Regular Article
Enhanced photodynamic inactivation for Gram-negative bacteria by branched polyethylenimine-containing nanoparticles under visible light irradiation

https://doi.org/10.1016/j.jcis.2020.09.106Get rights and content

Abstract

Antibiotic pollution has been a serious global public health concern in recent years, photodynamic inactivation is one of the most promising and innovative methods for antibacterial applications that avoids antibiotic abuse and minimizes risks of antibiotic resistance. However, limited by the weak interaction between the photosensitizers and Gram-negative bacteria, the effect of photodynamic inactivation cannot be fully exerted. Herein, photosensitizer chlorin e6-loaded polyethyleneimine-based micelle was constructed. The synergy of electrostatic and hydrophobic interactions between the nanoparticles and the bacterial surface promoted the anchoring of nanoparticles onto the bacteria, resulting in enhanced photoinactivation activities on Gram-negative bacteria. As expected, an eminent antibacterial effect was also observed on the Gram-positive bacteria Staphylococcus aureus. The cellular uptake results showed that photosensitizer was firmly anchored to the bacterial cell surface of Escherichia coli or Staphylococcus aureus by the introduction of branched polyethylenimine-containing nanoparticles. The light-triggered generation of reactive oxygen species, mainly singlet oxygen, from the membrane-bound nanoparticles caused irreversible damage to the bacterial outer membrane, achieving enhanced bactericidal efficiency than free photosensitizer. The study would provide an efficient and promising antimicrobial alternative to prevent overuse of antibiotics and have enormous potential for human healthcare and the environment remediation.

Introduction

As one of the greatest inventions of the last century, antibiotics have been widespreadly applied in all aspects of human society, including in livestock, aquaculture, agriculture and clinic medicine, and so on [1]. However, excessive consumption and pervasive misuse of antibiotics worldwide has led to antibiotic pollution and resistant bacterial strains, which drastically threaten the natural environment and human healthcare [2], [3]. Massive research efforts have been devoted to developing alternatives to traditional antibiotics to solve this predicament [4], [5], [6]. Many promising candidates, including inorganic nanocomposites, polymers, and natural products have been thoroughly explored [4]. In recent years, light has emerged as an intriguing trigger for sterilization functions [7]. Photodynamic inactivation (PDI), one of the most promising and innovative methods controlled by light for antibacterial applications avoiding antibiotic abuse and minimizing risks of developing antibiotic resistance, has aroused enormous enthusiasm in the research community as an alternative antibacterial method to conventional antibiotics [8], [9]. PDI is a photo-regulated efficient antibacterial modality. Upon irradiation with light at the appropriate wavelength, photosensitizer (PS) molecules, known as a non-toxic dye, generate reactive oxygen species (ROS) which could cause severe damage to bacterial DNA and the cytoplasmic membrane so as to inhibit bacterial growth [10], [11].

Compared with traditional antibacterial drugs, PDI does not have a fixed ligand-receptor model. According to the positioning of photosensitizers (PS) in bacteria, PDI causes irreparable damage at multiple bacterial targets. PDI, which is characterized by a highly effective bactericidal effect, eliminates bacteria completely before they gain antioxidative damage mechanisms, so there is so there is little risk of the occurrence of drug resistance [12], [13]. However, there are inherent structural differences between the cell walls of Gram-positive and Gram-negative bacteria. Therefore, the outcomes of PDI on these two groups are also disparate [14], [15]. PS can relatively easily cross the cell wall of Gram-positive bacteria and subsequently kill the bacteria under light radiation. Due to their robust outer membrane barrier, Gram-negative bacteria demonstrate significant resistance to the penetration of various PS, which greatly hampers the efficiency of PDI [16], [17]. Many efforts have been made to find a way out of this dilemma and to achieve a highly efficient photodynamic antimicrobial outcome. For example, lipopeptide antibiotic polymixin B and ethylenediaminetetraacetic acid (EDTA) have been used as outer membrane-disrupting agents to improve the sensitivity of bacteria to photosensitizers [18], [19]. However, these agents often cause potential mammalian cell and tissue reaction or bring new byproducts to environment. Recently, cell surface engineering in the PDI research field has provided another idea to tackle this challenge [20], [21]. Cell surface engineering, i.e. surface modification of living cells with natural or synthetic polymers, allows for new opportunities in biomedical engineering and science [22]. The commonly used approaches of cell surface engineering are covalent binding, hydrophobic interaction, and electrostatic interaction [23]. There is a wide range of applications for cell surface engineering, including targeted therapy, islet transplantation, blood transfusion, promotion of drug endocytosis, detection of the extracellular environment, and cell membrane imaging [24], [25], [26], [27], [28], [29]. Due to the superior advantages of cell surface engineering, it has been widely adopted as a versatile technique in antibacterial applications, supramolecular nano-assemblies, liposomes, polymer-based nano-agents, and many others [30], [31], [32], [33].

Amphiphilic polymers are a group of classic membrane anchors that can facilitate binding of engineered material to the cell surface by achieving effective surface modification of cells through hydrophobic interactions between their anchoring groups (e.g., alkyl chains and lipids) and the plasma membranes [34], [35], [36]. In this work, the amphiphilic polymer OC-PEI, which combines the hydrophobic and electrostatic interactions, was designed and synthesized. This polymer can self-assemble into micelles and load the PS Chlorin e6 (Ce6) in aqueous solution without any contaminating organic reagents, obtaining Ce6-loaded micelles (Ce6-OC-PEI). Ce6, characterized by low dark toxicity and a high yield of singlet oxygen species, can easily be extracted from algae, plants, and silkworm excrements and can be obtained in large quantities. Ce6-OC-PEI possess many superior qualities, such as broad-spectrum antibacterial activity, good bacterial affinity, low biotoxicity, and avoidance of drug resistance. The typical Gram-negative bacteria Escherichia coli (E. coli, ATCC No. 8739) was chosen as model to investigate the photoinactivation properties of Ce6-OC-PEI. Through hydrophobic and electrostatic interactions, Ce6-OC-PEI binds to Gram-negative bacteria to disturb the outer membrane permeability barrier. Dynamic growth curve results demonstrated that this agent achieved enhanced antibacterial performance than the group treated with free Ce6. Meanwhile, the excellent antibacterial effect was also obtained with the Gram-positive Staphylococcus aureus (S. aureus, ATCC No. 6538P), indicating that Ce6-OC-PEI can indiscriminately eliminate both Gram-positive and Gram-negative bacteria, namely, whether Gram-positive and Gram-negative bacteria are vulnerable to Ce6-OC-PEI upon laser irradiation. Finally, the dark cytotoxicity of the original OC-PEI and Ce6-OC-PEI were also investigated, indicating good biocompatibility in potential antibacterial applications.

Section snippets

Materials

N, N-dimethyl formamide (DMF) and triethylamine (TEA) were obtained from Guangzhou Chemical Reagent Factory (Guangzhou, China). Branched polyethyleneimine (PEI, 10 kD) was provided by Innochem Technology Co., Ltd (Beijing). Octanoyl chloride was purchased from Alfa Aesar. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), PS Chlorin e6 (Ce6) and dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma Aldrich Chemicals (St. Louis, MO, USA). 1-N-phenylnaphthylamine

Preparation and characterizations of the polymers

The self-assembly of the amphiphilic polymer (OC-PEI) and photosensitizer loading for PDI of E. coli by Ce6-OC-PEI upon light irradiation are illustrated in Scheme 1. The amphiphilic polymer OC-PEI was obtained from PEI and OC by an amidation reaction. Its chemical structure was verified by 1HNMR (SI, Fig. 1). The molecular weights and molecular weight distribution of OC-PEI polymers was analyzed by GPC. The Mw was found to be 14 kDa with a PDI of 1.24. The substitute degree of OC in OC-PEI was

Conclusions

In summary, in order to improve the previously reported weak interaction between the PS and Gram-negative bacteria [14], [16], [19], in this work, a PDI-based amphiphilic polymer was designed and synthesized through simple one-step chemical conjugation from commercially available reagents. Our results show that we achieved a highly efficient photodynamic antimicrobial outcome without the need for pretreatment with chemical or biological agents, such as CaC12, tris(hydroxymethyl)

CRediT authorship contribution statement

Qian Wang: Conceptualization, Methodology, Writing - original draft, Funding acquisition. Dandan Zhang: Investigation, Software. Jin Feng: Software, Data curation. Tingli Sun: Formal analysis. Cailing Li: Resources, Investigation. Xiaobao Xie: Project administration, Validation. Qingshan Shi: Project administration, Validation.

Declaration of Competing Interest

The authors declare no competing financial interest.

Acknowledgments

This work was financially supported by GDAS’ Project of Science and Technology Development (2020GDASYL-20200103030). GDAS' Project of Science and Technology Development (2017GDASCX-0102). Guangdong Science and Technology Program (2017B030314045).

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