Screening and enhancement of the antimicrobial activity of some plant oils using liposomes as nanoscale carrier

Liposomes have the ability to enclose hydrophilic or lipophilic materials. Bioactive macromolecules become more stable when they are entrapped within liposomes resisting environmental changes, allowing maintenance of the antimicrobial molecules and increasing their effectiveness and constancy thus can be used for food preservation. The aim of this study was to screen food samples for microbial contamination and to examine the antimicrobial activity of selected six ready-made plant oils which were; clove, black seed, thyme, garlic, rosemary and green tea against the isolated microbes from food samples and other selected microbes. Also to examine the possible enhancement of the antibacterial property of clove oil and tetracycline versus Escherichia coli when they were encapsulated into distearoyl phosphatidylcholine (DSPC) liposomes as a nanoscale carriers. Results of the antimicrobial action measured by minimum inhibitory concentration revealed that all six oils had antimicrobial action when facing at least one of the tested microbes. However only clove oil could inhibit the growth of all tested microbes. Moreover encapsulation of clove oil into DSPC liposomes enhanced its antibacterial action by 10 times when examined to inhibit the growth of E. coli. Also the antibacterial activity of liposome encapsulated tetracycline was improved by 8 times. Results of characterization of formulated clove oil liposomes by measuring their Zeta potential and their sizes implying that clove oil might be enclosed within the hydrophobic portion of the two layers of the liposome. Analyzing data of Fourier Transform Infrared Spectroscopy showed that clove oil was detected in the interfacial area of the liposome. Analyzing results of Differential scanning calorimetry and measuring phase transitions suggested that liposomes encapsulating clove oil had a membrane fluidization effect. Some plant oils like clove has antimicrobial activity which enhanced with liposomal encapsulation and thus reduces the needed concentration to give the desired actions.


Background
Lately there is great concern of using natural alternatives as food preservative to keep food safety, healthy and clear from pathogen contamination A lot of herbs and spices have antimicrobial activity more or less due to holding essential oil constituents which also called volatile oils that embrace different antimicrobial modes of action (Granados-Chinchilla et al. 2016;Ambrosio et al. 2017). While essential oils have antimicrobial action towards a great variety of pathogens transmitted by food still there is a problem in applying essential oils in food industry because may be more concentration of certain essential oil is obligatory needed to get the same antimicrobial result in food than the concentration required for in-vitro cultural media which may alter taste of food products in an undesirable way (Burt 2004;Songsong 2011).
Liposome is a spherical-shaped envelope synthesized as one or more phospholipid bilayer. Liposomes possess

Open Access
Bulletin of the National Research Centre *Correspondence: mghaggag@gmail.com 1 Microbiology and Immunology Unit, Allergy Lab, Research Institute of Ophthalmology (RIO), 2 El Ahram Street, Giza 12557, Egypt Full list of author information is available at the end of the article Page 2 of 14 Haggag et al. Bull Natl Res Cent (2021) 45:38 unique character being amphiphilic molecule possessing both hydrophilic and lipophilic preference because it is composed of hydrophobic bilayer surrounding hydrophilic inner core which allow solubilization, incorporation and encapsulation of both hydrophobic and hydrophilic drugs (Li et al. 2019). The structure of liposomes is more or less like that of biological membranes and hence liposomes are easily incorporated into cell membranes and adhere to them (Shafaa et al. 2011;Zou et al. 2012). The advantage of liposome entrapment is stabilizing the encapsulated bioactive macromolecules against various environmental and chemical traumatic factors as exposure to extreme pH and temperature, enzymatic digestion, presence of reactive chemicals and high ion concentration consequently providing protection, keeping maintenance of the antimicrobial molecules and increasing their effectiveness, reliability and strength hence can be used for food preservation (da Silva Malheiros et al. 2011). Formulation of antibiotics into liposomes prolong the time of antibiotic delivery and thus giving a better chance for dealing with infections that are intracellular. Moreover liposomes can disguise the taste of bitter compounds (Cipolla et al. 2016). However very recently a study made by Hashemzadeh et al (2020) stated that one of the problems in using liposomes as drug carriers is their longterm instability.
The aim of our study was to examine the antimicrobial ability of six plant's oils against selected isolates of Gram-positive and Gram-negative bacteria and yeast and to examine the possible enhancement of the antimicrobial activity of clove oil when loaded into liposomes as nanoscale carriers. Moreover to analyze the formulated clove oil liposomes characterizing their size, size-distribution, thermotropic changes and conformational changes.

Food samples and microorganism isolation
The present study involved 45 food samples which were collected from local markets located in Helwan, Cairo Governorate, Egypt. Samples varied form 18 milk samples, 16 yoghurt samples, 5 spices samples, 2 juice samples, 2 processed meat, one mayonnaise sample, and one tahina sample and were screened for microbial contamination by adding 10 g of tested sample to 90 ml sterile buffered peptone water, mixed well, incubated at 37 °C for 24 h then cultured onto Nutrient agar, Blood agar, MacConkey's agar, Eosin methylene blue (EMB) agar, MacConkey sorbitol agar and Sabouraud dextrose agar plates.
Escherichia coli were identified by its characteristic pink colonies on MacConkey's agar. While on EMB agar appeared as green metallic colonies sheen under reflected light (Fig. 1a). Pathogenic E. coli grew as colorless colonies on MacConkey sorbitol agar (Fig. 1b), while non-pathogenic E. coli colonies were red in color. Gram stained films showed G-ve bacilli (Fig. 1c). E. coli was confirmed by giving positive Indole test, positive methyl red and negative Voges-Proskauer (VP) tests. Motility test was positive by hanging drop method.
Salmonella was identified as red colonies with or without black centers on Xylose-Lysine Deoxycholate (XLD) agar (Fig. 2a), isolates have been identified as Gram-negative bacilli (Fig. 2b) which were motile and gave negative indole test.
Candida albicans grew as white colonies on Yeast Extract Glucose Chloramphenicol (YEGC) agar (Fig. 3a). Colonies were identified by Gram stained film that  et al. Bull Natl Res Cent (2021) 45:38 showed Gram positive budding oval cells (Fig. 3b) and by positive germ tube test.

Source of microorganisms used in this study
Escherichia coli, Salmonella and Candida were isolated from the tested food samples showed in (Table 1)

Source of plant oils used in this study
Six ready-made oils of clove (Syzygium aromaticum) oil, black seed (Nigella sativa) oil, thyme (Thymus vulgaris) oil, garlic (Allium sativum) oil, rosemary (Rosmarinus officinalis) oil and green tea (Camellia sinensis). All test oils were purchased from El-captain company "CAP Pharm" for extracting natural oils, herbs and cosmetics (www.elcaptain.net Info@elcaptain-eg.com). Product descriptions are available in the packages (Fig. 4). Oils were filtered using Nylon Olimpeak ™ Syringe Filter (Teknokroma Italy) 0.45 µm and stored in sterile falcon tubes at 4 °C till use.

Inoculum preparation
A loopful of the selected microbe was inoculated into 5 ml nutrient broth and incubated at 37 °C for 24 h, from this 24 h culture 0.2 ml was taken and added to 19.8 ml sterile nutrient broth and incubated for 3-5 h to standardize the culture to 10 6 cfu/ml according to Abalaka et al. (2012).

Antimicrobial activity assay
The antimicrobial activities of plant oils were measured using: The well diffusion method as described by the British Pharmacopoeia (2010). After sterilization of nutrient agar medium, it was left to cool to 48 °C then 1 ml of the bacterial suspension (10 6 cfu) was added to 100 ml nutrient agar medium and thoroughly mixed by rotary motion of the flasks. Immediately the seeded medium was poured into Petri dishes. A sterile cork borer 6 mm diameter was used to perform wells in each plate then 100 µl of sterile oil extracts were applied to each well and plates were kept on bench for one hour at room temperature to permit diffusion of the extract before incubation at 37 °C for 24 h. Finally measuring the diameter of inhibition zones in millimeters was done (Fig. 5).

MIC and MBC determination
In the present study MIC assay was done by broth macrodilution method by inoculating the organism into small microcentrifuge tubes containing two-fold dilutions of each antimicrobial agent (tested oil extract). After incubation at 35 °C for 24 h the lowest concentration of the antimicrobial agent that prevents visible growth of the organism is the MIC (CLSI 2012). MIC is considered as the logarithmic difference in population (Log DP) which is calculated by the following equation: Log DP = Log (N/No) = (Log N) − (Log No) Where N is the bacterial population after incubation for 24 h and No is the initial population. MIC is considered as the lowest concentration preventing visible bacterial growth (Log DP ≤ 0). The minimum bactericidal concentration (MBC) is defined as the concentration where 99.9% or more of the initial inoculums are killed (Log DP ≤ − 3) Pei et al. (2009).

Preparation of liposomes
Liposomes were prepared using high purity distearoyl phosphatidylcholine (DSPC), with a molecular weight of 790.161 g/mol (99% pure), Tris buffer extra pure 99% with a molecular weight of 121.14 g/mol (LOBA CHEMIE, INDIA). DSPC: tested extract of clove oil or tetracycline at molar ratio 7:4 were used to prepare neutral multilamellar vesicles (MLVs) using the method described by Shafaa et al. (2011).
Required mass of DSPC and tested extract of clove oil were calculated based on the molecular weight of each and selected molar ratio thus to prepare 5 ml of 1 mg/ml liposomal clove oil; 33.62 mg of DSPC were mixed with 5 mg clove then transferred to 50 ml round bottom flask, 10 ml of absolute ethyl alcohol was added and flask was shacked to dissolve all lipids in the solvent. Using rotary evaporator under vacuum to evaporate the solvent with the aid of a suitable flow of nitrogen to obtain a film of deposited lipids onto the wall and the bottom of the flask. The solvent was removed slowly to permit appropriate integration of components. To ensure complete evaporation of the solvent the flask was kept under vacuum for 12 h because the presence any traces of solvent is harmful for the assembly of domains (Ibrahim et al. 2011) Based on desired extract final concentration add suitable amount in milliliter of buffer 10 mM Trizma adjusted to pH7. Mechanical shaking of the flask was done for 1 h at 45 °C. Then the flask was thoroughly flashed with nitrogen stream and immediately closed.
Liposomal entrapment of tetracycline (source of tetracycline was the Arab Drug Company ADCO, Egypt) was done parallel to liposomal clove oil encapsulation thus to prepare 5 ml of 4 mg/ml of liposomal tetracycline; 15.56 mg of DSPC were mixed with 5 mg tetracycline in ethanol and evaporated to form a thin dried film under the same condition as before. The hydration of the dry lipid thin film was achieved with Tris base buffer at tetracycline to lipid molar ratio = 4:7. Control empty liposomes were made by only fractions of DSPC. Storage of liposomal dispersion at 4 °C until use ( (Masserini et al. 2002;Shafaa et al. 2011;Jurado et al. 2016;Chen et al. 2016).

Characterization of prepared liposomes
Prepared liposomes were characterized in terms of encapsulation efficacy, zeta potential, vesicle size, morphology, Fourier transform infrared spectroscopy, and differential scanning calorimetry analysis.
Entrapment efficiency (EE) of liposomes was assessed by measuring the amount of free un-encapsulated clove through UV-spectrophotometric technique at 237 nm (resonance absorption peak of clove oil) with reference to a previously constructed calibration curve.
EE is the percentage of the drug that is successfully entrapped into the nanoparticles. It was calculated by using the following equation: where Ci is the concentration of the initial clove added (also called total conc Ct) and Cf is the concentration of free clove (Ibrahim et al. 2011;Chen et al. 2016).
Zeta potential is the outcome of all charges present on the surface of any material when distributed into dispersion medium. It is a very reliable parameter that reflect the degree of electrostatic repulsion between the charged particles in the dispersion and hence it is a clue for stability of colloidal dispersions and its value allowing to access quality control and improving product performance (Perrotta et al. 2016). All samples were diluted in the ratio 1:10 (v/v) with double distilled water and the mean standard deviation of three determinations were reported. Evaluation of the zeta potential of liposomal preparation was done by Laser Zeta-meter (Nanotrac Wave II, Microtrac, USA) by Nawah Scientific according to Mozafari et al (2014).

Morphological characterization
Liposome suspensions were imaged, using a transmission electron microscope (TEM). Dilution of samples 10 times was done using distilled water. Equivalent volumes of both ammonium molybdate 2% solution and the diluted sample were mixed and left for 3 min at room temperature. A drop of this mixture solution was placed on a copper mesh for 5 min then by using filter papers the excess liquid was drawn off. Examination of the mesh was done using JEOL 1200-EX II TEM (JEOL Ltd, Tokyo, Japan) in the Central Lab at Ain Shams University according to Mozafari et al (2014).
Fourier Transform Infrared Spectroscopy (FTIR) spectra of lyophilized blank, and clove oil encapsulated into liposomes were measured from 400 to 4000 cm -1 by FT-IR spectrometer 4100 (JASCO, Japan), was done in the Micro Analytical Center at Cairo University.

Differential scanning calorimetry (DSC) analysis
The thermal behavior was studied by (DSC-60, Shimadzu, Japan) calibration was done using indium to examine the thermal performance of lyophilized samples of empty and clove-loaded multilamellar liposomes. Samples of 5 mg was placed in standard aluminum pans and sealed for analysis. The temperature range of 2-250 °C was covered by the thermogram for each sample at a scanning rate of 10 °C/min which was done in the Micro Analytical Center at Cairo University.

Results
In the present study screening of 45 different food samples for microbial contamination revealed the presence of 17 E. coli isolates, 5 Salmonella isolates and one C. albicans isolate.
Results of screening of the antimicrobial activity of six tested plant oils against six microbial isolates including the three microbes isolated from food samples measured by well diffusion method (Table 2) and by minimal inhibitory concentration (MIC) ( Table 3).
Results revealed clove was the only plant oil that showed antimicrobial activity against all the tested isolates and hence it was selected for testing the effect of liposomal encapsulation.
Results of antimicrobial activity enhancement by liposomal loading: According to the equation mentioned earlier MBC is when Log DP ≤ − 3 Pei et al. (2009) therefore from the bar chart (Fig. 6a, b) the value of Log DP of E. coli as − 4.30 represented the MBC of clove oil which reached at 5 mg/ml meanwhile this value could be reached with liposomal clove concentration of 0.5 mg/ml only, and hence the antimicrobial activity of clove oil was enhanced by 10 times when clove oil was carried on liposomes (5/0.5 = 10). Also encapsulation of tetracycline into liposomes increased its antimicrobial activity as shown in (Fig. 7a, b) where Log DP of E. coli as − 8.04 represented the MBC of unloaded tetracycline which reached at 1 mg/ml while LogDP as − 5.04 represented MBC of liposomal tetracycline which reached at 0.125 mg/ ml. Thus the antimicrobial activity of tetracycline was enhanced 8 times when tetracycline was carried on liposomes (1/0.125 = 8).

Liposomes characterization
Encapsulation efficiency (EE) of clove was measured to be 22.068%. The absorbance at different concentration of clove oil was measured using a UV-Vis spectrophotometer at 237 nm. A standard curve was made by plotting absorbance against concentration. The concentration of the non-encapsulated clove oil in the supernatant was measured using this curve. The encapsulation efficiency was calculated as the mass ratio of the amount of the clove oil entrapped into liposomes and this calculated ratio was used in the liposome preparation (Fig. 8).
Zeta potential analyzer was used to determine the electro-kinetic surface potential for empty and Table 2 The antimicrobial activity of studied plant oils against different microbial isolates using well diffusion method  encapsulated liposomes and showed that clove encapsulation increased in zeta potential about fivefold compared to empty liposomes. DSPC liposomes and clove loaded liposomes zeta potential were measured to be − 18.6 ± 25.0 and − 117.5 ± 85.2 mV, respectively. The morphology and size of blank and clove oil encapsulated liposomes were examined by TEM images that showed well dispersed and not aggregated round shaped empty and encapsulated vesicles had a diameter around 232.33 ± 10.63 nm and 979 ± 27 nm, respectively (Fig. 9).
A stack plot of the FTIR spectra of pure lyophilized DSPC liposomes compared with clove oil loaded DSPC liposomal sample in the region 400-4000 mm are presented in (Fig. 10). FTIR spectra of blank lyophilized DSPC liposomes were analyzed. Information on the order/disorder state by means of acyl chain flexibility of the lipid membranes was presented by symmetric, and anti-symmetric stretching vibration of the CH 2 in the acyl chain were located at wave numbers 2851.24, and 2919.7 mm respectively. CH 2 bending vibration peak was located at 1469.49 mm, while symmetric, and antisymmetric PO 2 − stretching vibrations were located at 1095.37, and 1247.72 mm, respectively. OH stretching, and bending vibrations were observed at wave numbers 3422.06, and 1636.30 mm, respectively. Carbonyl stretching vibrations C=O was located at 1738.51 mm.
Amide I band due to C=O stretching vibration of the peptide group in 1636.30 mm (CH3) 3 N + asymmetric stretching in 970.019 mm. While both Aliphatic phosphates P-O-C stretch, and (CH3) 3 N + symmetric bending spectra were not detected.
FTIR spectra of lyophilized clove oil loaded DSPC liposomes showed no change in both symmetric, and anti-symmetric stretching vibration of the CH 2 in the acyl chain to be 2851.24, and 2919.7 mm respectively. Reduction in wave number values were detected in case of CH 2 bending vibration, symmetric PO 2 stretching vibration, and amide I band due to C=O stretching vibration of the peptide group to be 1468.53, 1093.44, 1635.34, and 1635.34 mm respectively. Wave number values increased in case of Anti-symmetric PO 2 stretching vibration, OH stretching vibration, Carbonyl stretching vibrations C=O, and (CH3) 3 N + asymmetric stretching to be 1249. 65, 3429.78, 1739.48, and 970.983 mm respectively. While Amide II band due to N-H bending with contribution of C-N stretching vibrations was not detected.
DSC analysis of lyophilized pure DSPC and clove loaded DSPC vesicles were presented in DSC thermograms (Fig. 11). Upon dehydration, pure DSPC vesicles showed a major endothermic peak at 79 °C and the pretransition temperature was around 64 °C. Whereas lyophilized clove oil loaded liposomes exhibited noticeable broadening and a decrease in temperature to 72 °C and the pre-transition temperature also shifted from 64 to 54 °C.

Discussion
In the present study screening of 45 different food samples varied form milk, yogurt, cheese, spices, processed meat, and juice were screened for microbial contamination revealed 17 samples were contaminated with E. coli (37.8%) and 5 food samples were contaminated with Salmonella (11.1%) and only one food sample was contaminated with C. albicans. Both bacteria were found mainly in milk and milk goods. Oliver et al (2009) reviewed food safety and its hazards joined to utilization of unprocessed milk and showed that E. coli and salmonella were the most common contaminants of milk and its products.
The mechanism by which essential oils can combat microbes has not been fully explained. Essential oils do not aim a specific target in the cell nevertheless they targets many goals in the cell. When essential oils enter the cell wall penetrating the cytoplasmic membrane of pathogens they cause structure disruption of their polysaccharides, phospholipids and fatty acids layers increasing their permeability (Chaieb et al. 2007;Bakkali et al. 2008). This increased permeability is accompanied by ions depletion, inhibition of the proton pump, decreased ATP synthesis, inhibition of respiration and hence cell death of the pathogen whether it is G + ve or G − ve bacteria (Nazzaro et al. 2013;Mohammed et al. 2019).
Clove is the common name of Syzygium aromaticum family Myrtaceae. Eugenol is the main component of clove's essential oil which owes its antibacterial effect by making the cell membrane more permeable and interrelate with its proteins and that was proved by many different studies to be due to increased movement of potassium and ATP out of bacterial cells (Hyldgaard et al. 2012). Moreover when clove essential oil enters the bacterial cell it inhibits DNA synthesis and proteins necessary for bacterial growth implying damage at the molecular level rather than only physical damage (Xu et al. 2016).
In the present study results of screening of the antimicrobial activity of six ready-made oils: clove (Syzygium aromaticum) oil, black seed (Nigella sativa) oil, thyme (Thymus vulgaris) oil, garlic (Allium sativum) oil, rosemary (Rosmarinus officinalis) oil and green tea (Camellia sinensis) measured by well diffusion method against six microbial isolates: Escherichia coli, Salmonella typhi, Candida albicans, Staphylococcus aureus, Klebsiella and Pseudomonas proved that all tested oils had antimicrobial tendency when facing at least one of the above mentioned six microbes. However clove oil was the only one that has antimicrobial affinity against all of the tested microbes. Nzeako et al (2006) in their study reported that C. albicans was the most sensitive organism to the antimicrobial activity of clove oil and thyme than S. aureus, Pseudomonas aeruginosa, E. coli and Salmonella species. They also concluded that whatever the country of origin of herbs is had no effect on their antibacterial benefit. However Ncube et al. (2008) concluded that the variety  et al. Bull Natl Res Cent (2021) 45:38 of the plant parts used in extraction as well as the extraction method led to various antimicrobial effects. The antibacterial effects of clove oil and rosemary oil were among many other oils studied by Prabuseenivasan et al (2006) against S. aureus, E. coli, K. pneumoniae, P. aeruginosa and two other bacteria. Their results showed inhibition zone rang for clove oil from 16 to 17 mm against all four bacteria and that of rosemary was 12 to 23 mm also against the four bacteria. These results of clove oil and rosemary oil were greater than the results in this study.
Regarding the antibacterial activity of black seed (Nigella sativa) oil, quite large number of studies have been established by various researchers to explore the pharmacological action of black seed oil as stated by Safhia et al. (2014) the black seed oil commercially available was selected for their study without dilution or chemical alteration, Staphylococcus aureus, Escherichia coli and Klebsiella planticola were among other tested bacteria. Inhibition zone of S. aureus was 14 mm which was the most significant result when compared to Ciprofloxacin disc (5 µg/disc) while that of E. coli was non-significant however Klebsiella showed no zone of inhibition. The result in the present study as 13 mm agrees with their results regarding S. aureus only. Approximate results to ours were reported by Mohammed et al.  concerning the antibacterial action of black seed (Nigella sativa) oil towards Staphylococcus aureus where inhibition zone diameter was 11.5 mm. They explained the antimicrobial effect of black seed oil to be attributed to thymoquinone which is a volatile oil of these seeds that characteriscally has free rotation bonds in the CH 3 group which qualify thymoquinone and other constituents of black seed oil to change their shapes according to the available bacteria and easily cross or enter their boundary leading to their death. While earlier Bakathir and Abbas (2011) explained the positive inhibition of black seed to S. aureus may be due to thymoquinone and melanin. Various techniques for encapsulation were used in food industry to deliver some active compounds such as additives, colors, enzymes and preservatives. However their incorporation into liposomes as nanoparticles is distinguished by the unique liposomal characteristic which is the capacity to entrap both hydrophobic and hydrophilic particles. Liposomes also have the advantages of being biocompatible, biodegradable, non toxic and non immunogenic. For all these reasons liposome nanocapsules have considered as one of the tempting persuasive package material in pharmaceutical and food manufacturing (Ghorbanzade et al. 2017;Corrêa et al. 2019).
A comparative study was done by Anderson and Omri (2004) between three types of formulated liposomes considering encapsulation efficacy as a crucial factor in liposome parameters they reported that DSPC liposomes showed the greatest encapsulation efficiency between the three formulations which might be due to their shorter hydrocarbon chain (C 14 ) DSPC liposomes had also shown the most drug retention.
Regarding measurement of zeta potentials which is one of the standard characterization techniques to evaluate the final conclusion of nanoparticle surface charge character whether cationic, anionic or neutral character (Smith et al. 2017). That was clearly explained by Paolino et al. (2006) and Elmeshad et al. (2013) who stated that the magnitude of zeta potential is the clue for the potential stability of nanoliposomes composition and estimation of in-vivo fate of these nanocarriers. Because when zeta potential increases it leads to more repulsion between particles resulting in more stable colloidal dispersion. In other words when all particles in suspension have a large negative or positive zeta potential, then the particles have a propensity to repel each other leaving no chance for their aggregation together.
In the present study blank DSPC liposomes showed negative zeta potential as (− 18.6 ± 25.0 mV) in accordance with Makino et al (1991).
Results reported by Elmeshad et al. (2013) stated that the nanoliposomal formulations had average zeta potential with a range from 23.3 ± 1.2 to 35.8 ± 0.3 mV.
In the present study clove loaded liposomes had higher negative zeta potential (− 117.5 ± 85.2 mV) which may be explained by the presence of hydroxyl (OH − ) group on the clove surface liposomes that might be physically associated by the surface without upsetting the packing membrane suggesting that clove oil might be caught in the hydrophobic fraction of the liposomal bilayer. The presence of Hydroxyl (OH − ) group on the clove surface with more negative zeta potential acted as stabilizing material.
In the present study TEM images showed well dispersed and not aggregated round shaped empty and encapsulated vesicles had a diameter around 232.33 ± 10.63 nm and 979 ± 27 nm respectively. The large size of clove oil encapsulated liposome might be explained considering the existence of phospholipid PO 2 − group in the polar head of DSPC liposomes might prevent the clove hydroxyl (OH − ) group from getting close to the PO 2 − group because of the electrostatic repulsive force between the DSPC PO 2 − group and the clove hydroxyl (OH − ) group thus weakening the interactions between phospholipid PO 2 − and clove hydroxyl (OH − ) group. The presence of hydroxyl (OH − ) group on the clove surface liposomes that might be physically associated by the surface without upsetting the packing membrane suggesting that clove oil might be caught in the hydrophobic fraction of the liposomal bilayer. These findings show that the presence of clove oil in liposomes increased the spacing between the adjacent bilayer, resulting in larger-sized liposomes compared to the control empty liposomes.
While results reported by Elmeshad et al. (2013) showed the opposite as the loaded nanoliposomes was possessing the minimum vesicle size (532 ± 26 nm) which was a surprising finding and was explained by the hydrophilic nature of the entrapped material in their study which lead to its segregation inside the aqueous phase rather than being trapped within the hydrophobic part of the liposomes.
FTIR studies were done to examine the effect of clove oil on the DSPC liposomes. The spectrum of the DSPC liposomes (Fig. 5) exhibited the major characteristic bands demonstrating the symmetric and anti-symmetric stretching vibrations of the CH 2 inside the acyl chain (2850 and 2920 cm −1 respectively) the OH stretching and bending vibrations (3470 and 1640 cm −1 respectively) the carbonyl stretching vibration C=O (1734 cm −1 ) the CH 2 bending vibration CH 2 (1470 cm −1 ) and the symmetric and anti-symmetric PO 2 − stretching vibrations (1090 and 1220 cm −1 respectively) same results were reported and published in a previous study by one of our authors with Mady et al. (2012) also these results are in accordance with Mady and Elshemey (2011). Haggag et al. Bull Natl Res Cent (2021) 45:38 In the present study incorporation of clove oil into the DSPC liposomes showed no significant variation in the frequency of the symmetric and anti-symmetric CH 2 stretching bands within the acyl chain observed implying that clove oil does not alter the number of gauche conformers. While FTIR measurements reported by Mady et al (2011) revealed that gold nanoparticles made significant alterations in the frequency of the CH 2 stretching bands suggesting that gold nanoparticles increased the number of gauche conformers and generate a conformational change within the acyl chains of phospholipids.
An important finding that could be observed was that the CH 2 bending vibration modes that were located at 1469.49 cm −1 were affected by the encapsulation of clove oil into DSPC liposomal preparation as wave number was lowered to 1468.53 cm −1 . This suggested the presence of ordering outcome within acyl chain packing in the gel forms of phospholipids.
The interaction between clove oil and DSPC liposomal head group was monitored by means of the PO 2 − antisymmetric stretching band, which is positioned at 1247.72 cm −1 . The wave number was moved to higher values from 1247.72 to 1249.65 cm −1 after the encapsulation of clove oil in DSPC liposomes. This suggested the absence of hydrogen bonding between clove oil and liposome head group. According to previous investigational observations lowered frequency values are exhibited an enhancement of existing hydrogen bond strengthening or in creation of new hydrogen bonding between the constituents Severcan et al. (2005). These results suggesting that clove oil was located in the interfacial region of the membrane.
In the present study analyzing data of clove oil interaction with glycerol backbone close to phospholipids head group within the interfacial region the C=O stretching band and the wave number deviation of this band revealed shift of wave number value of C=O group towards higher value from 1738.51 to 1739.48 cm −1 regarding liposome sample containing clove oil without any proof of hydrogen bonding formation where the amount of hydrogen-bond formation was observed in the glycerol backbone region of the DSPC molecule by alteration in the contours of the ester C=O stretching. Analyzing these data suggesting dryness concerning these functional groups in the interfacial area of the lipid membranes. Consequently any effects in the spectra of this region can be assigned to an interaction between clove oil and the polar/a polar interfacial region of the membrane. Clove oil tends to decrease hydrogen bonding formation in the interfacial region of DSPC liposome suggesting the existence of the free carbonyl groups in the arrangement. In other words clove oil is likely to substitute some H 2 O molecules from the interfacial region and lead to an increase in the number of free carbonyl groups.
When using liposomes for drug encapsulation the preparations is a mixture of entrapped and unentrapped drug fractions and science Entrapment Efficiency (EE) is the percentage of the drug that is successfully entrapped into liposomes so the first step for EE determination was to separate the entrapped drug inside the liposomes from the free one. However the process of separation and measurement of concentrations are heavy, tiring and taking long time moreover the separation procedure itself might be unsuccessfully completed. (Laouini 2012;Bano 2000). Differential scanning calorimetry (DSC) is very efficient method that does not need a separation step meanwhile it is able to access more than one parameter of liposomal encapsulation as EE, size, drug-lipid interactions and partition coefficient so that achieving quality control of liposomal encapsulation (Bakonyi et al 2017).
In the present study results of encapsulation efficiency (EE%) of clove was 22%. This may be attributed to the lipo-solublized state of the drug owing to its entrapment within multiple lipoidal domains of liposomal vesicles which enables a sustainable release of clove oil as slowly leaking out of the clove compared to its free form in the vicinity of the bacteria in culture which might explain the enhancement of the antibacterial activity by 10 times.
Differential Scanning Calorimetry (DSC) was mainly applied to determine the thermodynamics of biological macromolecules measuring phase transitions, conformational changes and temperature dependence (Chiu and Prenner 2011).
In the present study incorporation of clove oil into liposomes exhibited noticeable expansion and move to lower temperature 72 °C of the main characteristic endothermic peak of unloaded DSPC that was existed at 79 °C which suggested that clove oil had a significant consequence on the acyl chains of DSPC bilayer and that its presence lowered the mutual transition of the lipid acyl chains. The decreased temperature of the major DSPC transition process showed that clove oil assimilation was more likely due to the configuration of acyl chains in a disorganized and free state. The pre-transition temperature was found to be shifted from 64 to 54 °C indicating that liposomes encapsulating clove oil had a membrane fluidization effect.

Conclusion
Using liposomes as nanoscale carrier increased the efficacy of the antimicrobial activity of clove oil by 10 times than unloaded clove oil and that of tetracycline by 8 times than unloaded tetracycline when they were tested against Escherichia coli. Thus encapsulation of the antimicrobial materials into liposomes enhances their action