Nanoantibiotic polymers typically need to be formulated into nanoparticles for full usage of the therapeutic antimicrobial properties. These polymers do however hinder the growth of bacteria through one of the nAbts mechanisms. While there are most likely many others that are not identified, examples of some well-known polymers are listed. Chitosan is a partially deacetylated chitin that has been shown to possess a wide spectrum of antibacterial activity [ 21, 22]. Chitosan’s antimicrobial mechanisms include increasing the permeability of the microbial wall or chelation trace metals [ 23, 24]. Gelatin is derived from type I collagen composed of glycine- and proline-rich repeating units [ 25– 27]. Gelatin has been shown to damage the cell membranes of Staphylococcus aureus [ 26, 27]. An additional class of polymeric materials are peptides, which have been developed as a treatment against various microbes including drug-resistant strains [ 28– 30]. For an example, stearylated melittin, is a well-known cationic antimicrobial peptide, which creates pores in the microbe membrane [ 31]. Furthermore, some synthetic and natural polymers may be considered as nAbts based on their abilities to act as matrices or vectors for varied deliveries. An example of this is dextran or acetylated dextran. Though dextran has not been shown to demonstrate any innate antimicrobial properties, it is an easily modifiable polymer that can aid in antimicrobial therapy [ 32, 33].

Gold-based nanoparticles have been used to treat bacterial infections using heat generated from photo-thermal effects. Gold’s antimicrobial activity is caused by strong electrostatic attractions to the negative charge bilayer of the cell membrane [ 34, 35]. Silver, on the other hand, has been used since ancient times as an antimicrobial. Amongst all the different types of metallic nanoparticles, silver NPs have proven to be the most effective against various types of microorganisms [ 10]. Silver NPs work by interfering with the respiratory pathway and cell division. Additionally, the release of silver ions further enhances antibacterial activity [ 4, 10, 36]. Titanium dioxide (TiO ₂) NPs are also well known for photocatalytic antimicrobial activity [ 5, 37] as demonstrated by the strong bactericidal activity of TiO ₂ upon receiving irradiation with near-UV light and UV-A. TiO ₂ NPs work by producing ROS such as free hydroxyl radicals and peroxide. Finally, zinc oxide NPs are nontoxic and biocompatible metal oxide NPs that have been widely adopted throughout the biotechnology and consumer goods industries [ 3, 38]. They are antibacterial against some important foodborne pathogens [ 3, 10], disrupt lipids and proteins of the bacterial cell membrane, and promote the generation of hydrogen peroxide and Zn ⁺² ions [ 10].

Fullerenes—and more specifically, colloidal C ₆₀ aggregates (nC ₆₀) in water—have been recently discovered to have beneficial antimicrobial properties [ 39]. Suggestions for the antibacterial mechanism for nC ₆₀ includes photocatalytic ROS production and lipid peroxidation in the cell membrane [ 10]. Other modifications of fullerenes allow for the destruction of microbes by insertion into the cell wall, which is then followed by the disruption of the cell membrane structure [ 3]. Additionally, single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) have been used for a multitude of antimicrobial applications [ 40, 41]. CNTs destroy gram positive and gram negative bacteria through irradiation mediated oxidative stress which affects the membrane integrity and metabolic activity of the bacteria [ 3, 42].

In addition to using stimuli-responsive linkers to combine conventional antibiotics and nAbts, these linkers are able to provide a unique advantage over current antibiotic strategies: they can be effectively tuned to certain infections or sites. To note, all organisms exist with a specific microenvironment around them. These environments are dynamic and deeply influenced by the organisms within them. Such phenomena can be observed in bacterial colonies, both commensal and pathogenic [ 43]. In the case of pathogens, environmental cues usually determine the virulence factor. The virulence factor of an infection is an intrinsic trait that is usually measured in terms of morbidity or mortality [ 44]. For most microbial infections, these virulent factors are responsible for efficient multiplication in the host with such attributes as adherence to host tissues, production of host-specific toxins, invasion into host cells, and resistance to the host defense mechanisms. Furthermore, a large proportion of these virulence factors have specific environmental signals such as temperature, osmolarity, pH, oxygen, carbon dioxide, amino acids, and iron levels, as demonstrated in Fig.  3. Pathogens may use these factors as signals to detect their environment, for instance, cueing them as to whether they are in the gut versus the lung or intracellular versus extracellular environments [ 44]. By taking advantage of these environmental cues, highly effective and multi-purpose nAbts can be created.

Fig. 3

Environmental regulator of virulent factors matched with stimuli classification. A schematic diagram of virulent factors that microbes possess which can be regulated by environmental signals which can be classified as chemical, biological, or physical

pH-responsive linkers have a unique advantage against microbes that thrive in highly acidic environments, whether extracellularly in the stomach (pH = 1–3) and gastrointestinal tract (pH = 5–8) or intracellular in the phagolysosomes (pH = 4.5–5) and macrophages [ 45– 47]. Additionally, chronic infections and wounds have pH values between 5.4 and 7.4 and are another potential site for the application of this therapy [ 45]. Examples of microbes that thrive in an acidic environment include Helicobacter pylori, Agrobacterium tumefaciens or Vibrio cholera [ 45, 46]. This specificity allows for a direct exploitation of the stimuli-response in certain tissues or in a cellular compartment. The key element for pH-responsiveness is protonation/deprotonation caused by charge distribution over ionizable functional groups such as carboxyl or amino groups [ 46] listed in Table  2. Polymers that contain carboxylic, sulfonic acid or amino groups such as poly( l-histidine), poly(methacrylic acid) (PMAA),and poly(acrylic acid) (PAA) [ 48], have been commonly used for their pH-responsive functionalities. pH changes induce a phase transition in pH-responsive polymers very abruptly, which can aid within intracellular compartments or with rapid release of drug cargos [ 46, 48].

Changes in an organism’s temperature have been correlated with dramatic changes in the expression of virulent factors. Additionally, multitudes of diseases have been known to manifest temperature changes [ 44, 45]. For example, Salmonella typhimurium, Bordetella pertussis, Escherichia coli, Vibrio cholera, and Shigella sp. have been shown to rely on temperature dependent environmental signals [ 44]. Normally, a temperature-dependent response is characterized by a critical solution temperature where the hydrophobic and hydrophilic interactions abruptly change within a small temperature range [ 45, 49]. This change induces the disruption of intra- and intermolecular electrostatic and hydrophobic interactions and results in collapse or expansion (a volume phase transition) [ 45, 47, 50]. Materials containing this group either become insoluble upon heating, classed as lower critical solution temperature (LCST), or become soluble upon heating, classed as upper critical solution temperature (UCST) [ 50]. LCST materials, Table  3, are usually based on N-isopropylacrylamide (NIPAM), N-vinylcaprolactam (NVCl), methylvinylether (MVE) [ 45, 47, 50– 52]. Whereas typical UCST systems are based on a combination of acrylamide (AAm) and acrylic acid (AAc) [ 47, 50].

While light might not be an environment dependent stimulus, it is an excellent external stimulus that can be applied to antimicrobial therapy. By combining UV exposure and drug release, light-responsive systems become very advantageous, particularly for antimicrobial systems because light can be applied instantaneously and under specific conditions with high accuracy [ 45, 48]. The wavelength of the light producing laser is tuned to near-infrared which is less harmful and has deeper penetration in tissues than visible light. Most photo-responsive materials possess light-sensitive functionalities, Table  3, such as azobenzene, spiropyran or nitrobenzyl groups [ 51, 52]. Examples of light-responsive antimicrobials can be seen in studies using modified TiO ₂ as a photocatalyst against such pathogens as E. coli [ 53, 54]. Additionally, the use of azobenzene- based antimicrobial compound has aided in the creation of polymer films active against S. aureus and C. albicans [ 55].

When trying to add redox-response functionality, researchers have typically focused on disulfide groups as these are unstable in a reducing environment [ 46, 48, 51, 56]. There is a redox potential (~100–1000 fold) that exists between extracellular and intracellular environments. The extracellular space environment is oxidative while intracellular is reductive due to glutathione concentration. Disulfide links degrade when exposed to glutathione or cysteine [ 52]. This degradation allows researchers to tune for environmental signals. At times, the redox reaction will alter the hydrophobic and the hydrophilic properties of a molecule, leading to a responsive swelling and de-swelling [ 51, 52] which is another responsive that can be taken advantage of for a nAbts therapy.

All organisms create special enzymes to help them thrive in their microenvironments. For example, bacteria in the colon produce reductive enzymes or hydrolytic enzymes capable of degrading various types of polysaccharides [ 57]. Researchers can take advantage of this particular characteristic since most infection sites will overexpress a certain enzyme, and responsive functionality can be applied to the material of interest [ 44]. Usually, in enzyme-responsive systems, enzymes are used to destroy the polymer or its assemblies [ 57]. Enzyme-responsive systems have many advantages, the largest being that they do not require an external trigger for their decomposition, Table  4. They also exhibit high selectivity and work under mild conditions [ 57]. For example, designing linkers that are responsive to pyroglutamyl-peptidase I [ 58, 59], a protease found in S. aureus, can allow for the creation of a nAbts that would selectively release at that infection site. The major drawback of enzyme-responsive systems, however, is the difficulty in establishing a precise initial response time [ 57].

Applying stimuli-responsive functionalities to the design of nAbts opens up the possibility of creating a multipurpose antimicrobial with an environmental dependent release. Additionally, the opportunity to include a targeted drug release functionality is presented. Thus, there is a synergist circumstance where the drug can find renewed efficacy against a drug-resistant organism.

While nAbts alone exhibit excellent antimicrobial properties, an even more effective therapy can be engineered by combining the properties of nAbts with that of conventional antibiotics. nAbts can target one mechanism of a microbe whereas conventional antibiotics can be used to address another, thus creating a combinatory therapeutic system that can tune to a number of scenarios. For example, it has been shown that there is a cooperative antimicrobial effect occurs when chitosan is combined with sulfamethoxazole to combat drug-resistant Pseudomonas aeruginosa [ 60]. Additionally, chitosan-capped gold nanoparticles coupled with ampicillin presented a twofold increase in efficacy [ 61]. A similar trend is observed when various aminoglycosides (streptomycin, gentamycin, neomycin) were conjugated to gold NPs [ 62]. Silver NPs has also been shown to significantly increase the antibacterial activities of drugs such as ampicillin, kanamycin, erythromycin, and chloramphenicol when tested against E. coli, S. typi, S. aureus, and M. luteus [ 63]. The clinical potential for such combinatory uses of nAbts and small drug molecules against drug-resistant infections, can also be aided by the addition of a stimuli-responsive linker, this is exemplified by the improved efficacy found in such attempts [ 4, 26, 35, 40, 64].

Another advantageous nAbts design is one that places various stimuli into one package. These will require the use of different stimuli-responsive linkers with two or more nAbts. For instance, a thermo-responsive group like NIPAM is combined with pH-responsive functionality for the purpose of designing a multi-environment NP [ 47]. These could be very useful in topical applications where one release will happen at body temperature, followed by another stimulus-triggered release intracellularly or at an inflammatory site. Moreover, a multi-stimuli nAbts can be advantageous for treating difficult intracellular drug-resistant microbe as illustrated in Fig.  4. Adding a stimuli-responsive functional group to a nAbts such as a gold nanoparticle or a carbon nanotube can be accomplished by different methods from surface coating using emulsion to reduction-driven synthesis [ 63, 65]. For polymeric systems, the method that dictates the addition of stimuli-responsive functional group is determined by the specific group being conjugated. For an example, conjugating a pH responsive functionality such as succinyl or acetyl to chitosan has been done by various groups with relative ease [ 66– 68]. An alternative pairing is one that places a light-responsive nanoparticle with one that has a different antimicrobial mechanism. For example, doping TiO ₂ with silver creates a particle that can destroy bacteria by photocatalytic inactivation and generation of silver ions [ 36, 69, 70]. These particles have been shown to have excellent light-independent antimicrobial activities against E. coli, S. aureus and P. aeruginosa [ 69]. A similar interaction can be observed when gold is combined with TiO ₂ NPs. While not necessarily used for antimicrobial therapy yet, various groups in the cancer research field are already applying the strategy of multi-stimuli-responsive particles [ 71].

Fig. 4

a Formation of a stimuli-responsive nanoantibiotics particle: a combined system can be created by combining conventional antibiotics with nanoantibiotics. Stimuli response can aid in the synergistic efficacy by allowing a particle to display multiple therapeutic effect based on the stimulus applied. b Proposed therapeutic effect of stimuli-responsive nanoantibiotics against intracellular infection. To target intracellular microbes, conventional antibiotics are combined with nanoantibiotics with stimuli response. Upon endocytosis, these stimuli-responsive nanoantibiotics (sr-nAbts) are triggered to release after certain environmental triggers. With pH triggered release, the particle unpacks to escape the endosome, target the microbe with 1 drug and nAbts. Based on the infection, a second trigger can release another drug and use an additional nAbts mechanism

Various nAbts, particularly metallic and carbon-based ones, have severe toxicity generated from prolonged exposure. Although silver NPs provide many benefits, prolonged exposure to soluble silver-containing compounds may produce an irreversible pigmentation in the skin (argyria) and the eyes (argyrosis) in addition to other toxic effects [ 10]. Some studies provide a direct contradiction to this, claiming minimal concentration dependent toxic effects [ 10, 72]. These concentration dependent toxicities to mammalian cells have been shown in other studies using metallic nanoparticles [ 73, 74]. Similarly, while nanotubes and fullerenes have been shown to be potentially toxic, there are some contradictions which suggest that the toxicity may be due to solvent contaminants during preparation [ 39, 75]. For one, a suspension of nC ₆₀ prepared without THF lacked toxicity [ 76]. Moreover, nC ₆₀ prepared without using any polar organic solvent lacked any acute or subacute toxicity in rodents [ 77]. And although these potential side-effects limit their applications, their use should not be disregarded entirely. A complete elucidation of nanoparticle toxicity needs to be ascertained before extensive manufacturing induced exposure.

In general, there has been increased scrutiny over the toxicity of nanoparticles due to increased use in various industries. Not only in the application, but also in the manufacturing of nanoparticles as those who manufacture the nanoparticles will experience the most exposure. Silver NPs have been analyzed to determine their toxicity when manufactured. Based on a continuous 3-day exposure assessment, it was evident that workers were exposed to high levels of nanoparticles on a day-to-day basis; similar results were also found for other metallic and carbon NP manufacturing [ 78]. While the exact toxicities of long-term overexposure are still completely unknown, a recent study by Das et al. [ 79] looks at the potential toxicity to mammalian germ cells and developing embryos from engineering nanoparticles such as gold, silver, fullerenes, and chitosan. They summarize various toxicities based on nanoparticle uptake and internalization mechanisms. The major emphasis of the study is that while some nanoparticles may not have any acute toxicity, there is a possibility that there is some long-term toxicity or effect to germ lines to consider. The authors disclose that a large percentage of these toxicities are mostly dependent on NP size and surface modifications. Taking those variables into consideration when designing and engineering nAbts can aid in ameliorating the possible toxicities that may arise.

Scale-up for creating nanoparticles requires sophisticated techniques [ 80]. Two key pathways to generate nanoparticles are through chemical or mechanical routes [ 37]; these can also be considered as bottom-up or top-down manufacturing approaches respectively, Fig.  5 [ 81]. A bulk material may be dissolved chemically into molecular entities to yield a distinct molecular intermediate form of the material. That intermediate is then reacted kinetically or processed further through the use of stabilizing agents such as emulsifiers. Generation of silver nanoparticles from silver nitrate is an excellent example of a chemical route [ 37]. Alternatively, mechanical energy can also be applied to a bulk material to split it into smaller particles. This usually requires heavy machinery such as a mill. Regardless of the chosen pathway, most particles are modified, whether by some type of surface modification or another type of customization, before further use [ 37].

Fig. 5

Manufacturing methods of nanoparticle. Nanoparticles can be manufactured in large scales either by bottom-up or top-down manufacturing methods

Another method used to industrially generate nanoparticles is an emulsion type system [ 82, 83]. This is a bottom-up synthesis approach that has some similarity to chemical synthesis route. Instead of distinct intermediates being formed, the emulsion route allows for the formation of nanoparticles based on an oil–water system. The versatility of the reactor system, where starting materials may simply be mixed, serves as an advantage for this system. A disadvantage of the emulsion system is that it will be limited to only certain types of nanoparticles made with materials that are soluble in an organic phase or an aqueous layer, and thus, it cannot be widely used.

The other end of the spectrum for scaling and manufacturing is the top-down approach of templated systems [ 84, 85]. Using cast molding generated from polydimethylsiloxane (PDMS), different shapes and sizes can be generated for a material that can be cured and dried, usually a polymeric material. While most nAbts are not applicable to this technique, there might be a way of combining the stimuli-responsive linkers or polymers that will allow for the templated-assisted system to be leveraged. The main advantage of using a template-like system is the ability to make many uniform nanoparticles very rapidly. This would reduce the cost of manufacturing for pharmaceutical applications while being able to maintain consistent quality. This consistency is something that the bottom-up approach may lose. The downside is the limited source materials that can be used in the template-assisted systems.

One thing is certain, when industrial scale manufacturing of nAbts comes into question, attempting to use just one system may not be adequate enough to serve a broad therapeutic need. Entities that decide to venture into commercializing nAbts for therapeutic applications need to consider which systems they are trying to target, and what would be the safest and most economical way to accomplish that goal.