http://informahealthcare.com/drd ISSN: 1071-7544 (print), 1521-0464 (electronic) Drug Deliv, Early Online: 1–9 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2013.863409

CRITICAL REVIEW

Nanoliposome-based antibacterial drug delivery Somayeh Hallaj-Nezhadi1 and Maryam Hassan2 Immunology Research Center & Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran and 2Faculty of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran

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1

Abstract

Keywords

Although most bacterial infectious diseases can be treated successfully with the remarkable array of antibiotics, the microbial pathogens continue to be one of the most critical health challenges worldwide. One of the common efforts in addressing this issue lies in improving the existing antibacterial delivery systems since inefficient delivery can lead to poor therapeutic outcome of the administered drug. Recently, nanoliposomal systems have been widely used as promising strategies to overcome these challenges due to their unique set of properties. This article tries to briefly summarize the current studies that have taken advantage of liposomal nanoparticles as carriers to deliver antibacterial agents. The reviewed investigations demonstrate the immense potential of liposomal nanoparticles as carriers for antibiotic delivery and highlight the latent promise in this class of vehicles for treatment of bacterial infections. The future of these promising approaches lies in the development of more efficient techniques for preparing liposomal nanoparticles with great potential in effective and selective targeting of antibiotics to bacterial cells for eradication as well as the highest safety for human host.

Antibiotic, antimicrobial agents, drug delivery, liposome, nanoparticle History Received 9 October 2013 Revised 2 November 2013 Accepted 4 November 2013

Introduction

The privileges of nanoparticles as drug carriers

The use of antibiotics as powerful tools to combat bacterial infections has saved many lives and rescued the world from the fatal infectious diseases since antimicrobial agents were introduced. The word ‘‘antibiotics’’ was first used in 1941 by Selman Waksman to describe antimicrobial agents produced by microorganisms (Clardy et al., 2009). Though most bacterial infectious diseases can be treated successfully with the notable range of antibiotics, the microbial pathogens still have the capacity to be one of the world’s most critical public health threats. Indeed, development of bacterial resistance is one of the biggest challenges that extremely restrict the benefits of antibiotics in controlling infections. In addition to the introduction of new effective antibiotics (Hassan et al., 2012a,b), improving the existing antibacterial delivery systems can be practical for addressing this issue since inefficient delivery can cause unsatisfactory therapeutic effects of the administered drug. One of the recent strategies to overcome these challenges is development of nanostructured liposomal systems as novel and encouraging approaches to deliver antibiotics. In this article, after a brief look at the benefits of particles in the nanometer scale as well as liposomes as drug carriers, we will provide a review to update the reader on the recent state of liposomal nanoparticles for antibacterial drug delivery.

Encyclopedia of Pharmaceutical Technology defines ‘‘nanoparticles’’ as solid colloidal particles ranging in size from 1 to 1000 nm (1 micron) (Hallaj-Nezhadi et al., 2013b). In fact, nanoparticles have many benefits for pharmaceutical purposes: They are able to cross blood–brain barrier, interact with and cross mucosal surfaces, cause deeper tissue penetrability, and escape endo-lysosomal compartments (HallajNezhadi et al., 2013a). Also, nanoparticles possess the ability of targeting the drug into the site of action and therefore reducing side effects, controlling the drug release at the active site and enhancing the drug uptake (Nahaei et al., 2013). In addition, nanoparticles can be utilized to modify the kinetic profiles of drug release (Barzegar-Jalali et al., 2008; Faraji & Wipf, 2009). According to the Ostwald–Freundlich equation, the saturation solubility enhances with decreasing particle size below approximately 1 mm. Hence, nanoparticles show improved saturation solubility in addition to increased surface area which leads to a further increase in dissolution rate based on the Noyes–Whitney equation. On the contrary, the solubility of normally sized (above 1 micron) particles is a compound-specific constant which depends only on the temperature and the solvent (Muller & Keck, 2004; HallajNezhadi et al., 2010). Furthermore, the stability of the drugs can be improved by encapsulating the pharmaceutical agents inside nanoscale vehicles. This may provides an opportunity to reconsider potential drugs ignored in the past due to their poor pharmacokinetics (Alexis et al., 2010).

Address for correspondence: Somayeh Hallaj-Nezhadi, Immunology Research Center & Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. Email: [email protected]

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S. Hallaj-Nezhadi & M. Hassan

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The advantages of liposomes as drug carriers One of the most promising strategies for antimicrobial drug delivery is to encapsulate the antimicrobial agents into lipid constructs called liposomes. They were first introduced by Bangham in 1965 and are of the most widely used carriers for antimicrobial drug delivery due to their unique properties (Coune, 1988; Swenson et al., 1988; BakkerWoudenberg & Lokerse, 1990). Liposomes are tiny spherical structures composed of amphipathic lipids arranged in one or more concentric bilayers with aqueous phase inside and between the lipid bilayers (Figure 1). The lipids commonly used in preparation of liposomes are found in the human body and are mostly from commercially available lipid molecules. These lipids are also generally approved by the FDA, for instance, EggPG (egg yolk phosphatidylglycerol) and DSPC (1,2-distearoyl-glycero-3-phosphocholine) (Alexis et al., 2010). Liposome-based delivery systems have demonstrated several benefits: They are able to carry both lipophilic and hydrophilic drugs (in lipid bilayers and aqueous compartments, respectively) and protect them from degradation. Liposomes comprised of natural lipids are biocompatible, biodegradable and less immunogenic. They are also low toxic due to change in the pharmacokinetic and biodistribution, and increased therapeutic index (Goyal et al., 2005; Mugabe et al., 2006a). Liposomes have numerous potential pharmaceutical applications and can be safely administered by different routes of administration (Pierre & Dos Santos Miranda Costa, 2011). They can be also utilized in dermatology as a local drug depot in combination with penetration enhancers (de Leeuw et al., 2009; Pierre & Dos Santos Miranda Costa, 2011; Puglia & Bonina, 2012; Rahimpour & Hamishehkar, 2012). Furthermore, as antimicrobial drug delivery carriers, liposomes are able to enhance antibiotic concentrations at the site of infection (passive or active targeting), increase bactericidal efficacy (via fusion with the bacterial membrane), improve drug uptake and decrease the toxicity of potentially toxic antimicrobial agent (Fresta et al., 1995; Sachetelli et al., 2000; Mugabe et al., 2006b; Halwani et al., 2008, Drulis-Kawa et al., 2009). Targeted liposomal drug delivery can be achieved by either passive or active methods. Passive targeting is based on the accumulation of drug or drug delivery vectors at a special site owing to pharmacological factors or physicochemical properties (size or molecular weight) (Vasir et al., 2005). Active targeting is achieved by changes in liposome structure through various kinds of targeting moieties, e.g. monoclonal

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antibodies (MAb), fragments, peptides, growth factors, glycoproteins or carbohydrates (Immordino et al., 2006). The conventional liposomes that consist of natural phospholipids and cholesterol possess very high systemic plasma clearance and are rapidly captured by mononuclear phagocytic system (MPS) after intravenous administration (passive targeting) (Pinto-Alphandary et al., 2000). In general, larger liposomes will be more quickly taken up by the MPS. Hence, the size of liposomes is a main factor in their disposition in the body (Ishida et al., 2002). Furthermore, the surface of the conventional liposomes can be modified using hydrophilic polymers to avoid MPS uptake and increase the systemic circulation time of the liposomes. Polyethylene glycol (PEG) as the most widely employed polymer, provides a steric barrier against various interactions at the surface of liposomes with other components in the biological environment, i.e. decreases the recognition by MPS and inhibits the penetration of plasma proteins that could destabilize the liposomes (Toh & Chiu, 2013). Active targeting of drug carriers has also been studied for liposomes containing antibiotics; one example is rifampicin loaded liposomes containing ligands [maleylated bovine serum albumin (MBSA) and O-steroyl amylopectin (O-SAP) as the specific ligands for alveolar macrophages] for direct targeting of an aerosol formulation to the infected alveolar macrophagess (Pinheiro et al., 2011). Another example includes the delivery of rifampicin and isoniazid using liposomes containing an active targeting ligand O-SAP (Pinheiro et al., 2011). Despite all the advantages of liposomal drug delivery systems, they are known to possess some limitations such as stability, reproducibility, encapsulation efficiency, size distribution, sterilization, short half-life, as well as leakage and fusion of encapsulated drugs (Toh & Chiu, 2013). Overall, considering the privileges of nanoscale carriers and liposomes, development of liposomal nanoformulations for antibacterial drug delivery seems to be promising. To date, antibiotic delivery via liposomal nanocarriers as one of the most encouraging strategies has been accomplished through different preparation methods and various lipids. In this article, in detail, we summarize the recent studies that have taken advantage of liposomal nanoparticles as carriers to deliver antibiotics in the following section.

Liposomal nanoparticles as carriers for delivery of antibacterial agents Liposomal nanoparticles larger than 100 nm

Figure 1. Basic liposome structure for drug delivery.

Mugabe et al. (2005) developed nanoliposomal gentamicin formulation and evaluated the in vitro stability and antibacterial activity against resistant strains of P. aeruginosa. Different lipid compositions (1,2-dimyristoyl-sn-glycero3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC) and cholesterol (Chol) were used to encapsulate gentamicin into liposomes via a sonication method. The average particle size of liposome-encapsulated gentamicin with different lipid compositions was found to be between 408  28 and 418  21 nm, with the polydispersity

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DOI: 10.3109/10717544.2013.863409

index ranging from 0.59  0.009 to 0.74  0.007 and encapsulation efficiency of 4–5%. All liposomal formulations released 40% of the encapsulated drug in 48 h in normal human pooled plasma at 37  C with no significant difference in the release kinetics of gentamicin in relation to different liposomal formulations. The antibacterial effect of the obtained formulations was studied on non-mucoid and mucoid strains of Pseudomonas aeruginosa isolated from sputum of pulmonary infected cystic fibrosis patients. The minimum inhibitory concentrations (MICs) of all nanoliposomal gentamicin for two highly gentamicin-resistant mucoid and non-mucoid clinical strains of P. aeruginosa were reported to be significantly lower than that of free gentamicin (1–2 versus 256–512 mg/L). The prepared nanoliposomal formulations were able to significantly enhance the susceptibilities of the clinical strains of P. aeruginosa which were considered resistant to gentamicin (MIC416 mg/L). The nanoliposomal formulations increased the susceptibility of the used strains to gentamicin, from highly resistant to either intermediate (MIC58 mg/L) or susceptible (MIC54 mg/L). This investigation is considered as the first report on nanoliposomal formulations enhancing gentamicin antibacterial activity against gentamicin-resistant clinical strains of P. aeruginosa; however, the related mechanism was not studied. The same research group subsequently focused on the preparation method for liposomes with high yield entrapment of aminoglycoside and macrolide antibiotics along with favorable stability in both storage and physiological conditions (Mugabe et al., 2006a). They prepared liposomes via a modified dehydration–rehydration vesicles method (using freeze-drying) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and Chol. This method resulted in liposomes with smaller particle size (163.37–259.83 nm) with no significant difference regarding the type of the encapsulated drug. Interestingly, nanoliposomes produced in this study were found to be smaller than those reported in the author’s previous study (Mugabe et al., 2005; 408  28 to 418  21 nm; p50.05). They stated that this was as a result of the preparation protocols, i.e. sonication versus freeze/dry methods; in the previous study, the sonication method (Mugabe et al., 2005) produces a mixture of small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs); while in this study, the freeze/dry method removes the large vesicles and leads to a uniform suspension of SUVs; in addition, sucrose was used to prevent vesicle fusion during freezedrying and dehydration processes. This modified dehydration–rehydration method, also highly improved encapsulation efficiency of the used antibiotics (e.g. 32.06  0.82% for erythromycin and 33  0.76% for gentamicin) compared to encapsulation efficiency (4–5%) of liposomes prepared using sonication method in the investigator’s previous study (Mugabe et al., 2005). The authors related this to the fact that during the dehydration process, hydrophobic forces decrease and result in intimate interaction of the SUVs with the drugs. Following the controlled rehydration process, this brings about the entrapment of relatively large amounts of the drugs in the aqueous compartments of the liposomes. In contrast, in the sonication method, such intimate interactions are not available and therefore, small portion of

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aqueous phase are encapsulated into anhydrous lipid phase. Furthermore, the prepared nanoliposomes were reported to be stable regardless of the experimental temperature (4 or 37  C) and retained more than 75 and 95% of the initially encapsulated drugs for 48 h at 37  C and 4  C, respectively. Also, no significant alteration in the liposomes’ size was detected during or at the end of the stability study. The authors concluded that the introduced preparation method can be used to overcome the low encapsulation efficiency of aminoglycoside and macrolide antibiotics in liposomes. In an effort to overcome Burkholderia cenocepacia’s resistance to commonly used aminoglycosidic antibiotics, the same researchers developed a nanoliposomal antibiotic formulation composed of DSPC and Chol (molar ratio 2:1) (Halwani et al., 2007). The obtained nanoliposomes exhibited encapsulation efficiencies of 52.08  5.4%, 27.72  1.14% and 28.08  1.54%, for amikacin, gentamicin and tobramycin, respectively. The formulations demonstrated significantly lower MICs against Burkholderia compared to the free drugs. For example, the MIC for liposomal tobramycin against B. cenocepacia M13643 was reported to be 16 mg/L in comparison with that of 512 mg/L for the free drug. They also investigated the mechanism of action of the prepared liposomes by three different methods, Transmission electron microscopy (TEM), fluorescence-activated cell sorter (FACS) analysis and lipid-mixing assay. The results together indicated the lipid contact of the liposomal bilayers and bacterial cell membranes. Finally, the authors mentioned that the prepared nanoliposomes with decreased MICs for highly antibioticresistant strains are able to overcome bacterial membrane impermeability via fusing with membranes and exposing bacterial ribosomes to aminoglycoside antibiotics. Rukholm et al. (2006) compared the bactericidal effect of liposomal gentamicin with that of free antibiotic against clinical isolates of P. aeruginosa. The liposomes prepared using DMPC and Chol [2:1] were 426.25  13.56 nm in size and showed encapsulation efficiency of 4.51  0.54%. The highly gentamicin-resistant clinical isolate displayed the most remarkable difference between the two forms of gentamicin with the MIC of 512 mg/L for free form compared to 32 mg/L for liposomal form of the antibiotic. The investigators attributed this considerable difference between the MICs to the possible fusion of the liposomes with the outer membrane of P. aeruginosa, and the consequent increase in the penetration of the antibiotic. The in vitro time-kill values for liposomal gentamicin at 1, 2 or 4 times the MICs were reported to be either equivalent to or better than those of the corresponding free antibiotic. For instance, the investigators observed that the liposomal gentamicin at 4  MIC showed an improved killing time by achieving complete bacterial eradication at 6 h compared with 24 h for free gentamicin. The authors concluded that the prepared nanoliposomal gentamicin formulation possessed superior antibacterial effect against P. aeruginosa with an improved killing time and prolonged antimicrobial activity. Drulis-Kawa et al. (2006) studied the antibacterial activity of different formulations of cationic and anioionic liposomal meropenem on six drug-susceptible and two drugresistant P. aeruginosa strains. Various liposomal formulations were obtained via thin lipid film method using different

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lipids (e.g. 1,2-dioleoyloxy-3-trimethylammonium-propane (DOTAP), phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and octadecylamine (SA). The particles were in a size range of 107–152 nm and the encapsulation efficiency was reported to be between 3.7 and 7.2%. Among the cationic liposomes, the most efficient liposomal formulations against sensitive isolates were fluid liposomes prepared using PC/DOPE/SA [4:4:2] and PC/DOTAP/Chol [5:2:3], with the MICs 2–4 times lower than those of the free drug. In addition, anionic formulations exhibited lower antibacterial activity compared to cationic ones, due to the equal or higher MICs relative to the free drug. Moreover, no correlation was obtained between the zeta potentials of the cationic liposomes and the relative antimicrobial activities. Also, based on the killing curves of the P. aeruginosa (ATCC 27853 strain) exposed to free and encapsulated meropenem in a sub-MIC concentration (0.125 mg/ml), liposomal meropenem demonstrated significant inhibition of bacterial growth (p50.05) after 18 h of incubation relative to the control growth and free meropenem. This could be attributed to the effect of the liposomes on the interaction between bacteria and meropenem. Unfortunately, none of the tested lipid formulations indicated antimicrobial activities against isolates which are drug-resistant due to the low permeability. The capability of a new strategy for liposomes as a drug carrier by delivering two agents at the same time to prevent biofilm formation and bacterial resistance was studied by Halwani et al. (2008). They used liposomal formulation (Lipo-Ga-GEN) containing gentamicin and gallium metal (Ga) to improve gentamicin efficacy against clinical isolates of P. aeruginosa. Gallium has been shown to have an effective inhibitory effect against P. aeruginosa growth and biofilm formation. The experiments showed a notable difference between the MICs and MBCs of Lipo-Ga-GEN and free gentamicin for the antibiotic-resistant P. aeruginosa PA-48913 strain (2–4 versus 256 –4512 mg/L gentamicin). Also, in order to compare the minimum biofilm eradication concentration of the conventional drugs with that of LipoGa-GEN, P. aeruginosa biofilms were formed and studied. Quorum sensing (QS) molecules such as N-acyl homoserine lactone is considered as one of the communication tools for biofilm formation. On the basis of this study, Lipo-Ga-GEN was the only formulation that was able to completely eradicate biofilms and block QS molecules at a very low concentration (0.94 mg/L gentamicin). In addition, with due attention to the limitation of the clinical usage of Ga, its toxicity profile was studied by Trypan Blue assay on cultured human lung epithelial cells (A549). The authors stated that the Ga toxicity profile completely altered when liposomes were used and cell viability increased relative to free Ga. Finally, this new liposomal strategy (Lipo-Ga-GEN) with 337  35 nm size and ability of optimizing gentamicin and reducing the Ga toxicity could be considered as a promising approach for eradication of antibiotic-resistant P. aeruginosa isolates growing in a planktonic or biofilm community. Another application of nanoliposomes in antibiotic delivery was examined by Changsan et al. (2009) who investigated liposomes encapsulating rifampicin for delivery to the respiratory tract. The chloroform-film method was used to

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prepare the liposome suspensions containing different ratios of Chol and soybean L-a-phosphatidylcholine (SPC). Subsequently, liposome dry powders were prepared from liposome suspensions and their properties were examined. The liposomal particles were found to be between 200 and 300 nm. Fifty percent of rifampicin was encapsulated when the lipid content was high. Based on the Cryo-transmission electron microscope image of a liposome suspension, it consisted of a mixture of unilamellar and (predominantly) multilamellar vesicles. Vesicles containing higher Chol content exhibited better physical size stability (for 4 weeks). In contrast, liposomes containing lower Chol content aggregated following 2 weeks storage. The morphology of the resulting liposomes was investigated under cryo-TEM subsequent to the reconstitution of freeze-dried liposome powder (with mannitol as a cryoprotectant). Most vesicles were unilamellar in shape and larger than 500 nm in size. The authors mentioned that the increase in liposome size may occur in order to reduce the vesicle curvature, since the larger particles have less surface free energy and are more stable than smaller ones. Moreover, encapsulation efficiency of the liposomes decreased to 20–22% compared to 55% prior to freeze drying. These results may be explained by the change in vesicle size after reconstitution compared to the vesicles before drying; as the fusion process involves adhesion of the vesicles which may lead to leakage of the entrapped drug from the vesicles. They also found that rifampicin in dry powder formulations was noticeably more stable relative to both forms of aqueous solutions and liposomal suspensions of the drug (after being kept at 48  C for 6 weeks). On the whole, the formulated liposome suspension is suitable for dry powder development as it gave a high encapsulation efficacy (&50%) and improved stability. In addition, a crystalline powder with good aerosol characteristics was obtained. Liposomal nanoparticles smaller than 100 nm The feasibility of use of liposomes as carriers for delivery of lauric acid was examined by Yang et al. (2009). Propionibacterium acnes (P. acnes) is a bacterium that triggers inflammatory acne. Lauric acid demonstrated the strongest bactericidal activity against this bacterium in vitro relative to the other two examined free fatty acids: oleic acid and palmitic acid. Liposomes incorporating lauric acid (LipoLA) with a diameter of 100 nm prepared via vesicle extrusion technique using hydrogenated L-a-phosphatidylcholine (egg PC) and Chol. The obtained LipoLAs exhibited high drug encapsulation efficiency, 50–80%. LipoLA with 102 mg/mL LA was able to completely kill P. acnes confirming the maintenance of the antimicrobial activity of lauric acid after loading into liposomes. The researchers then addressed the interaction mechanism between LipoLA and the bacteria via labeling LipoLA with a FRET pair of chromophores and monitoring the FRET signal changes upon mixing LipoLA with the bacteria. They found that the LipoLA fused with the membranes of the bacteria and released the drug directly into the bacterial membranes. Overall, the developed liposomal nanoparticles offer innate, safe and efficient therapeutic medication for acne vulgaris and other P. acnes associated diseases.

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DOI: 10.3109/10717544.2013.863409

The same group in another investigation studied the antimicrobial activity of liposomal derivatives of oleic acid as innate bactericides against methicillin-resistant S. aureus (MRSA; Huang et al., 2011). In vitro studies showed that the oleic acid-loaded liposomes (LipoOA) with diameter of 80.6 nm and drug encapsulation efficiency of approximately 15% could decrease MBC value 12-fold compared to free oleic acid (with MBC value of 10 mg/mL) against MRSA. Besides, based on the preliminary skin toxicity study, the researchers observed no apparent epidermal cell apoptosis in the skin treated with LipoOA, proving good biocompatibility of LipoOA with mouse skin. The researchers also investigated the in vivo therapeutic efficacy of LipoOA against MRSA252 infections in ICR mouse skin after intradermal injection of MRSA followed by LipoOA injection into the dorsal skin of the mice. They observed no lesion in the mice treated with LipoOA, while lesion started to appear after 24 h on the back of the mice treated with bare liposomes as the negative control showing the proliferation of the infection. Also, for this group, epidermal rupture was reported after 48 h of MRSA252 injection based on the histology analysis of the skin. In contrast, for the skin of the LipoOAtreated mice intact epidermis structure was observed. In addition, for the LipoOA-treated mice, the amount of MRSA252 remaining in the skin was about 500 times lower compared to that in bare liposome-treated mice (p50.001). These results offer a new, effective and safe antimicrobial agent for the treatment of MRSA infections using liposomal nanoparticles. The same research group also prepared liposomal nanoformulation of linolenic acid (LipoLLA) and investigated its bactericidal activity against resistant strains of Helicobacter pylori (H. pylori; Obonyo et al., 2012). The liposomes were prepared via sonication and needle extrusion method using egg PC and Chol and were shown to have mean diameter of 88  3 nm and, surface zeta potential of 78  4 mV in deionized water. The authors observed that LipoLLA possessed an antibacterial efficacy comparable with free LLA in inhibiting both spiral and coccoid forms of H. pylori. Furthermore, LipoLLA was able to eradicate a metronidazole-resistant strain of H. pylori and seven clinically isolated strains regardless of their antibiotic resistance status. Drug resistance was not reported with the prepared LipoLLA at different sub-bactericidal concentrations under the experimental conditions, while a rapid drug resistance was observed with both metronidazole and free linolenic acid. Overall, this novel formulation can be considered as a promising antibacterial nanotherapeutic for treatment of antibiotic-resistant H. pylori infections. In one study reported by Gharib et al. (2012a), ticarcillinloaded nanoliposomes with positive, negative and neutral surface charges were prepared by extrusion method using egg lecithin and cholesterol. Stearylamine and dicetylphosphate were added to make cationic and anionic nanoliposomes, respectively. The encapsulation efficacies for antibioticloaded cationic nanoliposomes were found to be significantly higher (76%) than those of neutral (55%) and anionic (43%) nanoliposomes. The MIC values of ticarcillin against P. aeruginosa for free form (24) as well as cationic (3) and neutral (6) nanoliposomes were lower than anionic (48)

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formulation. On the basis of time kill studies, the killing rates of cationic nanoliposomes were reported to be higher compared to the other drug forms. Furthermore, the results obtained following in vivo study showed 100, 60 and 20% survival rates for ticarcillin-loaded nanoliposomes with positive, neutral and negative surface charges, respectively. Also, ticarcillin-loaded cationic nanoliposomes led to almost complete eradication of the bacteria from the spleens, livers and skins of infected animals which may be caused by the interaction between the cell membranes and optimal drug delivery. In another recent study, therapeutic efficacy of nanoliposomes encapsulating Epigallocatechin gallate (EGCG) against S. aureus was investigated by the same research group using the similar preparation process (Gharib et al., 2012b). The authors observed that the maximum entrapment efficacy and the slowest drug release rate are achievable using cationic nanoliposomal form. Cationic nanoliposomes also exhibited the highest killing rates relative to the free form, as well as neutral and anionic nanoliposomes. This indicates the considerable influence of formulation change in encapsulation efficacy and performance of the liposomes. The in vivo therapeutic efficacy of the formulations was also studied in burned mouse skin infected by MRSA. The experiments demonstrated 100, 70 and 30% survival rates, with positive, neutral and negative EGCG-loaded nanoliposomes, respectively. These findings suggest that the cationic EGCG-loaded nanoliposomes would be applicable for the treatment of MRSA infections. Li et al. (2013) recently described the use of flexible nanoliposomes for topical delivery of daptomycin (DAP-FL). They prepared these nanoliposomes via a dry film dispersion method using soy phosphatidylcholine and sodium cholate. The liposomal formulations indicated a small mean particle size (55.4 nm) with mean entrapment efficiency of 87.85  2.15%. Based on the in vitro skin permeation study using modified Franz diffusion cell, DAP-FL was able to diffuse rapidly and efficiently into the skin and could also lead to a significant inhibition in Staphylococcus aureus growth in a simulated receptor compartment. Moreover, in vivo studies showed that DAP-FL was able to permeate the skin and underlying structures, reach a maximum concentration rapidly and maintain high drug levels for several hours. In addition, the pharmacodynamic study in vivo revealed a non-significant difference in cell numbers of S. aureus (p40.05) between transdermal delivery of DAP-FL and the intravenous injection of daptomycin solution (as a positive control group). It is envisaged that this novel nanoliposomal formulation would be useful as a new approach in the clinical application of daptomycin. Ma et al. (2013) prepared the fluid liposomal encapsulated tobramycin using DPPC, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol sodium salt (DMPG) and/or DOPE via a dehydration–rehydration vesicle method. The antibacterial effect of the liposomes against P. aeruginosa exposed to subMIC of encapsulated tobramycin was evaluated. The fusion potential of liposomes containing DOPE (as a fusogenic lipid) enhanced to 20% under experimental conditions. Also, the MIC of liposome encapsulated tobramycin reduced while it was proportional to the degree of fusion of the liposomes with

Antibacterial agent

Gentamicin

Aminoglycoside and macrolide antibiotics

Aminoglycosides

Gentamicin

Meropenem

Gentamicin and gallium metal

Rifampicin

Lauric acid

Oleic acid

Delivery system

DMPC, DPPC, DSPC and Chol

DPPC and Chol

DSPC and Chol

DMPC and Chol

PC/DOPE/SA and PC/DOTAP/Chol

DPPC and DMPG

SPC and Chol

Egg PC and Chol

Egg PC and Chol

Activity

Particle size (nm)

Method

Reduced MICs for highly antibiotic-resistant strains, enhance the antibiotics’ penetration into the bacterial cells, stable liposomes with high yield entrapment of drug. Reduced MICs for highly gentamicin-resistant clinical isolate, improved killing time and prolonged antimicrobial activity. Decrease in MICs and significant inhibition of bacterial growth, cationic liposomes showed higher antibacterial activity compared to anionic ones. Stable liposomes delivering two agents together, decrease in MICs and MBCs, complete eradication of biofilms at a very low concentration, reduction in the Ga toxicity. Stable liposome, high encapsulation efficiency. Enhanced antimicrobial activity, fusion of LipoLA with the bacteria membrane. 12-Fold decrease in MBC value, good biocompatibility of liposomes, highly effective in curing skin infections and preserving the integrity of the infected skin. Thin lipid film method

Dehydration–rehydration technique

107–152

337  35

80.6

100

Extrusion method

The chloroform-film method Vesicle extrusion technique

Sonication

426.25  13.56

200–300

Dehydration– rehydration vesicles technique

In the 200 nm range

Mugabe et al. (2005)

References

15

50–80

50



3.7–7.2

4.51  0.54

Huang et al. (2011)

Yang et al. (2009)

Changsan et al. (2009)

Halwani et al. (2008)

Drulis-Kawa et al. (2006)

Rukholm et al. (2006)

Amikacin: 29.27  1.2, Mugabe et al. (2006a) gentamicin: 33  0.8, tobramycin: 22.33  1.5, erythromycin: 32.06  0.8 Halwani et al. (2007) Amikacin: 52.08 þ 5.4, gentamicin: 27.72 þ 1.1, tobramycin: 28.08 þ 1.5

4–5

EEb %

S. Hallaj-Nezhadi & M. Hassan

Methicillin-resistant Staphylococcus aureus (MRSA)

Propionibacterium acnes



Pseudomonas aeruginosa

Pseudomonas aeruginosa

Pseudomonas aeruginosa

Burkholderia cenocepacia

408  28 to Sonication method Highly gentamicin-resistant Significant decrease in MIC. 418  21 mucoid and non-mucoid clinical strains of Pseudomonas aeruginosa – Stable liposomes with high 163.37  38.4 Modified dehydration– yield drug entrapment. to 259.83  11.8 rehydration vesicles method

Targeted MOa

Table 1. Liposomal nanoparticles to deliver antibacterial agents.

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6 Drug Deliv, Early Online: 1–9

Doxycycline

Egg PC

b

MO, Microorganism. EE, Encapsulation Efficiency.

a

Staphylococcus aureus

Daptomycin

SPC and sodium cholate

Helicobacter pylori

Methicillin-resistant Staphylococcus aureus

Epigallocatechin Egg lecithin, Chol, gallate (Stearylamine for cationic and dicetylphosphate for anionic liposomes,)

Helicobacter pylori

Pseudomonas aeruginosa

Linolenic acid

Ticarcillin Egg lecithin, Chol, (Stearylamine for cationic and dicetylphosphate for anionic liposomes,)

Egg PC and Chol

88  3 Killing both spiral and coccoid forms of the bacteria via disrupting bacterial membranes, eradication of all strains of the bacteria regardless of their antibiotic resistance status, no bacterial resistance at various subbactericidal concentrations. 95.2  0.31 to Significant decrease in 96.1  0.14 MIC, higher killing rates for cationic nanoliposomes compared to the other drug forms, ticarcillin-loaded cationic nanoliposomes showed 100% survival rate and almost complete eradication of the bacteria from the spleens, livers, and skins of infected animals. 89.4  15 to Cationic nanoliposomal 93.4  0.31 form showed 100% survival rates and also the highest entrapment efficacy, the slowest drug release rate, and the highest killing rates relative to the other forms. 55.4 Enhance the ability of drug to permeate the skin efficiently, significant inhibition in Staphylococcus aureus growth. &75 The stabilization of the liposomes at acidic pH, higher antibacterial efficacy at all tested concentrations relative to free doxycycline. –

Thamphiwatna et al., (2013)

Li et al. (2013)

87.85%  2.15%. Dry film dispersion method

Extrusion method

Gharib et al., (2012b)

Cationic: 78, Neutral: 56, Anionic: 45

Extrusion method

Gharib et al., (2012a)

Obonyo et al. (2012)

Cationic: 76, Neutral: 55, Anionic: 43



Extrusion method

Sonication and needle extrusion method

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DOI: 10.3109/10717544.2013.863409

Nanoliposome-based antibacterial drug delivery 7

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S. Hallaj-Nezhadi & M. Hassan

bacteria. This indicates that the fluidity of liposomes is essential for fusion of liposomes with bacteria. More recently, Thamphiwatana et al. (2013) engineered advanced liposomal formulations for gastric antimicrobial delivery. The chitosan-modified gold nanoparticles were bound to the surface of negatively charged liposomes (diameter & 75 nm) which were resulted in the stabilization of the liposomes at gastric (acidic) pH, effective inhibition of drug release and liposome fusion with H. pylori bacteria. Once the acidity decreases to near neutral value (i.e. pH value at the mucus lining of stomach as the infection sites), gold nanoparticles detached from liposomes and led to free liposomes with both fusion, targeting bacterial membrane and drug release properties restored. The results obtained following in vitro study showed that gold nanoparticlestabilized liposome containing doxycycline possessed superior antibacterial efficacy against H. pylori bacteria at all tested concentrations relative to free drug. This novel liposomal antibiotic delivery system could be considered as a promising strategy for the treatment of stomach bacterial infections such as H. pylori infections. Taken together, these handful of investigations demonstrate immense potential of numerous liposomal nanoparticles as carriers for antibiotic delivery (summarized in Table 1), and highlight the latent promise in this class of vehicles for treatment of bacterial infections. The path ahead is summarized in the following section.

Future perspective The future of these promising approaches lies in the development of more efficient approaches for preparing liposomal nanoparticles with great potential in effective and selective targeting of antibiotics to bacterial cells (resistant to currently available drugs) for eradication as well as the highest safety for human host. The potency of the liposomal nanoparticles can be also improved via control of antibiotic release rate to make sure that the drug is released from the liposomes rapidly enough at the targeted site. In addition, the size of the nanoliposomes is required to be in a range (5200 nm) that avoids their quick clearance by the mononuclear phagocyte system, especially the resident macrophages of the liver (Kupffer cells), bone marrow, lung and spleen. Charge-neutral as well as saturated lipids should also be used for the formulation of the nanoliposomes since they may lead to reduced levels of protein binding and longer circulation half-lives (Maurer et al., 2001). Finally, greater understanding of the mechanism between bactericidal nanoliposomes and bacterial cells is required to optimize these antibiotic delivery systems.

Executive summary Taking into consideration, the benefits of liposomal delivery systems and in view of the fact that the unique advantages of drug nanocarriers have made them as powerful tools for drug delivery, liposomal nanoformulations for antibacterial drug delivery seems to offer great improvements in treatment of infectious diseases. To date, antibiotic delivery via liposomal nanoparticles as one of the most encouraging approaches has

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been accomplished through different preparation methods and various lipids. In this article, we have briefly summarized the recent studies that have taken advantage of liposomal nanoparticles as carriers for antibiotic delivery. A summary table (Table 1) is provided to list the major studies in this area.

Declaration of interest The authors report no conflicts of interest.

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Nanoliposome-based antibacterial drug delivery.

Although most bacterial infectious diseases can be treated successfully with the remarkable array of antibiotics, the microbial pathogens continue to ...
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