Nanomedicine to overcome antimicrobial resistance: challenges and prospects

Antimicrobial resistance (AMR) has become a global threat to human health and economic development, and causes millions of deaths each year [1]. AMR is the result of a naturally evolving process through genetic mutation or by acquiring resistance genes from another microbe that could limit uptake of a drug, modify a drug target, inactivate a drug and/or actively efflux a drug [2]. These are often associated with improper use of the antimicrobial agents during the prevention and management of the infections and associated diseases. Additionally, the biofilms formed by AMR bacteria have significantly contributed to the multidrug resistance [3,4]. Although AMR have also been found in fungi, viruses and protozoa, the most significant and consequential challenge is the fact that more bacteria have developed multiantibiotic resistance. Multiantibiotic resistant bacteria are more difficult to treat, the choices of antibiotics are limited, and often higher doses are required. Alternative and more effective medications against AMR are urgently needed.

The AMR threat may be overcome by introduction of effective vaccines and new antibiotics; however, these developments have encountered various challenges. The US FDA has only approved a small number of antibacterial agents in recent years [5], although intensive investigation has led some to late-stage clinical developments [6]. The WHO has listed 12 highly virulent and multidrug-resistant pathogens [7] that include Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. so-called ESKAPE bacteria, capable of ‘escaping’ the action of antimicrobial agents [8–10]. Currently, there is no effective vaccine for ESKAPE bacteria, and though a number of clinical trials have been conducted with most efforts on S. aureus vaccine candidates (Table 1, https://clinicaltrials.gov/), so far, the outcomes are rather disappointing [11]. On the other hand, ESKAPE bacteria have effectively developed multidrug resistance, through various mechanisms as summarized in Table 2 [2,12], including inhibition of cell wall synthesis, depolarizing cell membrane, inhibiting protein synthesis and nucleic acid synthesis, and interfering with other metabolic pathways through drug inactivation by bacterial enzymes, drug target mutation and modification, reduced permeability and elevated efflux [10,13]. These pathogens are also able to form biofilms that act to prevent immune response and antibiotic action, and become less sensitive to antibiotics: as a result they are responsible for many difficult-to-treat, reoccurring infections [14]. As time progresses, the number of antibiotics to treat ESKAPE pathogens declines. In response to those urgent needs, antibiotic combination has been widely studied [15,16] and a few are currently under clinical trials (see https://clinicaltrials.gov/). In addition, clinical trials, based on bacteriophage therapy [17], fecal microbial transplantation [18–20] and probiotic bacterial replacement [21] as alternative approaches, can also be found at https://clinicaltrials.gov/.

A high dose of antibiotics is often required to treat AMR bacteria. If the current antibiotics can be target-delivered to the infection site, an improved antibacterial efficacy and safety profile could be expected. Approaches with targeted delivery of broad-spectrum antibiotics include antibiotic–antibody conjugates (AACs) [22] and antibiotic nanoparticles, which may provide more effective treatment while minimizing pressure on other bacteria to become antibiotic resistant. These two strategies have been applied successfully as anticancer therapies, which have yielded FDA-approved antibody–drug conjugate (ADC) drugs [23] and anticancer nanomedicines [24]. Unlike the highly potent payloads used in ADC and uptake of ADC through endocytosis followed by drug release by lysosomal enzymes, resulting in effective cytotoxicity against cancer cells, AAC lacks all of those mechanisms. Therefore, AACs are less likely to become successful antibacterial drugs unless much more potent antibacterial agents are employed and rapid drug release is achieved, which would then provide the desired therapeutic effect. On the other hand, nanomedicines may provide an attractive alternative against the threat of AMR, since they are able to improve drug stability, prolong circulation, improve pharmacological properties and most importantly show efficacy as demonstrated by FDA-approved nanomedicines, for example, Doxil^®, a doxrubincin-liposome (1995), DaunoXome^®, a daunorubincin-liposome (1996), and Vyxeos^®, a daunorubicin/cytarabine-liposome (2017) [25]. However, the clinical development of nanomedicine for AMR pathogens has met a few challenges which remain today. As an example, although quick uptake of nanoparticles by macrophages has provided a basis for the treatment of intracellular bacterial infections, the rapid clearance of nanoparticles by the mononuclear phagocytic system (MPS) and reticuloendothelial system is still a significant challenge. Other challenges for antibacterial nanomedicine include overcoming other biological barriers, including vascular barriers and microenvironmental barriers such as biofilm for targeted delivery.

Adsorption of serum protein by nanoparticles forms a nanoparticle protein corona, which alters the size, shape and surface properties of nanoparticles, and often leads to rapid clearance. Some cancer nanomedicines are therefore formulated with PEGylated lipids to minimize their interaction with serum proteins to achieve a stealth effect thus providing more stability and longer circulation time. However, a new challenge has emerged since the discovery that anti-PEG antibodies could be generated, which can in turn interact with nanoparticles, therefore enhancing the rapid clearance by the immune response. Cancer nanomedicines overcome vascular barriers by taking advantage of highly porous capillaries between endothelial cells around abnormal tumor tissues for enhanced extravasation and sequestration into the tumor [26,27]. The stealth effect as well as the enhanced permeability and retention are believed to be essential characteristics for nanomedicine. Additionally, the diverse cellular, intracellular and microenvironmental barriers associated with patients are also found to be significant challenges. In order to address these challenges, personalized chemoimmunotherapy nanomedicines have been proposed as cancer treatment in the future [25,28,29].

Antibacterial nanoparticles

In order to be clinically successful, antibacterial nanomedicines need to be serum-stable to avoid rapid clearance by the immune system and be able to cross the vascular barrier effectively. Antimicrobial agents could then be efficiently delivered to the infection site. The approach needs the nanomedicine to penetrate biofilms, leading to antibiotic uptake by bacteria through fusion and/or diffusion, thus to achieve improved antibacterial activity and pharmacologic properties [30,31]. Serum-stable nanoparticles also provide a possibility to include those potent but toxic antimicrobial agents, so that the side effects can be minimized through targeted delivery without premature release [32]. In this respect, the combination of nanotechnology and antimicrobial agents is one of the most promising strategies to cope with AMR.

Most successful antibacterial nanomedicines so far are delivered directly to proximity of the infection site, including the nanomedicine Arikayce™, an amikacin liposome inhalation suspension approved by the FDA for the treatment of Mycobacterium avium complex lung disease [33,34]. Further clinical trials were conducted on Arikayce™ against P. aeruginosa in cystic fibrosis patients, but this did not show benefits compared with the control group [35]. Other nanoagents, particularly silver nanoparticles, were also tested [31,36]. Table 3 lists those clinical developments (https://clinicaltrials.gov/). The progress and current status in the field have been extensively described [37–42], including applications of both organic (liposomes, lipid-based nanoparticles, polymeric micelles and polymeric nanoparticles) and inorganic (silver, silica, magnetic, zinc oxide, cobalt, selenium and cadmium) nanosystems. Studies on metallic nanoparticles as carriers for antibiotics – for example, glycopeptide antibiotics and daptomycin, to treat multidrug-resistant pathogens, particularly S. aureus, as well as E. coli, P. aeruginosa and A. baumannii – were also described by Berini et al. [43]. The metallic nanoparticles are often coated with biocompatible polymers like PEG, chitosan, polyvinylpyrrolidone and poly(vinyl alcohol), as well as surfactants, proteins (such as bovine serum albumin) and oligonucleotides to increase both the stability of the nanocomposite and the antibacterial activity [44,45]. Although some metallic nanoparticles, such as the most extensively studied silver nanoparticles, can act as broad-spectrum antibacterial agents (like SilvaSorb^®), as well as carriers of antibiotics [36,46,47], the emergence of silver-resistant bacteria has been observed similar to the antibiotic resistance through the process of natural selection [48].

The ability of nanoparticles to penetrate biofilms, particularly those formed by ESKAPE bacteria, provides a possibility to overcome multidrug resistance with nanomedicine [49]. Antibiotics conjugated to nanocarriers [50] or encapsulated by a variety of nanoparticles all showed favorable antibacterial activities [36,44], by inhibiting biofilm formation and enhancing intracellular delivery. Despite clear evidence of their strong antibacterial efficacy, the metal oxide-based nanoparticles, which are only effective to address bacterial biofilms in wound dressing, surfaces of medical devices and dental materials [38], are unlikely to be applied to systematic infections due to safety concerns [51,52]. Meanwhile, the lipid-based nanoparticles have emerged not only as a topical treatment for bacterial infections [53–55], but have also been explored as antibacterial agents delivered systemically. Although the clinical outcome so far has been disappointing [56], hitherto, no lipid-based nanomedicine has been approved by the FDA for systematic treatment of bacterial infection.

In a critical review, Crommelin et al. assessed the current status of clinical development of nanomedicines, and analyzed the nanomedicine research from academia and industry over two decades [57]. The most successful family within the broad field of nanomedicine so far were liposomes, while other families of nanosized drug-delivery systems have not met their expectations. Based on lessons learned from cancer nanomedicine development, antibacterial nanomedicine will likely follow a similar path; therefore, the ‘liposome experience’ should be valued in the antibacterial nanomedicine development. With the success in anticancer nanomedicine development and successful introduction of mRNA-liposome vaccines [58], lipid-based nanoparticles, including liposomes and solid lipid nanoparticles, could be favorable choices for anti-AMR nanomedicine. To enhance the interaction with specific targets, the liposome surface might be modified with proteins, antibodies, carbohydrates or immunoglobulin fragments, in addition to pH-sensitive and thermosensitive liposomal vesicles. Those modifications, however, need to be carefully executed with consideration of their burdens to immunological and biological barriers. The significant differences in microenvironments between cancer and bacterial infections also need to be properly addressed in the nanoparticle engineering and formulation. A few reviews on antibiotic liposomes are listed in Table 4, with their titles and highlights. The major challenges in antibacterial nanomedicine engineering are similar to those encountered in cancer nanomedicine development. In order to translate a promising technology into clinical reality, antibacterial nanoparticles, like liposomes, should be properly designed with deep understanding of the biological interactions involved, and meanwhile formulated with a simple approach. The following discussion provides some challenges and prospects on future anti-AMR nanomedicine development.

Challenges & prospects

Immunologic barrier

Rapid clearance of nanoparticles by phagocytosis is a common problem faced during nanomedicine development [65]. Nanoparticles in circulation often adsorb various serum proteins on their surface to form coronas. If opsonin, immunoglobulins or complement protein are attached, their interaction with phagocytes will lead to a rapid clearance by MPS. Most nanoparticles are then taken up and accumulated in the spleen and liver. On the other hand, the adsorption of serum albumin may protect nanoparticles from phagocytic uptake, which could contribute to the stealth effect of nanoparticles [66–68]. Since the distinct corona protein may determine the outcome of serum stability of nanoparticles [67], it is plausible to engineer nanoparticles that are able to form a specific corona by refining surface charge and other surface properties. For example, it has been shown that opsonins interact more favorably with positively charged nanoparticles than those negatively charged, whereas neutral and slightly negative charged nanoparticles are observed with longer circulation time [69,70]. In addition, when a protein corona forms, the ligands on nanoparticle surface might be blocked as well, losing their targeting capability [71]. Furthermore, the nanoparticles of size larger than 200 nm can activate the complement system [72], which also leads to rapid clearance.

One common method to improve serum stability of nanoparticles is to incorporate PEG as a stealth coating. However, nanoparticle PEGylation does not completely prevent the protein adsorption and the recognition by macrophages and other immune cells. Exposure to PEG also leads to the production of anti-PEG antibodies. Those anti-PEG antibodies can, in turn, aid the rapid clearance of PEGylated nanomedicine. Since PEGylated agents are commonly used in medicine or nonmedicine, pre-existing anti-PEG antibodies have been detected in humans. Additionally, PEGylated nanoparticles have been shown to cause severe allergic reactions and anaphylaxis in a small number of patients, suggesting PEGylation may not be a long-term solution. Mitigation approaches include use of cleavable PEG [73], and with formulations of chitosan [74] and dextran [75] included in nanoparticles. In the face of the observed drawbacks to PEGylated nanoparticles, the alternative hydrophilic and nonimmunogenic coating agents would be preferred in antibacterial nanomedicine. It is plausible that carbohydrates could play such a role as they are hydrophilic and, in many cases, poorly immunogenic. Indeed, bacteria are covered with carbohydrate with a purpose to evade the innate immune system. Since the nanoparticle as a carrier may augment the immunogenicity of antigens on its surface, it is important that only nonimmunogenic carbohydrates be incorporated. In fact, human glycoantigens, from either glycoprotein or glycolipid, have been introduced onto the nanoparticle surface as recently described by Singla et al., where ciprofloxacin-loaded liposomes functionalized with host cell glycans improved the delivery of antibiotics to the site of P. aeruginosa infection, likely due to increased retention of the liposomes in the circulation [75]. Another option is to optimize the surface properties of nanoparticles through the use of novel lipids and formulation, so that only serum albumin is selectively adsorbed as a stealth coating to avoid the uptake by phagocytes and rapid clearance from circulation.

Since the interaction with serum proteins can bring significant changes to nanoparticles, such as size increase, surface charge neutralization, blocked ligands and even modified nanoparticle structure, one must be very mindful in the nanoparticle design by taking consideration of all those possibilities, and if possible, take advantage of those changes.

Vascular barrier

The nanoparticles circulating in the bloodstream need to cross the vascular barrier to achieve targeted delivery. It has been observed that the size, shape and surface properties all affect the attachment of nanoparticles to the blood vessel wall [76,77], and the complicated process is not fully predictable [78]. Optimized particle size (<200 nm) exhibited in vitro the greatest extent of vascular stability as well as the lowest extravasation [79]. Since the abnormal growth of tumors leads to formation of small pores between the endothelial cells [80], nanoparticles can traverse these pores and reach the interstitial sites in tumors and deliver the loaded drug to cancer cells.

Similarly, during infection, bacteria produce toxins and other virulence factors that can disrupt the barrier function of the endothelium to enter and exit the bloodstream. Those damaged endothelial cells, weakened cytoskeleton and broken junctions between endothelial cells would passively allow nanoparticle extravasation. Additionally, endothelial cells overexpress adhesion molecules during infection, such as selectins that bind to certain carbohydrates presented on the leukocyte glycoprotein, which facilitates leukocyte to leave the blood vessel and enter the site of infection. Thus, in order to achieve active extravasation, nanoparticles may be modified with those carbohydrates, and it is known that sialylated and sulfated carbohydrates on glycoproteins, glycolipids and proteoglycans can be relevant ligands for selectins [81].

In an early review by Forssen and Willis functional liposomes for targeted delivery against cancer were discussed, which included surface modifications with antibodies, peptides, carbohydrates, aptamers and cell surface receptors [82]. Folate receptors, adhesion molecules, extracellular matrix molecules and selectins were all suggested as targets for site-directed cancer therapy. In one example, Maruyama et al. [83] provided evidence that liposomes modified with a highly specific antibody to pulmonary endothelial cells and additional less than 10% GM1 ganglioside (immunoliposomes) resulted in slow MPS uptake and its improved accumulation in lungs. Kalita et al. [84] described how to harness glyconanoparticles' distinct optical, magnetic and electronic properties for disease diagnosis with improved magnetic resonance imaging, fluorescence imaging, etc., by adding carbohydrates on metal oxide nanoparticles. Discussion also included using glyconanoparticles to imitate viral particles (or host cells) to interfere with the virus–cell membrane interactions, thus protecting the host cells from infection. Under this context, properly designed nanoparticles with surface modification of carbohydrates may overcome not only the immunological barrier but also the vascular barrier. This could be a sensible approach to deliver nanoparticles to the infection site as illustrated in Figure 1.

Microenvironment & drug-taking-up barriers (heterogeneity & biofilm)

Bacteria create a unique microenvironment and often form biofilms, particularly in chronic infections. In addition to infective endocarditis, gum and wound-related infections, biofilms have been found in many diseases such as lung, colon, urethra, eye and ear infections. Once the nanoparticles pass through the immunological and vascular barriers, they still need to overcome those additional obstacles in order to deliver antibiotics to bacteria. The microenvironment is heterogeneous, depending on the type of bacterial infections, and often slightly acidic. Bacterial biofilms contain various enzymes, toxins and other molecules released from bacteria, such as DNA, proteins and polysaccharides [85]. It is this extracellular matrix that makes biofilm communities highly resistant to antibiotic treatment, as observed that bacterial cultures in intact biofilms are up to a 1000-fold more resistant to antibiotics than those cultures growing as planktonic (free-floating) forms [86], by limiting the penetration of antibiotics to reach the bacterium. Additionally, biofilms may lead to physiological changes in the bacteria, such as a drastic slowing of the growth phase leading to a state close to dormancy, and in some cases, bacteria can develop a resistant phenotype by altering drug targets for antibiotic activity [87]. Biofilms, such as those found in urinary tract and lung infections, as well as those in implanted medical devices [76,79], are often caused by ESKAPE bacteria [88]. For example, P. aeruginosa biofilm contributes to chronic lung infections in cystic fibrosis patients [89,90], while S. aureus, K. pneumonia, A. baumannii and Enterobacter spp. are all responsible for upper respiratory and lung infections and all but S. aureus are also responsible in many cases for urinary tract infections.

Certain nanoparticles, including liposomes, micelles and inorganic nanoparticles, have shown the ability to penetrate the biofilm and bacterial cell walls to kill bacteria by various mechanisms [91], which may help design and engineer more sophisticated nanomedicines against this adversary. The interaction between nanoparticles and biofilm is very complex and not fully understood, but it is believed that the interaction is primarily determined by their electrostatic characteristics. These features depend on the zeta potential of the nanoparticle after its corona formation and extravasation, and the charge of the biofilm matrix [49]. It is believed that the negatively charged matrix can interact with positively charged nanoparticles to allow further diffusion toward bacterial cell, such as those nanoparticles developed from metals or metal oxides, synthetic or natural polymers, or hybrids therein. However, since only lipid-based nanomedicines are clinically approved by the FDA, it will be more fruitful to pursue similar strategies through careful refinements of nanoparticle lipid compositions, the hydrodynamic size, surface charge and other properties, to ensure efficient biofilm targeting and matrix interactions, thereby enhancing their antibacterial efficacy. However, a delicate balance is required in nanoparticle formulation since negatively charged nanoparticles are preferred to overcome immunological barriers while positive charged nanoparticles interact better with biofilm. Although antibiotic-liposomes may be able to fuse with the bacterial membrane and release the drug directly into the bacteria [92], it should be remembered that the nanoparticles at this stage are no longer the same in both size and surface properties after passing through the immunological, vascular and other biological barriers. Thus, the consequent interactions with biofilm are very different from those observed in in vitro experiments. Lipid-based nanoparticles are attractive candidates for nanomedicines but improving their biofilm penetration is still a major challenge. Engineering antibiotic-liposomes which can be degraded within biofilm matrix could be one of the solutions. The approach results in release of antibiotics close to bacteria for their spontaneous diffusion.

Payload encapsulation

To date, only a few therapeutic agents have been formulated into lipid-based nanomedicines which provided clinical advantage by improving the circulation time and the pharmacology. The nanomedicines, for example, Doxil, Onivyde^® and Onpattro^®, are PEGylated stealth nanoparticles whereas DaunoXome, AmBiosome^®, Marqibo^®, Vyxeos and LEP-ETU are formulated as conventional liposomes (Table 5). Active ingredients are encapsulated within the aqueous chamber of liposomes [93,94] or carried in the lipid bilayer [95–98]. The encapsulation capacity of those nanomedicines, which is measured by lipid-to-drug weight ratio, varies from approximately 2.2:1 in Onivyde to 30:1 in LEP-ETU (Table 5). With an 8:1 lipid-to-drug ratio, Doxil's dosage for cancer treatments at 20–50 mg/m² requires roughly 40–100 mg of doxorubicin or close to 0.36–0.9 g of nanomedicine per treatment.

The cancer drugs encapsulated in the FDA-approved and experimental nanomedicines are highly potent, whereas antibiotics are often much less potent with variable sensitivity toward different bacteria (Table 6). This could pose a challenge for antibiotic liposomal nanomedicine, particularly if the efficiency of the antibiotic encapsulation is suboptimal. The encapsulation limitation may be addressed with novel lipid-based nanoparticles, formulated with biodegradable and biocompatible materials, instead of phospholipids. As choices of antibiotics have already been limited [103], antibacterial nanomedicines would likely include a combined drug regime to improve efficacy, reduce drug resistance and minimize toxicity. Additionally, the limitation on antibiotic payload could be mitigated if the nanoparticles have a more favourable serum stability and an effective ligand-directed extravasation to quickly reach and release antibiotics at infection site as we have discussed above (Figure 1).

Ligands for bacterial targeting or other receptors

Early anticancer nanomedicines were formulated without specific ligands. The improved efficacy is largely due to improved circulation time and passive extravasation to cancer location due to the fenestrated blood vessels. More cancer nanomedicines with ligands such as specific monoclonal antibodies and other targeting moieties are believed to be able to enhance targeted delivery. Although ligand-aided liposomes delivery has been explored, and encouraging results were obtained in animal models, its potential benefits in patients have yet to be clinically established [110–112].

Unlike cancer, bacterial infection progresses much more quickly and diversely, and it is not always possible to identify exactly the bacterium which causes infection in clinical settings. The lack of common receptors on bacterial surface and the presence of microbial biofilms have presented a serious challenge for bacteria-specific targeting, which seems difficult to overcome at this moment [113], even if bacteria-specific nanoparticles are developed with monoclonal and single domain antibodies, or other ligands by rapid assembly at bedside. When nanoparticle passes through the biofilm matrix, the ligand is no longer essential since the nanoparticle is already in the proximity of bacteria. Realistically, rather than directly targeting bacteria, the focus of targeted delivery should be more on enhancing the nanomedicine to reach the infection site through improving serum stability and active extravasation, which can be achieved, for example, through targeting adhesion molecules of the vascular endothelial cells (such as selectins), whose expression is often increased near the site of inflammation and infection. Nanoparticles that may degrade by interaction with biofilm matrix could also be considered as a means of targeted delivery.

Conclusion

Targeted delivery of therapeutic agents has been a long-sought necessity for the effective treatment of serious diseases with fewer side effects. Nanotechnology may provide such a platform to achieve this goal. Targeting cancer biomarkers, bacterial surface components and vascular adhesion molecules with functional nanoparticles is an attractive approach, but due to individual heterogeneity and changing disease states, optimal target selection would be a great challenge. Unlike cancer treatment, which is aided with precision diagnosis and regime optimization, antibacterial nanomedicine faces unique challenges. Acute bacterial infection must be dealt with quickly and decisively, often without knowing the nature of the bacterial infection. Therefore, the new paradigm in antibacterial nanomedicine should focus more on the engineering of stable, yet, biodegradable nanoparticles with stealth coating or the ability to acquire stealth through selective corona formation, such to resist the rapid clearance by immune system. Meanwhile the surface functional ligand which recognizes specific molecules on the vascular endothelium near the infection site remains a sound choice to facilitate vascular crossing and therapeutic delivery. This approach requires no information on what bacteria caused infection, and the nanomedicine with broad-spectrum antibiotics encapsulated may provide desired pharmacological properties and therapeutic outcome.

However, after over three decades of intensive research and clinical development, only a limited number of nanoparticles have translated into nanomedicines to benefit patients. In addition to those challenges described above, the toxicity, manufacture and cost-associated concerns on nanoparticles have to be addressed [114]. More importantly, a deeper understanding of interplay between engineered nanomaterials and biological systems is required. The expectation in the translation of nanoparticles to nanomedicines can only be met by properly designed and engineered nanoparticles that could navigate through complex physical, immunological and biological barriers, and finally deliver desired doses of antibiotics to bacteria. Each step poses unique challenges, and a concerted overall strategy is required to achieve positive clinical outcomes.

Future perspective

Nanomedicine is designed as a vaccine and therapeutic delivery system, promising to provide improved efficacy and reduced side effects. However, only a few in clinical development so far have met the expectation. Unlike proposed future treatment for cancer by individualized nanomedicine, nanomedicine for AMR must be readily accessible and affordable. Simplicity must be a goal for anti-AMR nanomedicine, which can be gradually achieved through further understanding of the immune responses toward nanomedicine by the human body, and the biological interactions interplayed among nanomedicine, heterogeneous microbes and diverse human systems. A relationship between nanostructure and function may eventually emerge, which could guide nanomedicine design.

In the near future, it is desired to engineer more robust nanoparticles with synthetic or natural biocompatible and biodegradable materials. They must provide better consistency and control in size and surface charges than current nanomedicines, with improved encapsulation, and be ready for in situ add-on as a multicomponent system for targeted delivery. More specifically, the antibacterial nanomedicine must overcome heterogeneous biofilm, the last and the most challenging barrier. As to clinical development, we believe that the focus of nanoparticle design should be more on taking advantage of the extracellular environment rather than on particular molecular structures. The delivery of therapeutics to bacteria can then be achieved through either fusion or diffusion.

Finally, similar to virus-like particles as a promising vaccine platform against various diseases, nanoparticles, particularly the lipid-based nanoparticles, could be built through a combination of biosynthesis and chemical synthesis as microbe-like particles, which can be potentially explored as effective vaccines against AMR, other than as vaccine/immunogen delivery systems.