Versatile antimicrobial peptide-based ZnO quantum dots for in vivo bacteria diagnosis and treatment with high specificity

Abstract

Abstract

The increased mortality caused by pathogenic bacterial infection calls for early infection diagnosis and effective antibiotic alternatives. In this work, we developed a fluorescent nano-probe named ZnO@PEP-MPA by conjugating BSA-stabilized ZnO quantum dot (ZnO@BSA) with UBI_29-41, an anti-bacteria peptide fragment, and MPA, a near infrared (NIR) dye. The nanoprobe ZnO@PEP-MPA exhibited low cytotoxicity and could discriminate the bacterial infection from sterile inflammation or cancer in vivo with high specificity and low detection limitation. Based on the platform of ZnO@PEP-MPA, a theranostic nanocomposite, Van@ZnO-PEP-MPA was firstly established by further decorating ZnO@PEP-MPA with Vancomycin, a kind of glycopeptide antibiotic, which demonstrated enhanced antibacterial activity and desirable biocompatibility both in vitro and in vivo. Furthermore, another antibiotic methicillin was immobilized onto ZnO@PEP-MPA, forming Met@ZnO-PEP-MPA and demonstrated significant improved capability to combat with the anti-methicillin-resistant-bacteria in comparison with free methicillin as a result of the increased cell membrane permeation mediated by ZnO@BSA-PEP-MPA. Therefore, ZnO-PEP-MPA reported in this work holds promising potential to realize efficient non-invasive diagnosis of bacterial infections, providing important guiding information for treatment, and can be employed as drug carriers for effective bacterial-targeting therapy, favorable to hurdle multi-drug resistance after being loaded with antibiotics.

Introduction

Bacterial infection as an arising medical and public concern has led to significant mortality and morbidity worldwide [1]. One of the challenges that hampers timely and effective treatment of bacterial infection is the difficulty to detect the bacterial infection at early stage in a sensitive, specific and non-invasive way and clearly discriminate bacterial infection against sterile inflammation, cancer or other pathological changes [2]. Meanwhile, another tough problem faced by current bacterial-infection therapy is the emergence of drug resistant microbes such as methicillin-resistant staphylococcus aureus (MRSA) and Vancomycin (Van) resistant enterococci enzyme [3], [4] which is initially stemmed from antibiotics abuse and may lead to the failure of antibiotic therapy. Each year in the United States, at least 2 million people become infected with bacteria that are resistant to antibiotics and at least 23,000 people die each year as a direct result of these infections. Many more people die from other conditions that were complicated by an antibiotic-resistant infection [5]. Currently, only indirect imaging modalities are clinically available for the diagnosis of bacterial infection, as exemplified by urine or blood test, which visualizes the bacteria by optical microscopy. Radiolabelled antibiotics, peptides, leukocytes though were intensely investigated to directly localize bacterial-infected site, their further movement to clinic was stymied by inherent limitations in specificity and resolution (∼ 0.5 cm) of the radiologic imaging modality and radioactivity itself [1], [6], [7], [8], [9]. By contrast, optical imaging can non-invasively monitor the lesion sites in real-time at high resolution without the concern of radiation-related risks [10]. Therefore, it would be desirable to develop an easy-touse clinical optical imaging tools which is able to realize direct and early diagnosis of bacterial infection [11].

Near infrared (NIR) fluorescent imaging (700–900 nm) as one of the novel optical imaging technique possesses advantages such as non-invasive, low interference by intrinsic chromophores, deep penetration and is receiving more and more researchers' interest for disease diagnosis and therapy monitoring [12], [13], [14]. The application of NIR fluorescence imaging has been widely reported for cancer diagnosis and tumor surgery guidance in recent decades [12], [15], [16], [17], but the story to employ NIR-fluorescent molecular imaging probe for in vivo bacteria-infection detection just began and much work need to be explored [18], [19].

Molecular imaging probes are basically composed by high-affinity ligands and imaging reporter groups. Among various reporter groups, quantum dots (QDs), a sort of fluorescent nanomaterial, have been approved to own favorable merits such as broad excitation spectra, narrow emission spectra, tunable emission peaks and negligible photobleaching. Typically, Cd-free ZnO QDs are expected to have promising potential in biomedical field since ZnO was granted as safe by U.S. Food and Drug Administration (21CFR182.8991) for various biological applications including drug cargos, disease treatment and bacteria labeling [20]. In present study, ZnO QDs was adopted as the imaging reporter for in vitro investigation due to its suitable emission wavelength (550 nm), and hydrophilic indocyanine green (ICG) derivative (MPA), one kind of organic NIR-fluorescent dyes (λ_ex = 763 nm; λ_em = 820 nm) synthesized by our laboratory, was attached o ZnO QDs for in vivo bacteria imaging.

On the other hand, affinity ligands including antibodies [21], sugars [22], bacteria binding peptides [23], antimicrobial peptides [24], enzyme substrates [25], and antibiotic drugs [1] have been reported in terms of bacterial targeting. Particularly, antimicrobial peptides, produced by phagocytes, epithelial cells, endothelial cells, and many other cell types are of importance in innate immunity against infection by a variety of pathogens such as bacteria and fungi [26], [27]. UBI_29-41 (TGRAKRRMQYNRR, 1693Da), which is a cationic human antimicrobial peptide originally isolated from mouse macrophage cells, has displayed high accumulation in bacterial infection with encouraging specificity to targeted bacterial cells but not sterile inflammatory processes [28], [29].

In our research, UBI_29-41 and MPA were covalently onto BSA-stabilized ZnO QDs for bacteria-selective optical imaging, forming ZnO@PEP-MPA. The neglectable cytotoxicity of this fluorescent probe as well as its function to selectively detect and distinguish invasive infection from general inflammation and cancer were confirmed. Since the concept of “theranostics” suggesting a combination of diagnostics and therapy was proposed in 2002, nanomedicine platforms that integrate imaging and therapeutic function have procured considerable attention as the next generation of medicine [30]. Herein, we further covalently attached Van, a widely used anti-bacteria drug, to ZnO@PEP-MPA, and the anti-bacterial activity of formed conjugate (Van@ZnO-PEP-MPA) was examined in vivo and proved to be better than free vancomycin. In a similar way, ZnO@PEP-MPA was loaded with another antibiotic drug methicillin (Met) to form Met@ZnO-PEP-MPA, which tends to easily induce relevant drug resistance among bacteria. Gratifyingly, under the help of ZnO@PEP-MPA, Met@ZnO-PEP-MPA manifested enhanced inhibition effect against methicillin-resistant bacteria (MRSA).