Antibiotic resistance, primarily caused by the overuse and misuse of antibiotics, continues to pose a significant threat to the global public health, despite warnings about the problem from multiple agencies, including the United States Centers for Disease Control (CDC) and World Health Organization (WHO) [1], [2]. Several novel alternatives to conventional antibiotics have emerged in recent years, such as endolysins, bacteriophage-derived enzymes, antimicrobial peptides, and bacteriocins [3], [4], [5], [6]. In addition to these therapeutics, plant extracts including polyphenols such as catechins, have also been studied as potential antimicrobial treatments that have fewer side effects than traditional antibiotics [7], [8], [9], [10]. Although many of these molecules are promising, traditional antibiotics are still heavily relied upon in treating infections due to their wide availability, inexpensive cost, and proven safety. However, in recent years, not only has the discovery of new classes of antibiotics declined drastically, but the time interval between the introduction of new drugs and the emergence of resistance to them by pathogens has also significantly shortened [11], [12]. The very use of antibiotics causes a selective pressure that leads to resistance [2], [13], [14]; as long as antibiotics are used in clinical settings, the emergence of drug-resistant bacteria is an inevitable problem. Gram-negative bacteria are particularly problematic due to their complex cell wall, which consists of two lipid membranes that effectively prevent internalization of most antibiotics [15]. As a result, no new classes of antibiotics that are effective against Gram-negative bacteria have been discovered since the quinolone family was introduced in the 1970 s [12]. It is therefore imperative that we improve the administration of currently efficacious antibiotic agents to slow the development of antimicrobial resistance [16], while alternative treatments remain under development. The use of targeted delivery and controlled release systems is a promising strategy for antimicrobial treatments amid the antibiotic crisis, as the use of drug carriers for targeted delivery reduces the rate of acquired antibiotic resistance due to increased local concentration at the site of infection and decreased exposure in non-infected sites. Furthermore, reduced drug side effects and increased drug uptake can be achieved through the use of suitable antibiotic delivery systems [16], [17], [18], [19], [20].

Aggregatibacter actinomycetemcomitans is an oral Gram-negative bacterium that has been closely associated with inflammatory periodontal disease, particularly aggressive forms of periodontitis [21], [22]. A. actinomycetemcomitans possesses an arsenal of virulence factors that facilitate its pathogenesis and infection, one of which is a leukotoxin (LtxA), a membrane-disrupting toxin belonging to the repeats-in-toxin (RTX) family that specifically attacks human immune cells [23], [24], [25], [26]. A correlation between LtxA production and disease was first uncovered in the 1980’s when a majority (>90%) of juvenile periodontitis patients were observed to have high serum titers of antibodies against LtxA [27], [28]. Subsequent work found that LtxA could be extracted from five clinical isolates derived from the subgingival plaque of adolescent periodontitis patients [29]. Zambon et al. investigated 165 isolates from 91 patients and found that 64% of isolates obtained from juvenile periodontitis patients were highly leukotoxic, while none of the isolates obtained from healthy or adult periodontitis patients were highly leukotoxic [30]. Similarly, Haubek et al. examined 326 clinical isolates and observed that 38 were highly leukotoxic, and 36 of these were obtained from juvenile periodontitis patients [31]. More recently, in a study of 1023 clinical isolates, Haraszathy demonstrated that 73% of A. actinomycetemcomitans isolates from juvenile periodontitis patients were highly leukotoxic [32].

Without proper and timely treatments, these aggressive forms of periodontitis lead to the loss of connective and supporting tissue and damaged alveolar bone and eventually tooth loss, affecting patients physically and cosmetically [22], [25]. The traditional treatment for periodontitis is scaling and root planing (SRP), sometimes followed by periodontal flap surgery [33]. However, these mechanical treatments are not always effective since they are unable to suppress bacterial infections at the inflammation sites, thus allowing A. actinomycetemcomitans to persist [34], [35], [36], [37], [38]. As a result, antibacterial agents have been used systemically as a supplement to SRP to treat aggressive periodontitis. Although adjunctive antibiotic treatment has been reported to improve therapeutic outcomes [39], [40], [41], systemic antibiotics have drawbacks, including a risk for drug side effects [42] and increased development of antibiotic resistance [19], [43]. In particular, subtherapeutic concentrations in the blood and at the infection site [44] fail to eradicate the pathogen completely, contributing to the development of antibiotic-resistant species [45]. Over the years, several patient studies have reported resistance of A. actinomycetemcomitans to numerous commonly used oral antimicrobial agents, including amoxicillin, tetracycline, doxycycline, and metronidazole. Therefore, there is an essential need for targeted delivery strategies to treat A. actinomycetemcomitans-associated periodontitis to decrease the risk of antibiotic resistance [46], [47], [48], [49], [50].

Due to the immunosuppressive activity of LtxA and the observation that highly leukotoxic strains of A. actinomycetemcomitans, such as JP2, are more closely associated with disease [51], the mechanism of action of LtxA has been studied in detail [23], [52], [53], [54], [55]. After secretion by A. actinomycetemcomitans via a Type I secretion system [52], [53], LtxA interacts specifically with immune cells via recognition of an integrin receptor, lymphocyte function-associated antigen-1 (LFA-1) on the cell surface [23], [54]. In addition, LtxA binds to cholesterol on the target cell membrane through a cholesterol recognition amino acid consensus (CRAC) domain [55].

During its insertion into the plasma membrane, LtxA disrupts the integrity of the host cell membrane bilayer. A series of biophysical assays in liposomes demonstrated that LtxA-mediated content leakage occurred only when the liposome composition included lipids with a negative spontaneous curvature [56]. These types of lipids are able to form nonlamellar phases [57], such as the inverted hexagonal (HII) phase. The incorporation of the bilayer-stabilizing lipid, cholesterol sulfate, inhibited LtxA-mediated membrane disruption [56]. These results suggested that LtxA might mediate membrane disruption, not through pore formation, but via destabilization of the bilayer by promotion of nonlamellar phase formation. Subsequent work demonstrated that indeed, LtxA is able to promote the bilayer-to-HII phase transition in liposomes containing lipids with negative spontaneous curvature, in particular, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (N-methyl-DOPE) [56].

In this work, we were motivated by the association of LtxA and the pathogenicity of A. actinomycetemcomitans and aimed to use LtxA as a trigger for controlled antibiotic release from a liposomal delivery vehicle. We chose to use 100 nm unilamellar liposomes due to their structural resemblance to the host cell membrane, adequate encapsulation capacity, and advantageous circulation time [58]. Based on the high affinity of LtxA for cholesterol and its ability to disrupt lipid membranes in a specific manner, we hypothesized that a liposome encapsulating antibiotics and composed of cholesterol and an HII-forming lipid would be disrupted only in the presence of LtxA (Fig. 1), that is, in the presence of disease-associated strains of A. actinomycetemcomitans. We therefore synthesized liposomes composed of N-methyl-DOPE due to this lipid’s great responsiveness to LtxA [56]. Our results demonstrate that this “Trojan Horse” delivery system is effective in releasing antibiotics in a controlled, toxin-dependent manner.