INTRODUCTION:

Pseudomonas aeruginosa is a gram negative opportunistic pathogen that can cause serious fatal infections¹ and the mortality rate from its sepsis is high and exceeds the rates from all other Gram negative agents². P.aeruginosa has been one of the major causes of nosocomial infections (surgical site infection, urinary infection, septicaemia).

Although P.aeruginosa exhibits various drug resistance mechanisms such as enzymatic inactivation of drugs and target site alteration^3,4, its low permeability to drugs through the outer membrane contributes the major factor to antibiotic resistance⁵. Currently, aminoglycosides are potent antibiotics for treating pseudomonal infections by inhibiting the protein synthesis upon binding to the bacterial ribosomes⁶, but its use is limited due to serious ototoxicity, nephrotoxicity and low permeability⁷. Despite the discovery of new antibiotics, it is difficult to eradicate P.aeruginosa from intracellular infections. Delivery of antibiotics to infected cells can be improved by encapsulating the antibiotics into colloidal carriers and nanoparticles. In particular, Liposomes are fast becoming the carrier of choice for a number of ‘promiscuous’ drug candidates and the liposomal encapsulation of these drugs can help address most of these issues associated with drug delivery. The intracellular penetration of liposomes is effected through endocytosis and get into endosomes where degradation by phospholipases takes place⁸. Liposomes are well studied as drug carrier molecules for delivering antimicrobial agents because they usually provide a sustained drug release effect, minimize drug toxicity, and increase overall drug efficacy⁹. Natural antimicrobial agents, on the other hand, represent a new promising solution for treating drug resistant pathogens. Free fatty acids(FFAs) such as oleic acid, Linoleic acid and dehydrocrepenynic acid naturally exist in blood stream and plays a vital role in innate immune systems¹⁰. It has been reported that oleic acid have an extensive bactericidal effect and are less prone to be selected for resistance since they cannot be easily modified by the bacteria, thereby holding great promise to avert the development of bacterial resistance¹¹. The size of the liposomal vesicles significantly influences drug distribution. Large (>1µm), Multi Lamellar vesicles (MLV), formulations are usually not used as antibiotic carriers while small unilamellar vesicles (SUV) of ~100nm exhibited high efficacy in the eradication of bacterial pathogens.In this communication, we report the superiority of liposomaloleic acid (LipoOA) over free oleic acid exhibiting antibacterial activityon resistant strains of P.aeruginosa.

MATERIALS AND METHODS:

Materials:

Hydrogenated L-a-phosphatidylcholine (Egg PC), cholesterol, oleic acid were purchased from Sigma Aldrich (St Louis, MO). Phosphate buffered saline (PBS), Peptone water, Muller Hinton agar, antibiotics were obtained from Hi-media laboratory (India). Polycarbonate membrane was purchased from Millipore.

Culture:

Sputum from patients with pulmonary infections and pus from wound site were collected from patients hospitalized at Sri Ramachandra Medical Hospital (Porur, Chennai, India) using standard aseptical procedures and processed for isolation and identification of P.aeruginosa according to the standard microbiological technique¹².

Antibiotic sensitivity test:

Antibiotic sensitivity pattern of P. aeruginosa isolates against amikacin (30µg), cefepime (30µg), tobramycin (10µg), ciprofloxacin (5µg) and imipenem (10µg) was investigated by Kirby-Bauer disc diffusion method according to Central Laboratory Standards Institute (CLSI) guidelines¹³. Turbidity of broth culture was adjusted to 0.5 McFarland standards and streaked on Muller Hinton Agar (MHA) plates, incubated overnight at 37°C for 18-24 hours. Zones of inhibition were measured in mm and compared to standard chart and interpreted P.aeruginosa ATCC 27853 was used as quality control strain.

Preparation of LipoOA:

Liposomes were formulated with egg phosphotidyl choline (EPC) and cholesterol in a ratio of 8:1.5to which oleic acid at 5, 10, 25, 50, 100, 200, 400 and 800µg/ml was added respectively. Thin film hydration method was preferred for the preparation of liposomes¹⁴. The mixture was dissolved with chloroform in a round-bottomed flask and a thin film was formed upon rotating the flask in a rotary evaporator. The solvents were removed subsequently and remaining traces were removed by purging nitrogen gas for 10mins. The dried lipid film was rehydrated with 10ml of sterile phosphate buffered saline(PBS) at pH 7.2. Multilameller vesicles(MLVs) were obtained by sonicating (Frontline electronics) the lipid suspension for 3 min. Then a Ti probe was used to sonicate the MLVs for 1 minute at 20W to produce large lamellar vesicles (LUVs). These LUVs were extruded 12 times in a 100nm pore sized polycarbonate membrane filters for obtaining (SUVs). A corresponding amount of HCl or NaOH was added to maintain pH inside liposomes.

Characterization of LipoOA:

Size determination and encapsulation efficiency:

Malvern zetasizer was used to measure the size (diameter, nm) of lipoOA through dynamic light scaterring (DLS) and surface zeta potential(mV) through electrophoretic measurements respectively. The zeta potentials of liposomes were measured in water to eliminate the ionic strength effect from PBS. The encapsulation efficiency of OA in liposomes was determined using liquid chromatography mass spectrumLC-MS/MSn LCQ DECA Ion Trapsystem (LC Agilent Technologies, Santa Clara, CA; MS Thermo Finnigan). A fixed amount of LipoOA was dried by rota vapour and then dissolved in methanol for LC-MS measurements. Briefly, 30 mL samples were injected through a C18 (3 µm, 2.1 mm ID ×18.5 cm) column (Sigma Aldrich, St Louis, MO, USA). The mobile phase composed of 85 v% acetonitrile and 15 v% water/TFA (99.9:0.1, v/v) at a flow rate of 0.2 mL/min. The molecular mass of the effluent from the column was measured using negative ionization. The acquired LC/MS chromatogram of OA was compared with a linear standard curve of OA at different concentrations to calculate the amount of OA encapsulated in LipoOA.

Analysis of liposome-oleic acid by SEM:

The surface morphology of the liposomes and lipoOA was visualized by scanning electron microscopy (Zeiss Gemini Supra 55). The lipid samples were coated with gold ions using ion coater under the following conditions: 0.1 Torr pressure, 20mA current and 70 s coating time, using a 15 KV accelerated voltage.

In vitro antimicrobial activity of LipoOA against MDRPa:

To determine the antimicrobial activity of LipoOA against MDRPa, a fixed amount of PS13 (1x10⁶ CFU in 100 ml) was incubated with 1 to 270mg/l concentrations of LipoOA in PBS buffer and 2 to 2048 mg/l free oleic acid at 37°C for 5 h in a 96 well plate. After incubation, the mixtures were diluted 1:10 with sterile PBS and 5µl of the diluted suspension was spotted on cetrimide agar plate for overnight incubation at 37°C and MBC was determined.

RESULTS AND DISCUSSION:

Selection of resistant strain

A total of 27 P.aeruginosa strains were isolated from 110 specimens obtained from patients with pulmonary and wound infections. Five different antibiotics tested showed 100% resistance to both cefepime and amikacin and 97.14% were susceptible to to bramycin, ciprofloxacin and imipenem. One strain PS13 was highly resistant to all the 5 antibiotics tested. The percentage of resistant and susceptible strains on various antibiotics is shown in Table 1.

Table 1: Antibiogram ofP. aeruginosa (total number of strains tested – 27)

Antibiotics	Resistant (%)	Susceptible (%)
Amikacin	100	0
Cefepime	100	0
Ciprofloxacin	2.86	97.14
Imipenem	2.86	97.14
Tobramycin	2.86	97.14

Particle size, zeta potential and Encapsulation efficiency:

Liposomes have a wide array of uses that have been continuously expanded and improved upon ever since it was observed to self assemble into vesicular structures. These arrangements can be found in many shapes and sizes depending on lipid composition. Earlier researches on liposomes infer the knowledge that small liposomes (diameter, < 50 nm) are prone to fuse with one another and are highly unstable due to their high surface tension¹⁵. On the other hand, large liposomes (diameter, >200 nm) are usually stable but may have difficulty to penetrate through skin for topical drug delivery thus limiting them as a poor carrier molecule¹⁶. Liposomes with moderate size range (50–100 nm) will have relatively prolonged stability¹⁷ and possess good skin penetration ability¹⁸. Hence, LipoOA complex synthesized in this study with a size of about 86.1 ± 0.5nm (Fig.2) will be an ideal nanocarrier to fuse with bacterial membranes, and subsequently release the entrapped antimicrobial substances to the bacteria. The polydispersity index (PDI) of bare liposomes and LipoOA were 0.46 and 0.45 respectively, indicating the relatively narrow distribution of liposome sizes. The surface zeta potential of LipoOA was -60.1 mV in deionized water which in contrast for liposomes without OA was -6.9 mV (Table 2).

The encapsulation efficiency of OA in liposomes was determined by comparing the synthesized LipoOA with a standard curve of OA ranging from 5 to 250µg/mL. In our study, it was found that when 300mg/L OA is taken as the initial input concentration, the final concentration after incorporating OA into the liposomes would be 101.1 µg/mL corresponding to a drug encapsulation efficiency of 33.7%.

Table 2: Characteristics of liposomes and LipoOA

Formula-tion

Particle Size (nm)

Zeta potential (mV)

PDI

Entrapment Efficiency

Liposomes

43.5

-6.9

0.465

LipoOA

86.1

-60.1

0.454

33.7

Fig. 1: Particle size distribution of (a) Liposomes (b) LipoOA

Fig 2 : Stability of bare liposomes and liposomal oleic acid done through zeta potential (mV)

Morphology of LipoOA:

Generally, liposomes may suffer structural perturbations as a result of the high-vacuum conditions and the staining process required by some electron microscopy techniques¹⁹. Therefore, scanning electron microscopy (SEM) is a less frequently used imaging technique because analysis requires the sample to be dried or fixed before imaging. In our case, we tried to study the morphology of both bare liposomes and LipoOA using scanning electron microscopy. LipoOA formulation was found to be spherical, whereas liposome without oleic acid was in irregular in shape(Fig. 3). Unfortunately, the resolution of SEM analysis don’t provide detailed information related to the surface and the architecture of the nanoscale structures, as already pointed out by Mohammed et al²⁰.

Fig. 3: SEM images of the liposome in a mixture of phosphate buffer in the absence (a) and presence (b) of oleic acid.

Antibacterial activity of LipoOA against MDRPa

With an increasing number of antibiotic-resistant pathogens, the selection of antimicrobial treatment has become difficul²¹. In this study the engineered liposomes with oleic acid (LipoOA) at various concentrations (2 to 1024µg/ml) were tested against MDRPa strain.

Fig. 4: PS13 strain incubated with varying concentration of (a)free OAindicating the bactericidal activity on 1024µg/ml which is 15 fold more than (b)LipoOAat 67.4 µg/ml

Minimal bactericidal concentration (MBC) of LipoOA was determined for killing 99.9% of the target bacteria. The MBC value for lipoOA was 67.4µg/ml which is significantly lower than free oleic acid (OA) with MBC of 1024µg/ml (Fig. 4). In a similar study by Chun et al., (2011) it was reported that OA-loaded liposomes (LipoOA) could rapidly fuse into the bacterial membranes, thereby significantly improving the potency of OA to kill methicillin-resistant Staphylococcus aureus compared with the use of free OA²². This treatment process of resistant strain PS13 explains the enhanced antimicrobial activity of liposomal oleic acid may be due to rapidly fusing into the bacterial membranes delivering the lethal dose (OA), thereby suppressing the bacterial growth even before they develop any possible resistance mechanisms.

CONCLUSION:

Liposomes are currently used as carriers for drug delivery and are non toxic to humans²³.We have encapsulated oleic acid into liposomes composed of EPC and cholesterol in an attempt to enhance the antibacterial activity of oleic acid against an antibiotic-resistant strain of P. aeruginosa. Our data provide a compelling argument for pursuing liposomal oleic acid based therapy for use as a treatment for life-threatening pseudomonal infections.Greater control of drug leakage rates within disease sites and the use of targeted liposomes for intracellular delivery offer opportunities to increase the efficiency and specificity of liposome encapsulated agents. The challenge for the future will be to develop systems for loading lipoOA with various hydrophilic and hydrophobic antimicrobial drugs in the management of infections caused by P.aeruginosa.

ACKNOWLEDGEMENTS:

We acknowledge funding support from Indian Council of Medical Research (ICMR). The authors also wish to acknowledge the Department of Microbiology, Sri Ramachandra Medical Hospital (Porur, Chennai, India) for their help in collecting clinical specimens.

ETHICAL APPROVAL:

This study was approved by the Institutional Ethics Committee, Sri Ramachandra University, Porur, Chennai, India. Reference number: IEC-NI/13/FEB/32/18.

CONFLICT OF INTEREST:

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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Received on 08.03.2017 Modified on 20.03.2017

Research J. Pharm. and Tech. 2017; 10(7): 2114-2118.

DOI: 10.5958/0974-360X.2017.00370.5