Antibacterial Efficacies of Nanostructured Aminoglycosides

The widespread use of broad-spectrum aminoglycoside antibiotics is restricted from various clinical applications due to the emergence of bacterial resistance and the adverse effects such as ototoxicity and nephrotoxicity. The intensive applicability of nanoparticles in modern medicinal chemistry has gained the interest of researchers for modification of aminoglycosides as nanoconjugates either via covalent conjugation or physical interactions to alleviate their undesirable effects and bacterial resistance. In this context, various carbohydrates, polymers, lipids, silver, gold, and silica-attached aminoglycoside nanoparticles have been reported with improvements in physicochemical properties, bioavailability, and biocompatibility in physiological medium. Overall, this review encompassed the synthesis of nanostructured aminoglycosides and their applications in the development of new antibacterial therapeutics.


INTRODUCTION
Nanoscience and its associated technology represent revolutionary research in drug manufacturing, particularly, diagnostics, imaging, and/or drug delivery at the cellular or atomic level. The discoveries mainly focus on the enhancement of drug specificity to cells, the efficacy of targeted drug delivery, and suitability of carriers for drug distribution and drug toxicity reduction. The unique and well-defined pharmacodynamic and pharmacokinetic properties of nanomaterials 1 result from the effects of ultrasmall and well-behaved quantum size, large surface by volume ratio, shape, functionalized structure, surface charge, and unusual electrodynamic interactions with the biological system, among other parameters. Likewise, the tunable physicochemical properties of nanoparticles in addition to crystallinity, electronic states, surface instability, surface roughness, and radius of curvature contribute in different ways to greatly affect their medicinal properties. In this regard, we understand from the literature that nanostructured materials encourage the delivery of drugs to a preferred site of action in the living organism, overcoming biological barriers such as the blood−brain barrier as well as intestinal, nasal, pulmonary, and skin barriers. 2 Even though the involvement of nanostructure materials in different areas is well-recognized, the participation in the field of antimicrobials is highly significant. Well-known clinically used broadspectrum bactericidal aminoglycoside antibiotics (AAs) are active against aerobic Gram-negative as well as a few Grampositive microorganisms (Figure 1a) and are used to treat opportunistic infections with AIDS, cystic fibrosis, and cancer. Unfortunately, the extensive use of these aminoglycosides is impeded by significant dose-related toxicities, poor adsorption in the gastrointestinal tract (GIT), low solubility and permeability into the bacterial cell membrane, as well as the rapid rise of AA-resistant strains around the world. The looming threat of aminoglycoside resistance emerges because of enzymatic hydrolysis, target site modification, impaired entry to the bacterial cell, and active AA efflux. Furthermore, the global market for aminoglycosides was assessed at more than 1.1 billion USD in 2014, and it is predicted to grow at a CAGR (compound annual growth rate) of more than 3.0% in the coming years, owing to the rising prevalence of bacterial infections affected by Gram-positive and Gram-negative bacteria ( Figure 1B). Thus, access to aminoglycosides in a new state with antibacterial activity, low toxicity, and smaller susceptibility to aminoglycoside-modifying enzymes compared to those of their parent structures is required.
From a structural perspective, aminoglycosides are polycationic pseudo-oligosaccharides with an aminocyclitol/streptamine core linked to an aminated sugar. The primary binding sites of AAs are the hydroxy and amino groups, which engage in interaction with the A-site decoding area of the bacterial 16S rRNA, causing cell death by inhibiting protein synthesis. These groups are responsible for high-water solubility and limited lipid solubility, in addition to their easy synthetic modification. Several synthetic approaches to find the next generation of AAs with improved biological and therapeutic functions have been articulated mainly, fostering their uptake property modification or enzymatic alteration for the mechanistic studies toward the resistance of AAs with different multi-drug-resistant bacteria. 3 The benefits of current nanomedicine in an aspect of pharmacokinetic and pharmacodynamic characteristics could lead to more effective actions. 4 We have witnessed the unique and customizable features of nanomaterials that make the drugs easier to administer, eliminating some shortcomings of traditional antibiotic therapies. Thus, the use of nanomedicine and its associated technology could modify the existing aminoglycosides to yield nanostructured aminoglycosides for addressing the above-mentioned limitations of AAs. In this review article, the synthesis and applications of nanostructured AAs are presented.

SYNTHESIS OF NANOSTRUCTURED
AMINOGLYCOSIDES For the synthesis of nanostructured aminoglycosides, polymers, lipids, proteins, and nucleic acids, and carbohydrates (or saccharides) as major groups of biomolecules are explored in addition to silica-, iron-, silver-, and gold-based nanoparticles as carriers ( Figure 2). Even carbon dots and quantum dots are subjected to the synthesis of aminoglycoside nanoparticles.
3. AMINOGLYCOSIDE-ENCAPSULATED POLYMERIC NANOPARTICLES 3.1. Aminoglycoside-Loaded Carbohydrate-Supported Nanoparticles. Biocompatible/biodegradable naturally occurring biomolecules and carbohydrates assist in the preparation of nanotherapeutics because of their chemically well-defined structure, protein repellency, high water solubility, and no aggregation properties. 5 An additional behavior of the carbohydrates is their ability to self-assemble to form polymeric nanoparticles, which encapsulate/load the AAs into nanostructured materials.
Strategies and reactions for the loading of aminoglycoside to chitosan nanoparticles based on the shielding of the polyanionic characteristic of dextran to the polycationic characteristic of aminoglycosides ( Figure 3). 6a For AA-loaded chitosan (CS) nanoparticles (NPs), the difference of drug, chitosan, and dextran sulfate concentrations varied the size of nanoparticles in the range of 492.23 to 779.37 nm, with positive zeta-potential. With the representative loading efficiency of tobramycin (37.2%), gentamicin (Gent), and streptomycin (SM) (∼58 to 63%), in vitro, the nanoparticles sustain release >60% of drug into nanoparticles after 6 h in pH 1.2 buffer in a sustained behavior. Nano-SM has reduced bacilli growth by p = 0.01 in a mouse model infected with   Mycobacterium tuberculosis that was as good as subcutaneously injected aqueous streptomycin at the same dose (100 mg/kg). To crop more chitosan-based antibacterial films, Hari and colleagues 6b assorted streptomycin-loaded starch nanoparticles (SS-NPs) with 1% chitosan and 1% gelatin, and SS-NPs improved the crystallinity and controlled swelling of the chitosan−gelatin film. These antimicrobial films inhibit Escherichia coli by approximately 90% and Bacillus subtilis by 80%, with a sustained release (60%) of streptomycin for 10 days, indicating its clinical potentiality through oral administration.
Deacon et al. 7a accessed tobramycin-encapsulated alginate/ chitosan nanostructures that resulted in uniform size distribution with 6:1.5:1.5 ratios of alginate, chitosan, and tobramycin, as determined by scanning electron microscopy (SEM) and dynamic light scattering (DLS) analysis. With 45% encapsulation efficiency, in vitro antimicrobial activity of the nanoparticles against Pseudomonas aeruginosa PA01 is similar to that of free tobramycin with a minimum inhibitory concentration (MIC) 0.625 mg/L. In the in vivo model for P. aeruginosa infection, the survival rate is found to be 90% upon injection of nanoparticles, inferring low nanoparticle toxicity. Tobramycin nanostructures removed the lethal inoculum caused by P. aeruginosa and doubled the survival rates compared to those with free tobramycin. Adapting the ethanol injection approach, Monteiro et al. 7b prepared a chitosan nanofiber mesh (NFM) through the reaction of covalently immobilized SH groups of CS NFMs with liposomes. Gentamicin-loaded liposomes were formed with a 17% success rate ( Figure 4). The random test indicated the immobilization of liposomes (∼110 nm) on the surface of an electrospun CS nanofiber with a diameter between 100 and 500 nm. The preservation of electrospun CS NFMs and liposome structures was assigned from the SEM analysis of CS NFM before liposome immobilization and after liposome immobilization at the surface of an electrospun CS nanofiber. The drug−NPs displayed a sustained release of gentamicin during 16 h, accomplishing a steady state at 24 h. The released drug worked against Staphylococcus aureus, E. coli, and P. aeruginosa, with MICs 0.25, 0.83, and 0.50 mg/L, respectively, along with MBC 2.00 mg/L for all of the strains. Another example of chitosan/fucoidan nanoparticles (CS/F NPs) as carriers for gentamicin delivery was developed by Huang and co-workers. 8a The gentamicin-loaded CS/F NPs (Gent-CS/F NPs) with a positive zeta-potential had an entrapment efficiency (EE) > 90%, higher than that of the reported values by Balmayor et al. 8b from starch-conjugated CS NPs. Gent was encapsulated using the water/oil emulsion method (EEs from 55 to 67.2%), whereas for the tripolyphosphate cross-linked CS NPs, the EE of Gent loading was 61.7−87.2%. 8c With sustained release for up to 72 h with 99% from biphasic CS/ F NPs, the bacterial inhibition against Klebsiella pneumoniae by Gent-CS/F NPs (>96%) was lower than that of free Gent (after 48 h, 80−90%). The intratracheal injection of Gent-CS/ F NPs (0.27 mg/kg) had an area under the concentration− time curve/MIC ratio higher than that of the intravenous administration of free Gent (0.5 mg/kg), showing the improvement of antibacterial efficacy.
The kanamycin (KS) was encapsulated by the PEGylated water-soluble chitosan (WSC), that is, cationic deacetylated chitin NPs and PLGA-TPGS NPs. Interestingly, the KS-PEG-WSC NPs and KS-PLGA-TPGS NPs increased blood circulation while lowering dosage frequency. 9a The WSC layer showed a significant impact on the surface load, and zetapotential was reported to be near neutral at +3.61 mV. KS-PEG-WSC and KS-PLGA-TPGS NPs steadily and consistently released 91.56% in 14 days and 93.26% in 21 days, respectively.
For the transformation of injectable streptomycin to an oral version, streptomycin-encapsulated PLGA nanoparticles were made using the multiple emulsion technique. 9b The average particle size of the drug-loaded NP was found to be 153.12 nm, and the drug encapsulation efficiency was observed as ±4.08% with 14.28 ± 0.83% drug loading. Streptomycin was maintained in plasma for 4 days and in organs for 7 days after a single oral dose of SM-PLGA nanoparticles was applied to mice. Regarding the relative bioavailability, encapsulated streptomycin was 21-fold higher than that of injectable drugs. In M. tuberculosis H37Rv-infected mice, eight doses of the weekly oral streptomycin-loaded NPs were equivalent to 24 free streptomycin intramuscular injections.
Likewise, Akhtar et al. designed intravenous gentamicin to oral absorption through encapsulation to PLGA nanoparticles modified with chitosan following water-in-oil-in-water formulations (w/o/w). The nanoformulation shows sustained release with increasing residence time to 11.22 ± 0.42 h and with the higher elimination half-life value (∼6.23 h). 10a These particles were tested on plankton and biofilm crops of Gramnegative P. aeruginosa PA01 in vitro, as well as a peritoneal 96 h pattern of murine infections. With a MIC of 1.5 g/mL, free gentamicin inhibited bacterial growth, whereas the formulations of w/o/w and s/o/w (MIC of 3.0 g/mL) prevented the growth with efficacy lower than that of the free drug.
Gentamicin-AOT-loaded PLGA nanoparticles have a mean diameter ranging between 289.15 and 299.23 nm with a zetapotential of −3.7 to 0.4 and −3.6 to 0.7 mV, respectively. 10b In experimentally infected THP-1 monocytes, this gentamicin formulation reduced Gram-negative Brucella melitensis infection (>2-log10 reduction), whereas in vivo investigations showed effectiveness in the liver and spleen for up to 4 days in infected mice. Even though 14 doses of free gentamicin had no effect on infection, only four doses of gentamicin-AOT-loaded nanoparticles reduced splenic infection by 3.23 log in 50% of infected mice without causing any adverse effects.
Abdelghany et al. developed a controlled release gentamicin formulation in water/oil/water and solid/oil/water using PLGA nanoparticles for treating Pseudomonas infections. 10c Entrapment of a hydrophilic drug into a hydrophobic PLGA polymer can be increased by lowering the pH of the formulation, which reduces the hydrophilicity of the drug and thus improves entrapment, reaching levels up to 22.4 g/mg PLGA. These particles had a regulated release of gentamicin for up to 16 days under conventional incubation settings and have a MIC and MBC higher than that of free Gent.
Elfaky et al. 11a reported the gentamicin loading on nanostructured lipid carriers (NLC) containing TPGS surfactant to protect from gentamicin-induced nephrotoxicity. In vivo studies with three groups of rabbits for 10 days revealed differences in plasma creatinine, urea, sodium, potassium, and calcium between the control and Gent-NLC materials considerably lower than those with gentamicin, confirming the protective effect on kidney function. Amikacin-loaded PLGA nanoparticles were synthesized 11b by Sabaeifard et al. utilizing a solid-in-oil-in-water emulsion process with varied ratios of 50:50 PLGA/drug (100:3.5, 80:3.5, and 60:3.5) as well as a stabilizer (Pluronic F68) (0.5 or 1%). The drug encapsulation efficiency was 76.8%, and from the release kinetic study, 50% of the encapsulated drug was released within 1 h of incubation. No toxicity against RAW macrophages was observed in cell viability/cytotoxicity assays after 2 and 24 h of treatment. The slow-released drug from NPs showed activity 4 times lower than that of the free drug.
A synthetic block copolymer containing Pluronic-based core−shell nanostructures was synthesized by Ranjan and coworkers. 12a With a 20% antibiotic loading in their formulation, the toxicity and adverse effects of this core−shell nanostructure encapsulating gentamicin, as well as the percentage of viable bacteria in the liver and spleen, were reduced. The anionic homo-and block copolymers of poly(ethylene oxide-b-sodium acrylate) (PEO-b-PAA − Na + ) or poly(ethylene oxide-b-sodium methacrylate) (PEO-b-PMA − Na + ) were blended with PAA − Na + -based polyanions to get stable nanoplexes at physiological pH following gentamicin encapsulation through electrostatic interactions ( Figure 5). 12b Gentamicin was also incorporated (at a rate of 26% by weight) into macromolecular complexes with an average diameter of 120 nm and a zetapotential of −17 mV. These nanocomplexes can potentially enhance the attack of the intracellular pathogen Salmonella. The nephrotoxic profile of gentamicin nanoparticles was compared to that of free gentamicin by Jamshidzadeh et al. 12c Furthermore, Mugabe and colleagues developed 12d liposome-encapsulated gentamicin, which was less toxic than free gentamicin and showed high efficiency against dispersed Salmonella infections in mice. In rats and mice, the mean halflife of liposome-encapsulated gentamicin sulfate produced from egg phosphatidylcholine in serum was 4 times longer than that of free gentamicin in intravenous injection. This liposome encapsulation exhibited increased and extended activity in reticuloendothelial cells, particularly in the spleen and liver. In mice with acute septicemia, the liposomal formulation outperformed free medicines in terms of preventive activity.
Conjugation of gentamicin to the hydrophobic block polymer POEGMA-b-PVBA containing aldehyde groups through Schiff base formation with gentamicin produced an amphiphilic block copolymer closely related to polyethylene glycol. Core cross-linked star polymers reacted with NO to form a polymer containing a N-diazeniumdiolate (NONOate) group that release NO in a regulated manner for a few days and formed dispersed biofilms. The DLS results showed a good polydispersity index (PDI 0.1) and a number-average size (15 nm) that agree well with the TEM data ( Figure 6).
According to Nguyen et al., the conjugate effectively reduced the viability of P. aeruginosa biofilms by more than 90 and 94% (p < 0.0001) in planktonic phases, respectively, compared to free gentamicin (at 10 mM, only 7 and 5% reduction). 13a Amphiphilic diblock copolymers comprising a hydrophilic PEG block and a hydrophobic block containing enzymecleavable self-immolative side linkages was investigated by Li and associates. 13b After 4 h of penicillin G amidase (PGA) incubation, morphological changes of vesicles from transmission electron microscopy (TEM) analysis revealed the coexistence of PP2 LCVs with spherical nanoparticles, and in  the next 12 h, large compound vesicles (LCVs) were almost wiped out and formed hollow nanostructures, and after 24 h, only spherical nanoparticles remained. The released gentamicin from PGA-responsive large compound vesicle (PP2 LCV) bilayers against P. aeruginosa demonstrated the same growth inhibitory impact as free gentamicin at concentrations greater than 1.0 g/mL.
Self-polymerizable organic biopolymer, polydopamine (PDA), was tethered to gentamicin, kanamycin, and neomycin to obtain PDA−AA nanoconjugates under alkaline conditions (Tris buffer, pH 8.5) at 50°C via Schiff base formation and Michael addition reaction (Figure 7). 14a Out of these three nanoconjugates, PDA−kanamycin was the most potent pathogen active and less toxic in human embryonic kidney cells (HEK293), although more toxic to human glioblastoma cells (U87). Nanoconjugates had antibacterial and anticancer effects greater than those of free drugs, although they were toxic to the cell line.
Ultrathin nanocapsules were prepared successfully by Majumdar 14b upon coating of sodium alginate/polyallylamine hydrochloride (PAH) multilayers and loaded with gentamicin sulfate in the multilayer polyelectrolyte system to achieve a prolonged action of the drug. The average capsule size of the formulation was 400 nm, and the various parameters involved in the creation and optimization of the capsules were used. In capsular suspension, the maximal drug loading was reported to be 25.45% w/v (where 1.0 mL of capsular suspension contains 5000 capsules). In vitro, the total drug release was determined to be 65.91%. With each coating step, the capsule's zetapotential alternates between −19.7 mV (Na-alginate) and +29.8 mV (PAH), indicating multilayered particle development.
3.3. Aminoglycoside Antibiotic-Encapsulated Polymeric Nanogels. Polymer matrices are hydrogels that swell but do not dissolve in water. 15a Nanogels are nanoparticles made up of a hydrogel that has been physically or chemically cross-linked with hydrophilic polymer chains. Due to the tunable chemical and physical structure, good mechanical properties, biocompatibility, and high water content, these hydrogels have been extensively studied in the biomedical field.
Nanogels comprising polycationic chitosan and inorganic polyanion sodium tripolyphosphate was fabricated by Zabihian et al. 15b The nanocarriers were homogeneous in distribution and had a considerable polydispersity index (0.3), with an average size of about 250 nm (unloaded) and 493 nm (gentamicin-loaded). The drug loading efficiency ranged between 28 and 32%, and Gent gradually and continuously released (∼90%) over 24 h. In MIC and potency testing, the antibacterial activity of gentamicin-loaded nanoparticles was not significantly diminished. The gentamicin's rapid burst release from nanogels unravelled the use of PVP as a physical "reinforcing agent".

SILICA NANOPARTICLES FOR AMINOGLYCOSIDE
NANOHYBRIDS In 2008, He and collaborators developed native SiO 2 − gentamicin nanoparticles and gentamicin-loaded SiO 2 nanohybrids. The nanoparticles demonstrated the dose-dependent cell viability assay of SaOS-2 which is reduced both by SiO 2 − gentamicin nanohybrids and native SiO 2 NPs in the cell counting kit-8 (CCK-8) assay. 16a In the case of osteogenesis, the osteogenic differentiating capacity of SaOS-2 cells is not influenced by SiO 2 −gentamicin nanohybrids or native SiO 2 NPs at a concentration range of 31.25−125 μg/mL and the concentrations of 9.65 μg/mL for free gentamicin.
An additional SiO 2 −gentamicin nanohybrid for potential antimicrobial administration in orthopedic applications, developed by Mosselhy et al., 16b exhibited rapid release (21.4%) within the first 24 h and then 43.9% release in vitro in 5 days. This nanohybrid showed the most powerful antimicrobial activity against B. subtilis compared to that against P. fluorescens and E. coli. Filter-sterilized gentamicin has a MIC of 6.26 g/mL against P. fluorescens and E. coli, which is the quantity of gentamicin released from the 250 g/mL SiO 2 − gentamicin nanohybrids. Free gentamicin-treated bacterial cells were completely deteriorated rather than bacterial cells treated with SiO 2 −gentamicin nanohybrids, according to the TEM monographs.
Epoxy groups were introduced on the silica nanoparticles using 3-glycidyloxy propyl trimethoxysilane, followed by functionalization with gentamicin, neomycin, or kanamycin for the synthesis of aminoglycoside-conjugated silica nanoparticles (Figure 8) as described by Agnihotri et al. 16c The attachment was measured by the increase in average size from 160 to 223 nm from native silica nanoparticles to epoxy−silica nanoparticles, which increased further following conjugation  with aminoglycosides such gentamicin (256 nm), neomycin (298 nm), and kanamycin (269 nm). Limited cytotoxicity and antibacterial efficacy of functionalized silica NPs against Grampositive and Gram-negative bacteria as well as kanamycinresistant E. coli strain was found. Compared to native silica NPs, all the AA−silica nanoparticles showed significantly lower MICs but higher than that of free AAs. This may be attributable to the conjugation of aminoglycosides with active silica NPs in which a few primary amines are converted into secondary amines with charge density lower than that of primary amines.

AMINOGLYCOSIDE-COATED METAL NANOPARTICLES
5.1. Aminoglycoside-Coated Iron Nanoparticles. The synthesized magnetic nanoparticles (MNPs) prepared from coprecipitation of Fe 2+ and Fe 3+ iron salts in an alkali medium were coated with chitosan to form CS−MNPs, and subsequently, streptomycin was loaded to produce a strep-CS-MNP nanocomposite. X-ray diffraction was used to characterize CS−MNPs and nanocomposites. Later, the streptomycin-coated chitosan−magnetic (strep−CS−MNP) nanocomposite preparation 17a was modified, and the permanent magnet was used to isolate strep−CS−MNP. Initially, the nanocomposite displayed a rapid release, but it became slower over time and reached 100% after 350 min, following the pseudo-second-order model for this release. The nanocomposite, strep−CS−MNP, exhibited antibacterial activity against methicillin-resistant S. aureus (MRSA). El-Say and El-Sawy highlighted in a separate paper that polymeric NPs, as opposed to metal-based NPs, have several advantages, including low toxicity, biocompatibility, biodegradability, and environmental friendliness. Tobramycin was delivered stably and effectively by binding to alginate that had been functionalized with DNase I before being encapsulated in chitosan NPs. 17b Additionally, the antibacterial activity of the synthesized streptomycin-loaded chitosan-coated magnetic nanocomposites was assessed by El Zowalaty et al. 17c ( Figure  9). Grumezescu et al. 18a prepared a nanocarrier based on CS, poly(vinyl alcohol), and Fe 3 O 4 . Kanamycin was loaded into a water-dispersible metal oxide nanobiocomposite to increase active drug delivery, lowering the MIC of kanamycin by 2-fold (S. aureus) to 4-fold (E. coli) when compared to free kanamycin. The nanobiocomposite had a very minimal hazardous effect on eukaryotic cells in a cytotoxicity test.
Based on the electrostatic interactions of gentamicin with protonated chitosan and PEG (polyethylene glycol), Wang and colleagues 18b studied the preparation of chitosan/Fe 3 O 4 @ poly(ethylene glycol)−gentamicin NPs for drug entry through the bacterial membrane. PEG dicarboxylic acid was employed to improve the dispersity of Fe 3 O 4 NP as it contains adequate carboxyl groups for binding. In an acidic environment, the CS and PEG of Fe 3 O 4 @PEG-Gent were protonated to impart a positive charge to the NP surface, facilitating interaction with the negatively charged bacterial cell membrane and displaying greater antibacterial activity over the free drug. Functionalized magnetic nanoparticles (Fe 3 O 4 @PEG-Gent) are biocompatible with normal cells and effective against planktonic bacteria and biofilms. These nanocomposites could penetrate the mature S. aureus biofilm with the help of a magnetic field due to the superparamagnetic properties of Fe 3 O 4 NPs, leading to the successful delivery of gentamycin for biofilm eradication (Figure 10).

Aminoglycoside-Coated Silver Nanoparticles.
Caglayan and Onur devised a colorimetric silver nanoparticle sensor to determine aminoglycosides in milk. 19a The yellow color of silver transformed into orange and red in proportion to the amounts of analytes. The decrease in absorbance of silver nanoparticles at 394 nm was used to conduct quantitative measurements of AAs in milk. Gentamicin, tobramycin, and amikacin have linear ranges of detection at 20−60, 23−60, and 60−100 ng/mL, respectively. AgNPs were also made by Ghodake et al. 19b for colorimetric detection of aminoglycoside antibiotics in water, serum, and milk samples, with picomolar-level sensitivity to streptomycin. Habash et al. 19c explored tobramycin-loaded tiny citratecoated silver NPs to inhibit the formation of P. aeruginosa biofilm, in which the NPs significantly increased the interaction of tobramycin with the cell membrane and biofilm. The synergistic effect of tobramycin activity was greater for smaller  AgNPs (10−20 nm) at inhibiting biofilms working through cellular membrane disruptions, according to minimum biofilm eradication concentration experiment using clinical P. aeruginosa isolates, and this synergistic effect is likely a strain-dependent phenomenon. Due to the synergism with the aminoglycoside capping agent, the produced silver NPs outperformed those capped with citrate or SDS in antibacterial activity against E. coli and S. aureus, according to Kora and Rastogi. 20a The antibacterial and osteogenic properties of Gent-loaded AgNPs coated with silk fiber (SF) to address Tiimplant-associated infection and poor osseointegration issues were investigated by Zhou et al. 20b In this study, the SF-based film was precomposed using two methods: dip-coating chitosan (DCS) and spin-coating chitosan (SCS) barrier layers. The antibacterial activity of the multilayer coating with the SCS layer was good, whereas the improvement in the DCS coating was restricted. Furthermore, the pH-dependent release behavior of the Ag and the bioactive SCS layer enriched adhesion, migration, and proliferation of preosteoblast MC3T3-E1 cells as well as osteoblast difference. Katva and collaborators 21a presented that the combination of gentamicin and chloramphenicol with AgNPs has a superior antibacterial outcome in multi-drug-resistant Enterococcus faecalis compared to that with free antibiotics. Similarly, McShan et al. 21b noticed the synergistic effect of neomycin with AgNPs with an enhanced antibacterial activity at concentrations below the MIC of either the NPs or the antibiotic and dose-dependent Salmonella typhimurium DT104 growth inhibition is observed for neomycin−AgNPs with IC 50 0.43 μg/mL. 5.3. Aminoglycoside Antibiotic-Coated Gold Nanoparticles. Gold nanoparticles are widely acknowledged as attractive drug delivery possibilities because of their unique dimensions, varying surface functionalities, and regulated drug release. 22 The mixture of gold nanoparticles (AuNPs) and negative citrate ligands capped with polycationic ribostamycin aminoglycoside antibiotics showed unique properties. 22 The interaction between ribostamycin and AuNPs was examined at various doses using a combination of AuNPs of various sizes, and ribostamycin was determined using a dark-field optical microscopic study. Ribostamycin formed linear oligomers at higher concentrations, resulting in the formation of rod-like negative AuNPs. The antibacterial effect of ribostamycin, amikacin, and similar structural antibiotics may be directly related to the efficacy of the drug itself ( Figure 11). Wang et al. 23a synthesized unmodified gold nanoparticles for a sensitive and selective colorimetric biosensor to detect gentamicin, amikacin, and tobramycin antibiotics in milk and pharmaceuticals. Rad and co-workers 23b constructed gold nanoparticles coupled with aminoglycosides with a size of 10 nm to resist multi-drug-resistant, extensive drug resistance, and pan-drug-resistant bacteria. The antibacterial activity of the nanoconjugates of gentamicin and amikacin with gold against Acinetobacter baumannii isolates from burn wound infections was evaluated, and it was found that the conjugated amikacin had strong antibacterial activity (94.5%), whereas gentamicin had 50% efficacy. Payne et al. 23c loaded the kanamycin on the surface of AuNPs, which enabled the delivery of cytosol and bactericidal to S. epidermidis and Enterobacter aerogenes to prevent robust growth of multi-drug-resistant bacteria. The one-step synthesis of capped KS-AuNPs has dose-dependent widespread antibacterial activity against Gram-positive, Gramnegative, and kanamycin-resistant bacteria, though KS-AuNPs have amplified toxicity to primate cell line bacteria (Vero 76). According to transmission electron microscopy and fluorescence microscopy, KS-AuNPs increased their effectiveness by disrupting the bacterial membrane, causing cytoplasmic leakage and cell death. In all of the bacteria tested, the MIC was significantly lower when compared to that of free kanamycin.
Roshmi et al. 24a utilized Bacillus sp. SJ 14 (KJ451478) from the soil of jewelry sites for gold nanoparticle biosynthesis. The antibiotic-coated biogenic gold nanoparticles were tested against S. epidermidis 152 and vancomycin-bound AuNPs against S. hemolyticus 41, indicating that all AuNP-conjugated antibiotics had significantly lower MICs than their free forms, except for rifampicin-bound AuNPs. Mu and colleagues 24b developed gold nanoparticles conjugated with chitosan− streptomycin (CS−SM), which easily passed through biofilm and cell membrane barriers, inhibiting biofilm formation, and eliminating P. aeruginosa preformed biofilm. It can kill both Gram-positive and Gram-negative bacteria (Listeria monocytogenes, S. aureus, S. typhimurium, and E. coli). After RAW264.7 cells were treated with the CS−SM conjugate and L. monocytogenes was visualized with fluorescence microscopy, there was a substantial reduction in the number of beneficial bacteria in a time-dependent manner ( Figure 12).
In another experiment, CS−SM conjugates were produced for bacterial research. CS−SM-1 was formed by reduction of HAuCl 4 with NaBH 4 to form bare AuNPs, which were then mixed with CS, whereas CS−SM-2 was developed by chemical reduction of HAuCl 4 /CS mixtures with sodium borohydride. They show λ max at 531 and 545 nm in their absorption spectra, respectively. According to DLS measurements, the sizes of Figure 11. Interaction of self-assemble ribostamycin and citratecapped AuNPs. CS−SM-1 and CS−SM-2 were found to be 31 and 45 nm, with positive surface potentials of 18.7 and 25.0 mV, respectively. Bacterial TSB solutions (∼10 8 CFU (colonyforming units)) were embedded in 96-well polystyrene microtiter plates to allow biofilm formation. These CS−SM NPs have retained their ability to kill 300 biofilms and prevent Gram-positive bacteria from forming biofilms. In a similar concentration, CS−SM NPs also had better bactericidal effects on both Gram-negative and Gram-positive bacteria when compared to those of the CS−SM conjugate or free streptomycin. 24c,d Per the report of Bhattacharya and colleagues, 25a the conjugation of streptomycin and kanamycin to AuNPs enlarge the particle size of AuNPs, and according to the strength distribution plot, conjugated NPs are polydispersed, whereas the pure one is monodispersed. The MICs for drug conjugated AuNPs were significantly lower in all cases. Antibiotic stability improved as a result of the antibiotics' strong bond with AuNPs, which enhanced the antibiotics' bond energy. Nirmala Grace and Pandian confirmed amine group chelation of aminoglycosides with small AuNPs (15 nm). 25b The development of the gold−drug complex was guided by the replacement of citrate with aminoglycosidic antibiotics. The TEM images indicated nanoparticle aggregation with an average size of 15−20 nm, and the color change and shift in plasmon absorption to longer wavelengths at 670 nm in the UV−vis spectrum also established the same. The antibacterial activities through growth inhibition of S. aureus, Micrococcus luteus, E. coli, and P. aeruginosa with drug-coated gold nanoparticles were higher than those of pure gold nanoparticles. The nanoparticles enhance the antibacterial activity against E. coli.
Ahangari et al. 26a reported that gentamicin was conjugated onto AuNPs according to the method described by Huang. The color change from wine red to purple-blue of gold nanospheres of 10−12 nm diameter confirmed the conjugation. Biodistribution tests on BALB/c mice infected with S. aureus discovered that gentamicin-conjugated AuNPs were well retained in the bacterial infection site. The antibacterial activity of conjugated nanoparticles (0.09375 mg/mL) has more potential than free gentamicin (0.1875 mg/mL). To detect the kanamycin contamination in food, Sharma et al. 26b developed a highly sensitive "turn-off/turn-on" biosensor using the intrinsic peroxidase-like activity of AuNPs. Saha et al. 26c described the direct conjugation of streptomycin, kanamycin, and ampicillin to AuNPs. The TEM analysis revealed that the AuNPs conjugated to antibiotics produce larger particles. In SEM analysis, streptomycin conjugation has a rectangular rod shape, whereas kanamycin is stretched into star-like structures. For AuNP-conjugated streptomycin, the MICs were 7 and 14 μg/mL (50% reduction) and for kanamycin; these were 12 and 30 μg/mL (60% reduction) in E. coli DH5a.
6. QUANTUM-DOT-BASED NANOPARTICLES Li et al. 27 synthesized a polyamine-functionalized carbon quantum dots (CQDs) by applying a hydrothermal treatment of citric acid and branched polyethylenimine with different molecular weights following attachment of gentamicin through carbonization ( Figure 13). The high-resolution transmission electron microscopy revealed well-dispersed and narrow size distribution of particle with sizes ranging from 2.0 to 8.0 nm and a lattice spacing distance of 0.22 nm, which is like graphite (100) facets. The MICs of gentamicin sulfate-derived carbon quantum dots (CQD-Gents) by carbonization at 150°C are almost similar to MIC of gentamicin against S. aureus (0.18 μg/mL) or E. coli (3 μg/mL), though above 190−200°C, loss of antibacterial activity occurred. The toxicity of the synthesizing CQDs is low toward mammalian cells; even at a concentration of 2000 mg mL −1 , more than 91% of cells are viable, which is approximately 40000 times the MIC for S. aureus. After 10 min of treatment with CQD180, the cell morphology becomes irregular, and the bacterial membranes are destroyed in all directions, indicating the CQD180's ability to kill bacteria by disrupting their cell membranes, implying an additional mode of antibacterial action.
As a target to find bacterial biofilm, carbon dot PLGA-based hybrid nanoparticles (CQD-PLGA) as drug delivery systems were investigated by Huang et al. 28a and processed with modulation of CQD content in the formulation applying microfluidic method. The TEM and DLS data indicate the narrow size distribution, and the size and zeta-potential are in the range of 100 to 150 nm and −20 to −50 mV, respectively. The drug loading into the CQD-PLGA was approximately 12− 14%, and the encapsulated tobramycin delivered sustained release for up to ∼72 h. The near-infrared radiation releases 55% of the drug during 7 h. The nanoparticles' good biocompatibility with eukaryotic cells and low cytotoxicity and the chemo-photothermal therapy against bacterial biofilms might be useful in a future application.
The amikacin conjugation with fluorescent carbon dot (CQDs@amikacin) nanoparticles was performed by applying hydrothermal carbonization of amikacin and diammonium hydrogen citrate. The synthesized CQDs@amikacin are uniformly dispersed. The average particle size ranges from 1.5 to 4 nm, and the maximum size of CQDs@amikacin is 2.5 nm. Chandra et al. 28b have observed that there is no diffraction phase in the selected area electron diffraction pattern of CQDs@amikacin, conforming the particles as amorphous, and field-emission SEM images reveal its spherical morphology. The CQDs@amikacin detect E. coli in a linear range of 3.904 × 10 5 to 7.625 × 10 2 CFU/mL, with a detection limit of 552 CFU/mL, and here, amikacin works as a binding ligand toward E. coli. They also detect E. coli from apple juice, orange juice, and pineapple juice. Hence, this fluorescent carbon dot conjugated to amikacin might be used in the future to detect E. coli in other samples.

AMINOGLYCOSIDE-LOADED GRAPHENE
A new method by Pandey et al. 29 for the loading of gentamicin sulfate on a methanol-derived graphene (MDG) nanosheet was synthesized via the wet chemical route. At pH 3, the release of the drug was 62.75% following a diffusion-dominated Figure 13. Gentamicin sulfate-derived carbon quantum dots. release mechanism. The X-ray diffraction analysis resulted in a broad peak at ∼26°, that is, close to the interplanar spacing of the close-packed planes in graphite (0.34 nm) in addition to a new peak arising at ∼11°(0.74 nm spacing). The drug-loaded graphene nanoplatform reduced E. coli's growth, resulting in a viability loss of up to 82.2% compared to that with graphene alone (43.5% viability loss with 40 mg/mL). The gentamicin drug-loaded MDG nanomatrix demonstrated a strong antibacterial effect due to the synergistic effect of the drug and MDG. Furthermore, the controlled release provides a method for creating innovative graphene-based nanohybrids for the treatment of a variety of topical infections. Overall, the synthesis and bioactivities of the aminoglycoside-conjugated nanoparticles are summarized in Table S1.

CONCLUSIONS AND OUTLOOK
In this review article, we highlighted the research and studies on the preparation of nanostructured aminoglycoside antibiotics for future drug therapy. The use of nanoscale drug delivery devices is one of the burgeoning areas of research. Significant efforts continue in nanoparticle-based drug delivery that works via a similar mechanism of action; therefore, the research directions have moved toward the investigation of aminoglycosides to reduce toxicity, increase drug availability, and lower doses. Liposomes, lipids, carbohydrates, gold, silver, silicon, and other novel nanoparticle carriers are likely to play an important role in incorporating kanamycin, neomycin, streptomycin, gentamicin, and amikacin into the nanoparticles. Most of these AA-based nanoparticles are mainly studied in vitro, and a few are studied with in vivo assay; hence, more studies are essential for excellent clinical findings, particularly in terms of the lab to clinic transfer. We anticipate that the nanostructured aminoglycosides demonstrated by researchers around the world might escalate the development of new therapeutics.