• Composition and characteristics of liposomes
Liposomes are considered the most extensively evaluated antimicrobial drug delivery nanosystems. They are characterized by spherical structures made up of phospholipid bilayer(s) surrounding an inner aqueous space, ranging in size from 0.02 to 10 µm [28
]. The efficacy of antibacterial-loaded liposomes in biofilm eradication relies on the physicochemical properties of liposomes that control their stability and in vivo interactions [31
]. Moreover, liposomes are regarded as inclusive carriers for both hydrophilic and hydrophobic therapeutics. Large unilamellar vesicles including a large volume of aqueous phase are the best carrier for hydrosoluble agents, while hydrophobic compounds can be enclosed in the lipid bilayer of multilamellar or small unilamellar vesicles [32
For antibiotic delivery small unilamellar vesicles of ≃100 nm displayed high capability in the eradication of bacterial strains [34
]. Liposomes proved to be useful for the management of topical [35
], vaginal [36
], pulmonary [37
], and ocular [38
] bacterial infections.
• Advantages of antibiotics-loaded liposomes as drug delivery agents:
1- Better protection and enhanced antibiotics biodistribution.
Liposomes improved antibiotics pharmacokinetics and pharmacodynamics in a way where inclusion within the liposomal vesicles controls and sustains drug release, maintaining proper antibiotic level for a long enough time, on the contrary to free antibiotics administration that requires several doses per day thus minimizing patient adherence to therapy [39
]. In addition, encapsulation of antibiotics within liposomal vesicles safeguards antibiotics against the degradative effect of the defense mechanisms of the body, thus preserving their therapeutic response [40
In an attempt to enhance the stabilization of orally administered peptide antibiotics, vancomycin was encapsulated within liposomes containing specific tetra ether lipid. The results of in vivo study on Wistar rats expressed a strong enhancement in the oral bioavailability of vancomycin using the liposomal formulation (4.82 ± 0.56%), where the given oral dose of vancomycin reached the blood after one hour, which is considered a very good achievement for the oral administration of peptide antibiotics [41
]. Further, administrations of either dicloxacillin-loaded liposomes or dicloxacillin-loaded chitosan-coated liposomes were evaluated against MRSA infections. A significantly wider zone of inhibition of dicloxacillin-loaded liposomes compared to free drug and drug-loaded chitosan-coated liposomes (55.0 ± 1.70, 34.3 ± 0.5, 33.0 ± 0.89 mm, respectively) confirmed the better antibacterial activity of small-sized liposomes as well as better drug biodistribution. Nevertheless, testing formulations in vivo on an MRSA infected animal model is recommended [42
2- Selective biofilm targeting affinity.
The surface structure of liposomes specifies the type of interaction with the target bacterial biofilm. For nonspecific interactions, the charge of the liposome membrane plays a vital role. Consequently, the liposomes with positive charge showed the strongest interactions with the negatively charged bacterial biofilms. However, for a specific interaction with the target, liposomes are usually equipped with either proteins, antibodies, specific oligosaccharide chains, or immunoglobulin fragments that express an affinity to certain receptors located on the target biofilm, in addition to the possibility of formulating pH-sensitive or thermo-sensitive liposomes vesicles [31
To improve the gastrointestinal targeting affinity, Wenxi Wang et al. [43
] designed S-layer proteins coated positively charged liposomes. S-layer proteins are crystalline arrays of self-assembled protein located on the surface of bacterial cell, that have the ability to bond to cationic liposomes through their carboxyl groups, and then self-reassemble as a functional coat of liposomes. The authors revealed that coating liposomes with S-layer proteins results in significant improvement of the gastrointestinal adhesion property. Wheat germ agglutinin-conjugated liposomes with surface grafted cyclodextrin were developed to overcome oral infections. Two physicochemical variable drugs (ciprofloxacin and betamethasone) were successfully encapsulated and showed a prolonged co-drug release in saliva over a period of 24 h and a significant increase in oral cell survival against Aggregatibacter actinomycetemcomitans biofilm combined with reduced inflammation [44
Moayad Alhariri et al. [45
] tested the targeting efficiency of neutral and negatively charged gentamicin-loaded liposomes towards P. aeruginosa
) and K. oxytoca
) pathogenic strains. Surprisingly, it was found that anionic liposomes improved drug encapsulation and enhanced the targeting affinity of gentamicin to bacterial biofilm better than either neutral antibiotic-loaded liposomes or free gentamicin. This could be better interpreted based on the increased encapsulation efficiency of the positively charged antibiotic (gentamicin) within negatively charged liposomes based on the electrostatic interaction, followed by improved delivery of antibiotic-loaded negatively charged liposomes through a fusion mechanism that allows the direct injection of liposome-entrapped antibiotic into the cytoplasm of bacteria despite the repulsive forces.
3- Improved selectivity towards intracellular and extracellular bacterial strains.
Utilizing liposomes as drug delivery agents showed tremendous results in eradicating intracellular strains via enhancing antibiotic retention in the infected tissues, providing controlled drug release with minimal toxic effects, and maximizing the concentration at the infected area. For targeting macrophage infections, anti-tubercular drugs loaded within stealth liposomes with small interfering ribonucleic acid (RNA) were fabricated [46
]. The prepared system successfully inhibited the transforming growth factor-β1, eliminating the infection compared to the free drug.
For extracellularly multiplying bacteria, including Pseudomonas aeruginosa
, weakness of inhaled antibiotic for curing P. aeruginosa
infection accompanied with cystic fibrosis was reported due to poor drug permeation, inactivation by sputum, reduced efficacy against the protective biofilm, and shortened lung residence. Bilton et al. [47
] investigated the potential of inhalation suspension of amikacin-loaded liposome (ALIS) and inhalation solution of tobramycin (TIS) in an open-label, randomized, phase III clinical trial. The findings confirmed the hypothesis that ALIS was similar to TIS for curing chronic P. aeruginosa
infection accompanied with cystic fibrosis as shown from the comparable enhancements in forced expiratory volume in 1 s (FEV1%) and reductions in P. aeruginosa
sputum density that were identical in the 2 arms.
• Limitations of antibiotics-loaded liposomes as drug delivery agents:
Despite the significant improvements in antibiotics delivery using liposomes, these lipid vesicles also suffer from many drawbacks limiting their efficient usage.
Physical and chemical instability problems, that can be minimized by addition of antioxidants and/or freeze-drying [28
The possibility of antibiotic leakage from liposomes under physiological conditions, that can be controlled by adding cholesterol which lead to stabilization of liposomal membrane [48
The low loading capacity of liposomes compromises the liposomal usage as antibiotic delivery agent. This challenge can be solved by maximizing electrostatic attractions between liposomes and oppositely charged antibiotic molecules [49
Special sterilization techniques are needed due to the sensitivity of lipids to high temperatures [51
Fabrication techniques are very complex, expensive, and difficult to be scaled up [52
• Classification of liposomes:
Generally, liposomes have been categorized either based on their composition, vesicle size, bilayers number, and/or technique of preparation. In this context, the classification of liposomes according to their design and physicochemical characteristics into conventional, fusogenic, surface-modified, reactive liposomes encapsulating enzyme(s), antibiotic-metal co-encapsulating, liposomes-in-hydrogel, solid-supported liposomes, liposome-loaded scaffolds, and miscellaneous liposomes will be discussed [31
1- Conventional Liposomes
Conventional liposomes are regarded as bare liposomes, lacking any surface modulations. They are made up of phospholipids with or without cholesterol addition. Based on the surface charge of the used lipids, they can be grouped into uncharged, negatively charged, or positively charged liposomes of which positively charged liposomes expressed dramatic improvements in biofilm targeting due to the electrostatic attraction with the anionic biofilm surface. Interestingly, Arikace™®
are two examples of conventional liposome preparations of amikacin and ciprofloxacin, respectively, which are used for cystic fibrotic patients with P. aeruginosa
passed phases II and III of clinical trials and Lipoquin™®
passed a 14-day phase II trial, proving their tolerability, safety, improved biologic activity, and restoration of lung function [53
2- Fusogenic Liposomes
Fusogenic liposomes are well famed as Fluidosomes™. They are distinguished by relatively soft lipid bilayers compared to the rigid conventional liposomes. The presence of special lipid (phosphatidyl ethanol amine) that renders the vesicles more fluid encourage the reduction of the membrane transition temperature and destabilize the lipid packing [55
]. The enhanced anti biofilm activity of tobramycin Fluidosomes™ against many strains such as B. Cepacia (Burkholderia cepacia), S. maltophilia (Stenotrophomonas maltophilia), P. aeruginosa, E. coli (Escherichia coli)
, and S. aureus
at sub-MIC (minimum inhibitory concentration) levels were reported compared to the corresponding free antibiotic [56
]. Furthermore, Beaulac et al. [57
] elucidated the superior in vivo bactericidal activity of tobramycin loaded in the negatively charged Fluidosomes™ agianst P. aeruginosa
To realize the mechanism of fluid liposomes interaction with bacteria, Wang et al. [58
] realized that the bactericidal effect of tobramycin encapsulated fluid liposomes occur fast when bacteria is co-cultured with liposomes as a result of fusion process between liposomes and bacteria rather than the prolonged residence and release of antibiotic. This fusion process is dependent on degree of fluidity, temperature, pH, and presence of divalent cations as well as the properties of bacterial membranes.
3- Surface-Modified Liposomes
The application of surface-decorated liposomes, for example mannosylated liposomes, immune liposomes, and PEGylated liposomes, is among the proposed strategies for designing long-circulating liposomes to surmount biofilm-related infections. However, the strategy still suffers from many optimization challenges. Tatsuhiro Ishida et al. [59
] revealed the loss of long-circulating features of PEGylated liposomes following their intravenous administration to a mice model as evidenced by accelerated blood clearance. Although, polyethylene glycol (PEG) coat prevented this loss to some extent. The observation was attributed to the degree of PEGylation and the amount of lipid. Therefore, further studies will be imperative in order to design effective liposomal preparation suitable for clinical application.
To further examine the effect of the PEG coat on the anti-biofilm activity of liposomes, PEGylated anionic and cationic liposomes were formulated and tested against S. aureus
biofilms. Surprisingly, the results revealed the loss of anti-biofilm activity of liposomes after coating with PEG [60
]. On the other hand, rifampin-loaded cationic liposomes either with PEG coat or without had the same anti-biofilm activity towards to S. epidermidis
]. To explain this, the authors concluded the direct relation between incubation time and the anti-biofilm efficacy.
4- Reactive enzyme(s)-loaded liposomes.
The use of either one or more enzyme(s) loaded within liposomes represents one of the pioneer approaches in the field of anti-biofilm therapy. Moreover, encapsulation of enzymes within liposomes vesicles guarantees their adsorption and stay near to the biofilm surface. The antibacterial activity of reactive-enzyme(s)-loaded liposomes are governed by many factors, such as the enzyme entrapment efficiency, zeta potential, and phospholipid composition of liposomes [62
For example, endolysins enzymes were successfully encapsulated within cationic liposomes. Contrary to free endolysins that have limited activity, only towards Gm +ve strains, and unable to cross the outer membrane of Gm–ve ones, endolysins entrapped within liposomes could successfully cross bacterial membrane and reach their target peptidoglycan substrate, showing significant reduction in logarithmic growth of live cells of S. Typhimurium
and E. coli
Gm–ve biofilms [63
Another approach, which relies on the production of hydrogen peroxide or other oxidizing agents having antimicrobial properties upon contact of enzymes with certain substrate, was discovered. To test this, encapsulation of either a single glucose oxidase (GO) enzyme or coupled glucose oxidase-horse radish peroxidase (GO-HRP) enzymes within DPPC/PI liposomes was performed. The coupling of enzyme with glucose substrate leads to production of hydrogen peroxide, which yields oxy acids that have powerful antibacterial activities against oral Streptococcus gordonii biofilms. In addition, it was concluded that coupled enzymes containing liposomes were more effective than single enzyme formulation [64
Similarly, Jones et al. [65
] encapsulated chloroperoxidase and lactoperoxidase in combination with glucose oxidase enzymes within DPPC/PI liposomes. The reactive liposomes expressed significant antibacterial activity towards Steptococcus gordonii oral biofilm attributed to the reaction of hydrogen peroxide and oxyacids produced with glucose, chloride, or thiocyanate enzyme substrates.
5- Antibiotic-Metal Co-Encapsulating Liposomes
Certain metals, for instance gallium, bismuth, and bismuth-ethanedithiol, have shown promising antibacterial effects. Their activity period comes from affecting iron-metabolism, alginate expression, bacterial adherence, or interference of quorum sensing (QS) signaling and production of virulence factors [66
]. Following this approach, bismuth-ethanedithiol included in a tobramycin-loaded liposome preparation was fabricated by Alhariri and Omri [69
]. At sub-minimum inhibitory concentration (MIC), liposomes-loaded metal tobramycin formulation weakens QS signaling and reduces the production of virulence factors such as lipase, chitinase, and protease, compared to both free tobramycin or tobramycin-loaded liposome preparation. In vivo antimicrobial activity of metal-tobramycin incorporated liposome formulation in rats chronically infected with P. aeruginosa
showed significant count reduction of P. aeruginosa
6- Liposomes-hydrogel system
This policy involves the application of antibacterial-loaded liposomes after being incorporated within a suitable gel base to provide a unique and robust formulation. The hydrogel formulation maintains integrity of the liposomal structure, provides tunable release rate, better bioadhesion, and possibility of surface modification [70
]. For the first time, tetracycline HCl and tretinoin-loaded liposomes prepared by the thin film technique were incorporated in carbopol-based gel. The findings revealed enhanced extended release behavior of both drugs with an average 55% release of two drugs up to 24 h. Antibacterial efficacy of the prepared liposome in gel towards S. aureus
and Streptococcus epidermidis
biofilms has been confirmed. Therefore, it is an effective alternative option for treating Acne vulgaris
Hydrogels also offer capacity for prolonged release of antibiotics for infection control in wounds. Raj Kumar Thapa and colleagues developed collagen mimetic peptide tethered vancomycin-loaded liposomes hybridized to collagen-based hydrogels for the management of MRSA infections. The formulation achieved sustained antibiotic release and enhanced antibacterial efficacy with successful management of wound infection within nine days [72
]. In addition, an injectable, antibacterial, and self-healing multifunctional drug delivery system composed of adhesive liposomes loaded with bone morphogenetic protein 2 (BMP-2) incorporated into PEG hydrogels was successfully developed. The system could be used for the treatment of bone cavity damage and reduces the risk of postoperative infections. The presence of silver ions in the adhesive liposome PEG gel system showed effective inhibition of S. aureus
and E. coli
7- Liposomes supporting solid (SSLs) and liposome-loaded scaffolds (LLSs)
Liposome-supporting solid (SSLs) delivery relies on the loading of antibiotic-liposomes on solid particles surfaces. In this regard, the applicability of gentamicin-liposomes loaded onto particles of the calcium sulfate was tested. In vivo antibacterial study revealed the significant improvement of gentamicin SSLs more than gentamicin-loaded calcium sulfate and non-adsorbed liposomal gentamicin due to better targeting ability to the infection site [74
Targeting bacterial biofilm may be achieved by designing LLSs, whereas antibiotics containing liposomes can be further loaded onto artificial bone scaffolds. To validate this technique, gentamicin-sulfate liposomes have been impregnated onto beta-tri calcium phosphate granules. The in vitro release profile exhibited initial fast release of liposomal gentamicin from the scaffold matrix followed by more prolonged release of the free antibiotic from liposomes. The designed delivery system LLSs displayed significantly elevated anti-biofilm activity compared to free antibiotic [75
8- Miscellaneous liposomes
Miscellaneous liposomes such as biomineral-binding liposomes (BBLs) were proposed for treating device-associated osteomyelitis and for delivering antimicrobials to the skeletal muscles efficiently [76
]. The applicability of liposomes to develop antimicrobial surfaces for construction of efficient medical devices was also explored. Tobramycin-loaded liposomes were immobilized on gold-deposited stainless-steel surfaces and antibacterial efficacy was evaluated against S. epidermidis
) (American type culture collection; ATCC 35984 and ATCC 12228) strains. Antibiotic-liposome coated surfaces were found to possess good antibacterial activity especially for non-biofilm forming strains [77
]. Examples on the recent published studies of different types of liposomal preparations used to eradicate bacterial biofilm infections are illustrated in Table 1
Solid lipid-nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) represent the two main nanoparticles sub-types made up of lipids. SLNs are drug delivery systems in colloid form composed of high melting-stable lipids that were developed to beat the instability problems of liposomes [90
]. Techniques for fabrication of SLNs include; solvent emulsification-diffusion, supercritical fluid, microemulsion-based, and film-ultrasound dispersion method [91
SLNs are characterized by their nanosize range, thus bypassing uptake by reticuloendothelial system; provide high protective effect of incorporated drugs from degradation, offer great targeting, and controlled release opportunity. In addition to their biocompatibility and biodegradability, the possibility of easy scale up may be another advantage. However, their therapeutic application may be hindered by their reduced drug loading potential and possibility of drug ejection during storing [92
In the war against resistant bacterial infections, researchers explored the idea that the bioavailability of many antibacterial drugs were enhanced upon their incorporation within SLNs, such as clarithromycin, rifampicin, tobramycin, and ciprofloxacin [93
], in addition to many formulation patents reporting the oral use of SLNs loaded with anti-tubercular drugs [95
Nanostructured lipid carriers (NLCs) are considered as the advanced type of SLNs. NLCs composed of a rigid matrix blended with a liquid oil to form an unstructured matrix. Unlike SLNs, they form an imperfect core and an amorphous matrix for better drug loading ability and minimized drug escape from the matrix during storing [97
]. In addition to their high loading capacity for both hydrophilic and lipophilic therapeutics is their capability to pass through multiple biological barriers and efficiently deliver the enclosed therapeutic moieties [98
]. Furthermore, NLCs can be fabricated to be stimulated by various parameters such as pH and light for controlling the drug release [99
]. However, the literature on the usage of NLCs for delivering antibacterial drugs is limited (Table 2
). Therefore, there is an imperative requirement to further examine this system for improving antibacterial delivery.
Polymer-based nanosystems used against antibacterial resistance can be categorized into polymers that have themselves antibacterial properties and polymeric nanoparticles acting as antibiotic delivery systems [7
]. Polymer nanoparticles are among the class of organic macromolecule-based antibacterial drug carriers that have many advantages such as ease of fabrication, physical and chemical stability in physiological environment and under storage conditions, easily controllable physicochemical properties, and prolonged drug release with better targeting efficiency [123
Chitosan was proved to be the most efficient and versatile polymeric material from a natural source utilized for preparing antibacterial loaded drug carriers. The antibacterial activities of chitosan are dependent on numerous factors such as chitosan deacetylation degree and molecular weight, as well as the chemical structure and functionalization of chitosan molecules [124
]. There are variable mechanisms that illustrate the antibacterial activity of chitosan, most commonly depending on the electrostatic attraction between chitosan and anionic surface of bacteria that lead to changing the permeability of cell membrane. Leaking out of the bacterial components results in cell death [125
]. Examples of recent innovative studies done on antibiotic loaded chitosan nanoparticles are shown in Table 4
Other examples of natural polymers include dextran sulfate and chondroitin sulfate polysaccharides. Nanoparticles of either chondroitin sulfate or dextran sulfate were formulated with high encapsulation efficiency around 65% and size ranged from 100 to 200 nm. The results indicated that macrophages intracellular uptake of the antibiotic by using antibiotic-loaded dextran sulfate nanoparticles was 4-fold that of the antibiotic-loaded chondroitin sulfate nanoparticles. Further, enhanced anti-microbial activity against intracellular salmonella infections was confirmed [126
The main merits of natural polymers are being highly biodegradable and biocompatible. On the other hand, aforementioned merits may be originated in synthetic polymers too, for example PLGA (poly (lactic-co-glycolic acid) or PCL) (poly (ε
-caprolactone)). PLGA is a commonly used synthetic polymer. Recently, it has been utilized as basic excipient for antibacterial polymeric nanoparticles production [127
]. Clarithromycin antibiotic was successfully encapsulated within PLGA particles. The enhanced antibacterial efficacy against H. pylori
strains was evidenced from lower MIC values compared to free antibiotic. Although the mechanism remains unclear, it was postulated that PLGA-loaded nanoparticles could carry out either fusion or adsorption [128
]. PCL can be utilized for producing good antimicrobial drug delivery nanosystems because of its biocompatibility and biodegradability [129
]. For instance, a significant improvement of anti-tubercular rifampicin uptake into macrophages was observed after its encapsulation within PCL nanoparticles compared to free drug, thus improving its antibacterial efficiency towards M. tuberculosis
]. Recent literature on the application of polymeric nanoparticles against bacterial infections is described in Table 4