing are the two main challenges in treating wound and surgical site infections caused by multidrug-resistant (MDR) pathogens. The poor penetration rate and targeting ability of antibiotics leading to high mortality rates remain a public health problem worldwide [1]. Based on acquired antibacterial resistance recorded by the World Health Organization (WHO), Methicillin-resistant Staphylococcus aureus (MRSA- with 52.7% priority) and Escherichia coli (EC- with 76.5% priority) are the two most pathogenic Gram-negative and Gram-positive bacteria [2]. It is, therefore, urgently warranted to develop effective alternatives against these bacteria. Several researchers have focused on nanomaterials combined with naturally derived, short synthetic peptides as an alternative for conventional antibiotics [3], [4], [5].
Metal nanoparticles including Au, Ag, Cu, Bi, and Ni are at the forefront of fighting antibiotic-resistant bacteria. Metal nanoparticles have been used for the photolysis of bacterial membranes, controlled release of drugs under near-Infrarad (NIR) light trigger, and photothermal catalytic-antibacterial therapy [6], [7], [8]. Among metal nanoparticles, gold nanomaterials of varying shapes gained attention for their substantial light to heat conversion efficiency in the NIR region [9], [10], [11], [12], [13]. Gold nanorods (GNRs) are the most promising candidates among gold nanostructures, providing excellent photothermal conversion efficiency and penetration in biological windows I and II [14], [15]. Moreover, the surface modification of GNRs is relatively easy and can be covalently conjugated with antibodies and peptides through electrostatic interactions or metal thiol bonds [16]. For example, functionalized gold nanostructures, including DNA aptamer functionalized GNR [17], and NIR-driven Au-decorated polymer-metal protein microfibers [18], have been utilized for photothermal properties to kill MRSA in filters for sterilization.
Antimicrobial peptides (AMPs) are natural antibiotics that can be bacteriostatic or bactericidal and beneficial as targeting agents [19]. The surface charge and chemical composition of bacterial cells are different from mammalian cells. Therefore, the interaction of AMPs with human cells is weaker and shows minimal toxicity [20]. Gold nanocluster conjugated AMPs [21] and gold/silver nanohybrids stabilized with cationic dipeptides [22] were shown to have antibacterial and antifungal efficacy. Angiopeps are neuropeptides that penetrate the blood-brain barrier (BBB) hence useful for peptide-based drug delivery [23]. GNR-PEG-ANGIOPEP-2 has been extensively studied as a shuttle to transport GNR across the BBB and as a potent anticancer agent [24], [25], but no study to date has examined the ability of ANGIOPEP-2 to target bacteria.
Wound healing is a complex process that involves inflammation, proliferation, epithelialization, and remodeling at hemostasis. For a healthy wound healing process, fibroblasts arrive at the wound site within 24–48 h post-injury in response to inflammation. Prior research suggests that better wound healing is achieved by increased cell migration rate [26]. LL-37 can exert immunomodulatory functions, such as modulation of the pro-inflammatory response, cell differentiation, proliferation effects in angiogenesis, thus promoting wound healing [27]. A current review on topical antimicrobial peptide formulation development and prospects in wound healing reported that LL-37 is under clinical phase II/b by ProMore Pharma for leg ulcers [28].
The present study detailed the synthesis of two metal peptide conjugates with different functions. An antimicrobial peptide (LL-37) and a neuropeptide (ANGIOPEP-2) were conjugated with GNR and characterized. There are several advantages of the GNR-peptide nanoconjugates: (1) The synthesis process is simple and no significant toxicity, ensuring biocompatibility of the GNR-peptide conjugates. (2) Electrostatic interaction between peptides and bacteria provides targeting specificity. (3) GNRs endow strong NIR absorbance and high photothermal efficiency.
This is the first study to explore the ability of neuropeptide-conjugated GNRs to target bacteria and as an efficient photothermal antibacterial agent. Antibacterial efficiency, cell viability, and cell migration properties of the synthesized GNR-peptide conjugates were thoroughly characterized. We further analyzed the generation of ROS and bacterial cell integrity to uncover the possible mechanism of killing bacteria treated with GNR-peptide conjugates and NIR irradiation besides photothermal effect. The combination of several beneficial factors improved the healing rate after wound infection.
Section snippets
Materials
All chemicals were of analytical grade: hydrogen tetrachloroaurate (III) hydrate HAuCl4·3H2O, Alfa Aesar, 99.99%, (Ward Hill, MA, USA) hexadecyltrimethylammonium bromide (CTAB), Acros Organics, 99+% (Geel, Belgium), L-ascorbic acid (AA), (Mw 176.12,), 99%, Everdine technology (Chiayi, Taiwan), silver nitrate, (AgNO3), Alfa Aesar, (Mw 169.87), 99.9+%, (Ward Hill, MA, USA), sodium borohydride NaBH4, (Mw 37.83), 99%, Sigma-Aldrich, (Saint Louis, MO, USA), antimicrobial peptide LL-37 (LC-38,
Synthesis and functionalization of GNR and GNR-peptide conjugates
GNR-LC-38 and GNR-ANGI-2 were prepared as shown in Scheme 1. The seedless synthesis method was applied to prepare GNRs. The GNRs were modified with AMP or neuropeptides by ligand exchange via Au-S bonding. The peptides modified with cysteine ends in LC-38 and ANGI-2 were used to conjugate onto the surface of GNR-CTAB. As shown in Fig. S1, the conjugation efficiencies of LC-38 onto GNRs were 88.8, 83.7, 78.6, 89.7, 87.0, 60.0, 41.5, 20.3%, corresponding to 35.0, 23.0, 17.5, 8.75, 6.00 and
Conclusion
We successfully developed multifunctional gold nanorods combined with two different peptides, GNR-LC-38 and GNR-ANGI-2. We compared their efficiency in targeting and photothermal killing of bacteria and the possible enhancement of in-vitro cell migration in NIH-3T3 cells. In line with previous studies, the GNR-peptide conjugates provided the benefit of targeting bacteria with electrostatic interactions. Due to a redshift in the LSPR region, the GNR-LC-38 has shown enhanced photothermal
Supplementary information
The Supporting Information contains the quantitative analysis of LC-38 peptide in GNR-conjugates, TEM characterization of GNR-CTAB, XPS of GNR-CTAB, GNR-LC-38, elemental mapping of GNR-peptide conjugates, CD data of GNR-peptide conjugates, the temperature-responsive profile of GNR-CTAB, minimum inhibitory concentration of LC-38 against MRSA, cell viability assay of HEL-299 cells. Declaration of competing interests and acknowledgments is also included in the supplementary information.
CRediT authorship contribution statement
Sivasoorian Siva Sankari: Conceptualization, Writing – original draft, Software, Validation, Investigation, Formal analysis. Hans-Uwe Dahms: Writing – review & editing. Ming-Fong Tsai: Methodology, Investigation, Validation, Visualization. Yu-Lun Lo: Methodology, Investigation, Visualization. Li-Fang Wang: Conceptualization, Methodology, Writing – review & editing, Resources, Project administration.
Declaration of Competing Interest
The authors declare that they have no conflict-of-interest for this study.
Acknowledgments
The author would like to acknowledge Dr. Ruei-Nian Li (Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Taiwan) for the kind supply of E. coli bacteria. The authors further acknowledge the financial support received from the Ministry of Science and Technology of Taiwan (grant nos. MOST109-2320-B-037-017-MY3 and MOST108-2221-B-037-002-MY3). The authors are grateful for a grant by a Kaohsiung Medical University with grant nos. KMU-DK109001. We appreciate
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