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Phospholipid vesicles encapsulated bacteriophage: A novel approach to enhance phage biodistribution
Accepted Manuscript Title: Phospholipid vesicles encapsulated bacteriophage: A novel approach to enhance phage biodistribution Author: S. Singla K. Harjai K. Raza S. Wadhwa O.P. Katare S. Chhibber PII: DOI: Reference:
S0166-0934(15)30054-9 http://dx.doi.org/doi:10.1016/j.jviromet.2016.07.002 VIRMET 13048
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
22-10-2015 21-6-2016 3-7-2016
Please cite this article as: Singla, S., Harjai, K., Raza, K., Wadhwa, S., Katare, O.P., Chhibber, S., Phospholipid vesicles encapsulated bacteriophage: A novel approach to enhance phage biodistribution.Journal of Virological Methods http://dx.doi.org/10.1016/j.jviromet.2016.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Phospholipid vesicles encapsulated bacteriophage: A novel approach to enhance phage biodistribution S. Singlaa, K. Harjaia, K. Razab, S. Wadhwac, O.P. Katarec, S. Chhibbera,* a
Department of Microbiology, Panjab University, Chandigarh-160014, India.
b
c
University Institute of Pharmaceutical Science (UIPS), Panjab University, India
Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of
Rajasthan, India *
Corresponding author. Tel.: +91 172 2534141
E-mail address: [email protected] (S. Chhibber)
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Highlights
We developed liposome vesicles for the effective delivery of bacteriophages.
We examine percent entrapment, stability and in vivo distribution in mice.
Cationic liposomal formulation showed maximum encapsulation efficiency of 92%.
Transmission electron microscopy confirmed the entrapment of phages in liposomes.
Liposome entrapped bacteriophage was retained for longer duration in different organs.
Abstract Phage therapy has been at the centre of attraction for combating multi-drug resistant strains. However, less stability and rapid clearance of phage by mononuclear phagocytic system (MPS) restricts its use in humans. In the present study, aim was to develop a liposomal delivery system for bacteriophage that can assure efficient phage delivery and retention at the site of infection. Different ratios of cholesterol, lipids and surfactant along with different charge inducers were employed to prepare liposomes. Phage was then entrapped in the liposomes and characterized on the basis of morphology, size, entrapment efficiency and stability. Further, in vivo biodistribution of free phage and liposome entrapped phage was compared in different organs of mice. A cationic liposomal formulation showed maximum encapsulation efficiency of 92%. Transmission electron microscopy (TEM) confirmed the entrapment of phages in liposomes. Liposome preparation was found to be most stable at 4°C during storage. Liposome entrapped bacteriophage was retained for longer duration in different organs i.e. upto day 4 in blood, day 6 in liver, lungs and kidney, 14 days in spleen of mice as compared to free phage that became undetectable by 36th h in blood as well as lungs and by 48th h in all other organs.
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Keywords: K. pneumoniae; bacteriophage; liposome entrapment; bacteriophage delivery; biodistribution
1.
Introduction
Klebsiella pneumoniae (an opportunistic pathogen) is responsible for majority of nosocomial infections, mostly in immunocompromised persons. An increase in the emergence of multidrug resistance among K. pneumoniae isolates has renewed interest for alternative approaches for the treatment of K. pneumoniae infections. Various efforts have been made to alleviate this alarming problem. The one such approach has been the use of bacteriophage to eliminate specific bacterial pathogens (Mathur et al., 2003; Sulakvelidze et al., 2001; Thiel, 2004). The ability of bacteriophage to rapidly kill the infected bacteria, their specificity and their proven clinical safety makes them ideal, robust, safe and effective selfreplicating antimicrobial drugs of the future (Carlton et al., 2005; Hanlon, 2007). Although phages have been proposed as therapeutic agents, the technology has not yet been successfully developed. There are still some concerns which need to be addressed, like phage preparations do not arrive at the target site, rapidly removed from the circulation by cells of the mononuclear phagocytic system (MPS) and viability of phages need to be determined every time before using them for therapy. So, a suitable phage delivery system is required to ensure their prolonged survival, better phage retention at target site and reduction in their rapid clearance by MPS. Several nano-delivery systems composed of an assortment of different sizes, shapes, and materials, have been constructed. Among these, liposomes are highly investigated devices as these systems mimic the bio-membrane in terms of structure and bio-behavior.
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Liposomes are multi or uni-layered microscopic vesicular structures composed of phospholipid bilayers trapping the aqueous compartment (Raza et al., 2014; Uchegbu and Vyas, 1998). Since phospholipids are biocompatible and GRAS (generally recognised as safe) ingredients, therefore, they promise a safer and better delivery option. Liposomal phospholipid bilayers have the potential of merging with the cellular membranes that facilitate the release of their contents in the cell interiors (Mudshinge et al., 2011). Liposomes can load drugs of diverse nature irrespective of polarity, charge and size, which is a unique advantage. The optimization of liposomal composition is of significance so as to enhance the drug loading and decrease the drug leakage (Nallamothu et al., 2006). To the best of our knowledge, there are no traceable reports describing strategicallydesigned liposomal vesicles for the effective delivery of bacteriophages. The present study was undertaken to develop an appropriate vesicular carrier for bacteriophage so as to load maximum number of phage particles by keeping a check on other assets like stability and minimal phage leakage. We have also compared the in vivo stability of free phage and liposome entrapped phage.
2.
Materials and Methods
Materials
Phospholipon 90G (Soy phosphatidylcholine; PC) was a kind gift from M/s Lipoid GmbH, Germany. The growth media, antibiotic (amikacin), Tween 80 (T-80), Triton X-100 (TX) and protamine sulphate (PS) were purchased from M/s Himedia Laboratories, India. Cholesterol (CHOL), dicetylphosphate (DCP) and stearylamine (SA) were purchased from
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M/s MP Biomedicals, India. Chloroform and methanol used was of analytical grade. Double distilled water was employed throughout the study.
Methods
2.1.
Preparation of liposomes
Liposomal formulations were developed using well-established rota-evaporation technique (Bhatia et al. 2004). In brief, various ratios of PC and CHOL with different concentrations of T-80 were used (Table 1). PC, CHOL and T-80 (total mass of 100 mg) were dissolved in 10 mL chloroform-methanol mixture (2:1 v/v) in a 250 mL round bottom flask at room temperature. After removal of the organic phase by rotary evaporation under vacuum at 50 ºC and 70 rpm, a thin film was obtained on the inner wall of the flask. The film was desiccated overnight. Aqueous phase i.e. phosphate buffer saline (PBS), pH 7.2 was added to the thin film at 50 ºC and rotated for 10 min to detach the film from the glass wall. The obtained dispersion was sonicated in water bath sonicator for 30 min. The mass ratio of PC, CHOL and T-80 was selected on the basis of vesicle formation and vesicular stability.
2.2.
Introduction of charge inducer
DCP (negative charge inducer) and SA (positive charge inducer) were used in an attempt to increase the stability of liposomal formulation by electrostatic mechanism, keeping all
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other parameters of preparatory technique constant (Table 2).
The charge inducer
concentration was selected on the basis of liposomal dispersion stability.
2.3.
Selection of hydration temperature
Hydration temperature was optimized because phage dispersion is reported to be stable at 37- 40 ºC (Verma et al., 2009). Two sets of preparations were made with selected ratio of lipid, cholesterol and surfactant (F6, PC/CHOL) and with different charge inducers (F9 and F10, PC/CHOL/DCP/SA) in which both thin film formation and hydration was done at 50 ºC and 40 ºC.
2.4.
Entrapment of bacteriophage into liposomes
A fully characterized depolymerase producing lytic bacteriophage KPO1K2 (MTCC 5831) specific for K. pneumoniae B5O55 (MTCC 5832) isolated in our laboratory from sewage sample was used in the present study as aqueous phase for the entrapment of phage in liposomes (Verma et al., 2009). F6 (PC/CHOL), F9 (PC/CHOL/DCP) and F10 (PC/CHOL/SA) were used for the entrapment of bacteriophage into liposomes.
2.5.
Optimization of phage: lipid ratio based on entrapment efficiency
Total lipid (PC, CHOL, T80, SA/DCP) concentration is a crucial factor to decide the loading efficiency of liposomes. The purpose of this study was to optimize the lipid concentration so as to obtain the vesicles with maximum entrapment efficiency. Two sets of formulation having different concentration of total lipids i.e. 1% and 1.5% (molar percent)
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were made as described above. No significant decrease in phage titer was observed when free phage was sonicated for 30 min (data not shown).
2.6.
Plaque Assay
The titer of phage, expressed as PFU, was determined by using the DLA technique as described by Sambrook and Russell (2001). Briefly, 100 μl of phage was added to 100 μl of a bacterial suspension grown overnight at 37°C and 120 rpm. This solution was added to 5 ml of the top agar (nutrient broth with 0.6% Bacto agar), mixed gently, and poured into a 90-mm petri dish previously prepared with 25 ml of nutrient agar. The plates were gently swirled, dried for 10 min at room temperature, and then inverted and incubated at 37°C overnight.
2.7.
Characterization of liposomes
2.7.1. Morphology and structure of vesicles The morphological characteristics of liposomes were monitored viz. shape uniformity and structure by using optical microscope (Olympus CH20i) at suitable magnification.
2.7.2. Average particle size and polydisperity index The particle size and PDI of liposomes was measured using dynamic light scattering (DLS) technique using Beckman Coulter instrument (Delsa Nano C). The samples were appropriately diluted and placed in microcuvette. The conditions used for size measurement were – temperature, 25 ºC; viscosity, 0.887 cP and scattering intensity, 10784 cps.
2.7.3. Zeta potential Beckman Coulter instrument (Delsa Nano C) was used to measure the zeta potential values of developed vesicular samples. The samples were analyzed for ten cycles. 7
2.7.4. Transmission Electron Microscopy (TEM) Morphology and structure of phage-loaded liposome was determined with TEM. F10 (PC/CHOL/T80/SA) was used as a sample for TEM. Following negative staining with 1% aqueous solution of phosphotungustic acid (PTA), liposomes were dried on a microscopic carbon-coated grid. 4. The grid was viewed under Transmission Electron Microscope (TEM, Hitachi H 7500, Tokyo, Japan) at 80 kV at Sophisticated Analytical Instrumentation Facility (SAIF), Panjab University, Chandigarh. Photo-micrographs of liposomes were taken at suitable magnification.
2.7.5. Entrapment efficiency The phage-entrapment efficiency of liposome was determined by aggregation method followed by centrifugation. Total phage number (sum of entrapped and free phages) was determined by adding Triton X-100 (0.02% w/w) in a ratio of 1:1 v/v. Free phage was separated from liposomal phage by protamine aggregation method (Gulati et al., 1998). In this method, aqueous solution of PS was added to liposomal dispersion at a final concentration of 20 mg mL-1 of PS. It was mixed and allowed to stand overnight at room temperature. Subsequently, it was centrifuged at 7,900 x g for 10 minutes at room temperature. Number of free phages was determined in the supernatant using plaque assay. Phage entrapment efficiency was calculated using the following equation: Entrapment efficiency =
𝐸𝑜−𝐸1 𝐸𝑜
× 100
where E0 = total number of phages, E1 = number of free phages, and (E0-E1) represents the number of phages entrapped in the liposome formulation
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2.8.
Stability and Leakage studies
The plain liposome formulation and phage loaded liposome (F10, PC/CHOL/SA) was sealed in glass ampoules (in triplicates). The vials were stored at different temperatures viz. 4±2 ºC, 37±2 ºC and room temperature for a period of at least 3 months. Samples were withdrawn periodically after every 7th day and then size and percentage entrapment of phage was determined. 2.9.
Ethics Statement
The animal experiment protocols were duly approved by the Institutional Animal Ethics Committee (Approval ID: IAEC/156), Panjab University, Chandigarh, India. Animals were kept in clear polypropylene cages and fed on a standard antibiotic-free diet (Hindustan Lever Products, Kolkata, India) and water ad libitum. The temperature ranged between 18 and 22 ºC and the relative humidity was between 55 and 65%. Experiments on animals were completed according to the recent guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India which is in agreement with IACUC. All possible efforts were made to decrease the suffering of animals.
2.10
Toxicity testing of phage and liposome loaded phage
BALB/c mice, weighing 20–25 g of around 6–8 weeks age were employed for this study. Phage suspension toxicity was investigated by the method reported by Soothill (1992). Mice were injected with 0.25 mL phage suspension (109 pfu mL-1) by intraperitoneal (i.p.) route in three groups of five mice each. Three un-injected mice were retained as controls for this group. The mice were observed for signs of illness. Rectal temperature was taken hourly
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during the first 5 h after injection and then daily for the next 4 days. Similarly, the toxicity of liposome entrapped phage was also determined. Plain liposomal suspension, 0.25 mL, was also injected in another group of three mice which acted as control for liposome entrapped phage group. Humane endpoints were not used as none of the animals died.
2.11. Comparison of biodistribution of unentrapped (free) phage and liposome entrapped phage in mice For assessing the in vivo biodistribution of bacteriophage, a group of 18 mice (BALB/c, 20–25 g) was injected intraperitoneally (i.p.) with 0.5mL of phage preparation (109 pfu mL-1). Another group of mice (n=12) was injected with heat inactivated phage suspension that acted as a negative control. Similarly, for checking the in vivo biodistribution of liposome entrapped phage, a group of mice (n=36) was injected i.p. with liposome entrapped phage suspension (109 pfu mL-1). Another group of mice (n=24) was injected with empty liposome that acted as a negative control. After appropriate time intervals, five mice each (3 test and 2 control) from both groups (phage and liposome entrapped phage group), at one time point, were euthanized by cervical dislocation and their organs (lung, liver, spleen, kidney) were removed aseptically into sterile tubes. Phage titer was determined in the supernatant of the homogenized organs and blood. Homogenisation was done in homogenising buffer at room temperature and supernatant was stored in ice. Two mice were injected with sterile normal saline and observed till the duration of experiment.
2.12. Statistical analysis
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All results were expressed as mean ± S.D. obtained from experiments performed in triplicates. Comparisons between free and liposomal formulations were made by Student ttest, and P-values were considered significant when P < 0.05.
3.
Results
3.1.
Preparation of liposomes
Liposomes were prepared using different ratios of phospholipid and cholesterol. Effect of various product-affecting variables like speed of rotation, vacuum, hydration time and hydration media on the liposome formation were studied. The optimum rotational speed of the flask was noted to be 75 rpm, as it yielded a uniform thin film on the flask and subsequently uniform liposomal dispersion. The film was kept overnight under vacuum to achieve complete drying to avoid the formation of emulsion due to the presence of residual organic solvents. Prepared liposomes were observed under binocular microscope for lamellarity and shape. Light microscopy of the preparations (Fig. 1) showed that uniform liposomes with respect to (w.r.t.) size and lamellarity were obtained with PC:CHOL:T80 ratio of 9:1:2. The effect of PC:CHOL ratio was more pronounced at lower concentration of Tween 80. In general, there was increase in average globule size, as the PC:CHOL ratio was increased. However, at higher concentration of Tween 80, initially there was a milder increase followed by substantial decrease of size. Table 3 shows the average size of various liposomal formulations. The interaction between PC liposomes incorporated with varying quantities of Tween 80 surfactant was
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examined in order to elucidate the steric stability of empty liposomes. Experimental results showed that the average liposomal size decreased from 921.6 nm to 576.9 nm when the amount of Tween 80 of the liposome composition was increased from 0.1 to 0.2 (T-80: total lipids; molar ratio). When the ratio of T-80: total lipids was further increased from 0.2 to 0.3, liposomes gradually dissolved into mixed micelles. Therefore, the amount of T-80 used for further studies was kept 0.2 as molar ratio of T-80: total lipids. 3.2.
Introduction of charge inducer Sedimentation, observed mainly with neutral liposomes, was the main problem during
storage. Hence, to increase the further stability of liposomes, several charge inducers i.e. dicetyl phosphate (DCP) and stearylamine (SA) at different concentrations were incorporated into the liposomal bilayer membranes which reduced the chances of liposome-liposome contact due to repulsion as it provided electrical like charges on the surface. The charged liposomes thus showing no sedimentation may indicate better physical stability than the neutral liposomes. Light microscopy of all the preparations (Fig. 2) showed that uniform liposomes w.r.t. size and lamellarity were obtained with the PC:CHOL:T80:DCP/SA ratio of 9:1:2:0.5 and 9:1:2:1. Liposomal formulation having PC:CHOL:T80:DCP/SA ratio of 9:1:2:0.5 showed minimum size and polydispersity index (PDI) (Table 4).
3.3.
Selection of hydration temperature
Two sets of formulations were made with selected concentrations of components in which both thin film formation and hydration was done at 50 ºC and 40 ºC. The liposomal dispersion was found to be almost similar at 50 ºC and 40 °C in terms of average size and homogeneity (Fig. 3). Thus, 40 °C was selected for the preparation of liposome in further experiments.
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3.4.
Entrapment of phage in liposomes
Total lipid (PC, CHOL, T80, SA/DCP) concentration is a crucial factor in deciding the loading efficiency of liposomes (Nallamothu et al., 2006). No decrease in phage titer was observed when free phage was treated with 0.02% w/w of Triton X-100. So, 0.02% w/w of Triton X-100 was safely used with phage to determine total phage. Entrapment of phages increased with increasing total lipid concentration. At 1% total lipid concentration (molar percent), the entrapment efficiencies for negatively charged, positively charged and neutral liposomal formulations were found to be 28, 84 and 43% respectively. For these formulations, the entrapment efficiencies increased to 47, 92 and 71%, respectively when total lipid concentration was increased to the level of 1.5% (molar percent). The F10 (PC/CHOL/SA) formulation was finally selected on account of high physical stability (least aggregation potential), small particle size and relatively higher number of entrapped phages.
3.5.
Electron microscopy
The transmission electron photomicrographs of liposome entrapped phages clearly showed that phages were entrapped within liposome successfully (Fig. 4). Fig. 4(a) shows the morphology of plain liposomes. Fig. 4(b) and 4(c) shows liposomes entrapped phage. Fig 4(c) showed clear structure of KPO1K2 having a short non-contractile tail and icosahedral head with pentagonal nature.
3.6.
Stability study
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3.6.1. Micromeritics The stability of the two systems was determined by comparing the average size over the various storage time points. Fig. 5 presents the mean size of plain liposomes as well as liposome entrapped phages stored at 4 °C, RT and 37 °C. Plain liposomes and phage loaded liposomes were found to be reasonably stable at 4°C in terms of aggregation and size, over the studied storage period. A polydispersity index of 1 indicates large variation in particle size, a reported value of 0 means that size variation is absent.
3.6.2. Leakage on storage Phage leakage studies were performed to assess the ability of the prepared systems to maintain the loading efficiency over a period of time. The effect of different storage temperatures, i.e., 4 °C, RT and 37 °C on the leakage of entrapped phages has been depicted in Fig. 6. Liposome preparation was found to be most stable at 4 °C as there was insignificant reduction of 0.096 logs in the number of entrapped phages (P > 0.01) during entire period of storage. Preparation was found to be less stable at room temperature as there was significant reduction of 0.406 logs in the number of entrapped phages (P < 0.01) whereas it was highly unstable at 37 °C with 1.16 log reduction as compared to initial number of entrapped phages (P < 0.001).
3.6.3. Toxicity testing of phage and liposome loaded phage The phage KPO1K2 and liposome entrapped phage showed no toxicity in mice on intraperitonial injection. The mean rectal temperature at all time points in all the groups of mice injected with bacteriophage and liposome entrapped bacteriophage was 36.7 °C, which was comparable to the temperature of the control group, 37.1 °C. No symptoms of lethargy or
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sickness were noted in the test group during the period of observation. These preparations were thus considered safe for in vivo use.
3.6.4. Comparison of in vivo biodistribution of phage and liposome entrapped phage When mice were inoculated i.p. with 109 pfu mL-1 of bacteriophage suspension, blood titers of phage reached 6.35 pfu mL-1 within 1st hour of injection, which increased to 7.72 pfu mL-1 6 h post inoculation. Maximum phage titer in kidney, spleen, liver, lungs and blood was observed at 6 h post inoculation (Fig. 7a). The subsequent concentration of bacteriophage declined gradually to 2.39, 5.32, 3.73, 5.49, 5.28 pfu mL-1 in blood, liver, lungs, kidney and spleen, respectively 12 h post inoculation. Phage became undetectable by 36th h in blood and by 48th h in lungs, kidneys, liver and spleen. In mice inoculated i.p. with 109 pfu mL-1 of liposome loaded bacteriophage, maximum phage titer of 8.23 pfu mL-1 was observed in blood within 6 h of injection (Fig. 7b) whereas maximum titer of 8.06, 7.29, 7.71 and 7.75 in liver, lungs, kidney and spleen respectively was observed between 6 and 12 h post inoculation (Fig. 7b). This is in contrast to results obtained with free phage as titer started to decline at 12 h post injection instead of 6 h post injection. Concentration of phage fell gradually to 1.07, 3.28, 2.31, 2.01, 3.89 pfu mL-1 in blood, liver, lungs, kidney and spleen, respectively by day 3 of inoculation. Phage became undetectable by day 4 in blood and by day 6 in liver, lungs and kidney whereas phage persisted upto 14 days in spleen.
4.
Discussion
The increase in drug resistance among bacterial pathogens has limited the use of conventional therapies, leading to a search for newer therapeutic agents. Lytic bacteriophages
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with antibacterial property have been recommended as an adjunct to antibiotics. However, certain drawbacks associated with phage therapy have prompted the scientists to look for suitable phage delivery systems. Biocompatible nanomaterials particularly liposomes are preferred now-a-days due to their unique features like size and encapsulation efficiency. In the present study we attempted to adopt a novel approach by incorporating phage particles into liposome by altering physical attributes like lipid composition and surface charge. The bio-distribution of liposome entrapped phages was also evaluated. Preparation and optimization of plain liposomes by using different ratios of phospholipid and cholesterol along with different concentrations of Tween 80 was done. Uniform vesicles were obtained with the PC:CHOL:T-80 ratio of 9:1:2 and their physical stability, including electrostatic and steric stability, was improved by the addition of cholesterol and Tween 80. Though incorporation of cholesterol resulted in enhanced circulation time yet polysorbate 80 (T-80) was incorporated to increase the stability of the system. Sterically stabilized nano-liposomes have been reported to enhance their stability due to their slow clearance and sequestration on in vivo application. The average liposomal size decreased when the amount of T-80 was increased from 0.1 to 0.2. This change might be due to a steric repulsion among the T-80 surfactant molecules, which are exposed from the outer and inner leaflets of the liposomal bilayer membrane (Tasi et al., 2003). On increasing the ratio of T-80: total lipids from 0.2 to 0.3 gradual dissolution of liposome into mixed micelles were observed. This interaction between surfactant and liposomal membrane may be explained via Lichtenberg’s three-step model (Alino et al., 1991). Hence, in the final preparation, the amount of T-80 used for further optimization was 0.2 as molar ratio of T-80: total lipids. Liposomes are thermodynamically unstable and hence are likely to aggregate, fuse, flocculate and precipitate during storage. Increasing interparticle repulsion by modifying the
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surface charges of liposomes is likely to prevent aggregation. So, different charge inducers (SA and DCP) at different concentrations were incorporated into the liposomal bilayer membranes. Earlier researchers have reported that SA at less than 5% concentration (w/w) did not cause hemolysis (Yoshihara and Nakae, 1986). Hence, the formulation having low concentrations of SA and DCP i.e. PC:CHOL:T80:DCP/SA ratio of 9:1:2:0.5 was used for further experiments. Bacteriophage was entrapped within the characterized liposome formulation ratios using 1 % as well as 1.5 % total lipid concentration (molar percent). The results showed that entrapment efficiency of all formulations (neutral, negatively and positively charged) was increased following increase in lipid concentration. This increase can be attributed to increase in number of liposome mL-1 of the formulation. Maximum entrapment was found in positively charged liposome which may be due to the preferential electrostatic adsorption and encasement of negatively charged phage (zeta potential of -15.3 mV) (Anany et al., 2011). The transmission electron photomicrographs of liposome entrapped phage confirmed that phage was entrapped in liposome successfully. Ideal liposomal drug/phage carriers should release the contents only at target site and if such leakage is observed during the shelf life or during in vivo transit before the target site, the purpose of liposomal encapsulation is not achieved in totality (Nallamothu et al., 2006). Thus the ability of vesicles to retain the phage was assessed by keeping the liposomal suspensions at three different temperature conditions, i.e. 4 °C, 25±2 °C (Room temperature; RT) and 37±2 °C for a period of 8 weeks (Tiwari et al., 2000). Liposome preparation was found to be most stable at 4 °C as there was insignificant reduction in the number of entrapped phage during the entire period of storage. The results are in consonance with the previous findings of leakage from liposomes at higher temperatures (Parmar et al., 2010).
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These results indicate that in vitro release testing can be useful for quickly assessing batch-tobatch variation. Biodistribution pattern of phage and liposome entrapped phage was compared to assess whether entrapment increased the retentivity of phage or not. Maximum titer of free phage was retained in vivo only for short duration and became undetectable by 36th hour in blood as well as lungs and by 48th hour in other organs i.e. liver, spleen and kidney. Compared to this, when mice were inoculated with liposome entrapped phage, maximum titer of phage was achieved by 6 h and decreased level of titer persisted till day 4 in blood and day 6 in liver, lungs and kidney and upto 14 days in spleen. The macrophage phagocytic system (MPS) is reported to be the major site of liposome accumulation after systemic administration (Poste and Papahadjopoulos, 1976). Primary organs associated with MPS are liver, spleen, and lungs. The liver exhibited the largest capacity for uptake, whereas the spleen accumulated liposomes for longer duration as its tissue concentration was 10-fold higher than that of other organs. Enhanced biodistribution and bioretention of liposome-entrapped phage may be attributed to biocompatibility and biomembrane mimicking potential of liposome. In addition, the charge on liposomes affects the overall distribution of liposomal carriers as charged liposomes are reported to accumulate preferentially in the spleen, liver and lungs than neutral vesicles (Beaulac et al., 1997). Its longer bioretentivity in lungs and kidneys makes it a suitable candidate for the treatment of K. pneumoniae associated respiratory tract and urinary tract infections. In summary, the results of this study show liposome as potential carriers of phage and have the ability to not only overcomes the hurdles associated with phage therapy but it also increases their availabilty in vivo over a longer period of time. Hence, we speculate that liposome entrapped phage will act as an excellent antibacterial agent that will increase in concentration on interaction with its host. Their prolonged release kinetics in vitro coupled
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with their enhanced survival in blood and various organs on injection in mice proves their utility for human application.
Conflict of Interest The authors declare that they have no conflict of interest.
Acknowledgements We thank Ms. Shikha of the Institute of Microbial Technology (IMTECH), Chandigarh, India for the assistance in the operation of Delsa Nano C. Lead author also acknowledges Department of Science and Technology (DST), New Delhi for awarding her DST-INSPIRE Fellowship.
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Optimization through Drug Loading and In Vitro Release Studies. J. Pharm. Sci. Technol. 60, 144-155. Parmar, J.J., Singh, D.J., Hegde, D.D., Lohade, A.A., Soni, P.S., Samad, A., Menon, M.D., 2010. Development and Evaluation of Inhalational Liposomal System of Budesonide for Better Management of Asthma. Indian J. Pharm. Sci. 72, 442–448. Poste, G., Papahadjopoulos, P., 1976. Lipid Vesicles as Carriers for Introducing Materials into Cultured Cells: Influence of Vesicle Lipid Composition on Mechanism(s) of Vesicle Incorporation into Cells. Proc. Natl. Acad. Sci. (USA). 73, 1603–1607. Raza, K., Kumar, M., Kumar, P., Malik, R., Sharma, G., Kaur, M., Katare, O.P., 2014. Topical delivery of aceclofenac: challenges and promises of novel drug delivery systems. Biomed. Res. Int. 2014, 1-11. Sambrook, J., Russell, D.W., 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Soothill, J.S., 1992. Treatment of experimental infections of mice with bacteriophages. J. Med. Microbiol. 37, 258-261. Sulakvelidze, A., Alavidze, Z., Morris, J.G., 2001. Bacteriophage therapy. Antimicrob. Agents. Chemother. 45, 649–659. Tasi, L.M., Liu, D.Z., Chen, W.Y. 2003. Microcalorimetric investigation of the interaction of polysorbate surfactants with unilamellar phosphatidylcholines liposomes. Coll. Surf. A. 213, 7–14.
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Thiel, K., 2004. Old dogma, new tricks—21st century phage therapy. Nat. Biotechnol. 22, 31–36. Tiwari, S.B., Udupa, N., Rao, B.S.S., Puma, D., 2000. Thermosensiitive liposomes and localized hyperthermia– an effective bimodality approach for tumour management. Int. J. Pharm. 32, 214-220. Uchegbu, I.F., Vyas, S.P. 1998. Non-ionic surfactant based vesicles (niosomes) in drug delivery. Int. J. Pharm. 172, 33-70. Verma, V., Harjai, K., Chhibber, S., 2009. Characterization of a T7-Like Lytic Bacteriophage of Klebsiella pneumoniae B5055: A Potential Therapeutic Agent. Curr. Microbiol. 9, 274–281. Yoshihara, E., Nakae, T., 1986. Cytolytic activity of liposomes containing stearylamine. Biochimica. et. Biophysica. Acta. 854, 93-101.
22
LEGENDS
Figures Fig. 1 Photomicrograph (at 100 X) of different liposomal formulations prepared using different ratios of Phosphatidylcholine: Cholesterol: Tween-80 (PC:CHOL:T80). Bar = 10 µm Fig. 2 Photomicrograph of different liposomal formulations (at 100 X) having different concentrations of charge inducers. Bar = 10 µm Fig. 3 Average size and PDI of liposomal formulations prepared at two hydration temperatures. All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions Fig. 4 Electron micrograph of the liposomal formulation F10 (a) plain liposomes (b) white arrow represents the liposome entrapped phage (300000X). (c) White arrow represents the liposome entrapped phage (120000X) with clear pentagonal structure of phage Fig. 5 Average size of (a) plain liposomal formulation (liposomal formulation F10) (b) liposome entrapped phage (liposomal formulation F10)
at three different storage
temperatures i.e. 4°, RT and 37 °C. All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions Fig. 6 Number of liposome entrapped phages (Log10 pfu mL-1) on storage at different temperatures. All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions Fig. 7 Biodistribution of (a) KPO1K2 in various organs of BALB/c mice (n = 3) (b) liposome entrapped KPO1K2 (formulation F10) in various organs of BALB/c mice (n = 3). All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions
23
24
25
26
27
28
29
30
Tables Table 1 Different ratios of lipid, cholesterol and surfactant used for various liposomal formulations F1
F2
F3
F4
F5
F6
7:3:1
8:2:1
9:1:1
7:3:2
8:2:2
9:1:2
Formulation (PC/Chol) Mass ratio of PC:CHOL:T-80
Table 2 Composition of formulations containing different amount of charge inducer Formulation
Mass ratio of PC:CHOL:T-80:DCP/SA
F7 (PC/Chol/DCP)
9:1:0:1
F8 (PC/Chol/SA)
9:1:0:1
F9 (PC/Chol/DCP)
9:1:2:0.5
F10 (PC/Chol/SA)
9:1:2:0.5
F11 (PC/Chol/DCP)
9:1:2:1
F12 (PC/Chol/SA)
9:1:2:1
31
Table 3 Average size of various liposomal formulations prepared using different ratios of PC:CHOL:T80. All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions Formulation
Formulation ratio
Size (in nm)
(PC:CHOL:T80)
Zeta Potential (mV)
F1
7:3:1
706.4 ± 6.3
-4.31 ± 0.12
F2
8:2:1
892.5 ± 8.5
0.19 ± 0.01
F3
9:1:1
921.6 ± 7.1
-2.11 ± 0.02
F4
7:3:2
746.5 ± 6.7
-0.98 ± 0.03
F5
8:2:2
797.5 ± 3.5
0.07 ± 0.00
F6
9:1:2
576.9 ± 4.1
-0.11 ± 0.01
32
Table 4 Average size and PDI of various liposomal formulations prepared using different ratios of PC:CHOL:T80:DCP/SA. All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions Formulation
Formulation ratio
Size (in nm)
PDI
(PC:CHOL:T-
Zeta Potential (mV)
80:DCP/SA) F7 (PC/Chol/DCP)
9:1:0:1(DCP)
1006 ± 11.2
0.45 ± 0.12
-53.22 ± 2.17
9:1:0:1(SA)
1052.1 ± 13.7
0.367 ± 0.09
47.91 ± 1.97
F9 (PC/Chol/DCP)
9:1:2:0.5(DCP)
175.7 ± 4.9
0.238 ± 0.23
-31.65 ± 2.32
F10 (PC/Chol/SA)
9:1:2:0.5(SA)
120.7 ± 2.7
0.306 ± 0.17
39.04 ± 1.63
F11 (PC/Chol/DCP)
9:1:2:1(DCP)
426.9 ± 7.4
0.334 ± 0.28
-38.03 ± 2.81
9:1:2:1(SA)
540.8 ± 6.8
0.373 ± 0.14
29.76 ± 1.11
F8 (PC/Chol/SA)
F12 (PC/Chol/SA)
33
S0166-0934(15)30054-9 http://dx.doi.org/doi:10.1016/j.jviromet.2016.07.002 VIRMET 13048
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
22-10-2015 21-6-2016 3-7-2016
Please cite this article as: Singla, S., Harjai, K., Raza, K., Wadhwa, S., Katare, O.P., Chhibber, S., Phospholipid vesicles encapsulated bacteriophage: A novel approach to enhance phage biodistribution.Journal of Virological Methods http://dx.doi.org/10.1016/j.jviromet.2016.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Phospholipid vesicles encapsulated bacteriophage: A novel approach to enhance phage biodistribution S. Singlaa, K. Harjaia, K. Razab, S. Wadhwac, O.P. Katarec, S. Chhibbera,* a
Department of Microbiology, Panjab University, Chandigarh-160014, India.
b
c
University Institute of Pharmaceutical Science (UIPS), Panjab University, India
Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of
Rajasthan, India *
Corresponding author. Tel.: +91 172 2534141
E-mail address: [email protected] (S. Chhibber)
1
Highlights
We developed liposome vesicles for the effective delivery of bacteriophages.
We examine percent entrapment, stability and in vivo distribution in mice.
Cationic liposomal formulation showed maximum encapsulation efficiency of 92%.
Transmission electron microscopy confirmed the entrapment of phages in liposomes.
Liposome entrapped bacteriophage was retained for longer duration in different organs.
Abstract Phage therapy has been at the centre of attraction for combating multi-drug resistant strains. However, less stability and rapid clearance of phage by mononuclear phagocytic system (MPS) restricts its use in humans. In the present study, aim was to develop a liposomal delivery system for bacteriophage that can assure efficient phage delivery and retention at the site of infection. Different ratios of cholesterol, lipids and surfactant along with different charge inducers were employed to prepare liposomes. Phage was then entrapped in the liposomes and characterized on the basis of morphology, size, entrapment efficiency and stability. Further, in vivo biodistribution of free phage and liposome entrapped phage was compared in different organs of mice. A cationic liposomal formulation showed maximum encapsulation efficiency of 92%. Transmission electron microscopy (TEM) confirmed the entrapment of phages in liposomes. Liposome preparation was found to be most stable at 4°C during storage. Liposome entrapped bacteriophage was retained for longer duration in different organs i.e. upto day 4 in blood, day 6 in liver, lungs and kidney, 14 days in spleen of mice as compared to free phage that became undetectable by 36th h in blood as well as lungs and by 48th h in all other organs.
2
Keywords: K. pneumoniae; bacteriophage; liposome entrapment; bacteriophage delivery; biodistribution
1.
Introduction
Klebsiella pneumoniae (an opportunistic pathogen) is responsible for majority of nosocomial infections, mostly in immunocompromised persons. An increase in the emergence of multidrug resistance among K. pneumoniae isolates has renewed interest for alternative approaches for the treatment of K. pneumoniae infections. Various efforts have been made to alleviate this alarming problem. The one such approach has been the use of bacteriophage to eliminate specific bacterial pathogens (Mathur et al., 2003; Sulakvelidze et al., 2001; Thiel, 2004). The ability of bacteriophage to rapidly kill the infected bacteria, their specificity and their proven clinical safety makes them ideal, robust, safe and effective selfreplicating antimicrobial drugs of the future (Carlton et al., 2005; Hanlon, 2007). Although phages have been proposed as therapeutic agents, the technology has not yet been successfully developed. There are still some concerns which need to be addressed, like phage preparations do not arrive at the target site, rapidly removed from the circulation by cells of the mononuclear phagocytic system (MPS) and viability of phages need to be determined every time before using them for therapy. So, a suitable phage delivery system is required to ensure their prolonged survival, better phage retention at target site and reduction in their rapid clearance by MPS. Several nano-delivery systems composed of an assortment of different sizes, shapes, and materials, have been constructed. Among these, liposomes are highly investigated devices as these systems mimic the bio-membrane in terms of structure and bio-behavior.
3
Liposomes are multi or uni-layered microscopic vesicular structures composed of phospholipid bilayers trapping the aqueous compartment (Raza et al., 2014; Uchegbu and Vyas, 1998). Since phospholipids are biocompatible and GRAS (generally recognised as safe) ingredients, therefore, they promise a safer and better delivery option. Liposomal phospholipid bilayers have the potential of merging with the cellular membranes that facilitate the release of their contents in the cell interiors (Mudshinge et al., 2011). Liposomes can load drugs of diverse nature irrespective of polarity, charge and size, which is a unique advantage. The optimization of liposomal composition is of significance so as to enhance the drug loading and decrease the drug leakage (Nallamothu et al., 2006). To the best of our knowledge, there are no traceable reports describing strategicallydesigned liposomal vesicles for the effective delivery of bacteriophages. The present study was undertaken to develop an appropriate vesicular carrier for bacteriophage so as to load maximum number of phage particles by keeping a check on other assets like stability and minimal phage leakage. We have also compared the in vivo stability of free phage and liposome entrapped phage.
2.
Materials and Methods
Materials
Phospholipon 90G (Soy phosphatidylcholine; PC) was a kind gift from M/s Lipoid GmbH, Germany. The growth media, antibiotic (amikacin), Tween 80 (T-80), Triton X-100 (TX) and protamine sulphate (PS) were purchased from M/s Himedia Laboratories, India. Cholesterol (CHOL), dicetylphosphate (DCP) and stearylamine (SA) were purchased from
4
M/s MP Biomedicals, India. Chloroform and methanol used was of analytical grade. Double distilled water was employed throughout the study.
Methods
2.1.
Preparation of liposomes
Liposomal formulations were developed using well-established rota-evaporation technique (Bhatia et al. 2004). In brief, various ratios of PC and CHOL with different concentrations of T-80 were used (Table 1). PC, CHOL and T-80 (total mass of 100 mg) were dissolved in 10 mL chloroform-methanol mixture (2:1 v/v) in a 250 mL round bottom flask at room temperature. After removal of the organic phase by rotary evaporation under vacuum at 50 ºC and 70 rpm, a thin film was obtained on the inner wall of the flask. The film was desiccated overnight. Aqueous phase i.e. phosphate buffer saline (PBS), pH 7.2 was added to the thin film at 50 ºC and rotated for 10 min to detach the film from the glass wall. The obtained dispersion was sonicated in water bath sonicator for 30 min. The mass ratio of PC, CHOL and T-80 was selected on the basis of vesicle formation and vesicular stability.
2.2.
Introduction of charge inducer
DCP (negative charge inducer) and SA (positive charge inducer) were used in an attempt to increase the stability of liposomal formulation by electrostatic mechanism, keeping all
5
other parameters of preparatory technique constant (Table 2).
The charge inducer
concentration was selected on the basis of liposomal dispersion stability.
2.3.
Selection of hydration temperature
Hydration temperature was optimized because phage dispersion is reported to be stable at 37- 40 ºC (Verma et al., 2009). Two sets of preparations were made with selected ratio of lipid, cholesterol and surfactant (F6, PC/CHOL) and with different charge inducers (F9 and F10, PC/CHOL/DCP/SA) in which both thin film formation and hydration was done at 50 ºC and 40 ºC.
2.4.
Entrapment of bacteriophage into liposomes
A fully characterized depolymerase producing lytic bacteriophage KPO1K2 (MTCC 5831) specific for K. pneumoniae B5O55 (MTCC 5832) isolated in our laboratory from sewage sample was used in the present study as aqueous phase for the entrapment of phage in liposomes (Verma et al., 2009). F6 (PC/CHOL), F9 (PC/CHOL/DCP) and F10 (PC/CHOL/SA) were used for the entrapment of bacteriophage into liposomes.
2.5.
Optimization of phage: lipid ratio based on entrapment efficiency
Total lipid (PC, CHOL, T80, SA/DCP) concentration is a crucial factor to decide the loading efficiency of liposomes. The purpose of this study was to optimize the lipid concentration so as to obtain the vesicles with maximum entrapment efficiency. Two sets of formulation having different concentration of total lipids i.e. 1% and 1.5% (molar percent)
6
were made as described above. No significant decrease in phage titer was observed when free phage was sonicated for 30 min (data not shown).
2.6.
Plaque Assay
The titer of phage, expressed as PFU, was determined by using the DLA technique as described by Sambrook and Russell (2001). Briefly, 100 μl of phage was added to 100 μl of a bacterial suspension grown overnight at 37°C and 120 rpm. This solution was added to 5 ml of the top agar (nutrient broth with 0.6% Bacto agar), mixed gently, and poured into a 90-mm petri dish previously prepared with 25 ml of nutrient agar. The plates were gently swirled, dried for 10 min at room temperature, and then inverted and incubated at 37°C overnight.
2.7.
Characterization of liposomes
2.7.1. Morphology and structure of vesicles The morphological characteristics of liposomes were monitored viz. shape uniformity and structure by using optical microscope (Olympus CH20i) at suitable magnification.
2.7.2. Average particle size and polydisperity index The particle size and PDI of liposomes was measured using dynamic light scattering (DLS) technique using Beckman Coulter instrument (Delsa Nano C). The samples were appropriately diluted and placed in microcuvette. The conditions used for size measurement were – temperature, 25 ºC; viscosity, 0.887 cP and scattering intensity, 10784 cps.
2.7.3. Zeta potential Beckman Coulter instrument (Delsa Nano C) was used to measure the zeta potential values of developed vesicular samples. The samples were analyzed for ten cycles. 7
2.7.4. Transmission Electron Microscopy (TEM) Morphology and structure of phage-loaded liposome was determined with TEM. F10 (PC/CHOL/T80/SA) was used as a sample for TEM. Following negative staining with 1% aqueous solution of phosphotungustic acid (PTA), liposomes were dried on a microscopic carbon-coated grid. 4. The grid was viewed under Transmission Electron Microscope (TEM, Hitachi H 7500, Tokyo, Japan) at 80 kV at Sophisticated Analytical Instrumentation Facility (SAIF), Panjab University, Chandigarh. Photo-micrographs of liposomes were taken at suitable magnification.
2.7.5. Entrapment efficiency The phage-entrapment efficiency of liposome was determined by aggregation method followed by centrifugation. Total phage number (sum of entrapped and free phages) was determined by adding Triton X-100 (0.02% w/w) in a ratio of 1:1 v/v. Free phage was separated from liposomal phage by protamine aggregation method (Gulati et al., 1998). In this method, aqueous solution of PS was added to liposomal dispersion at a final concentration of 20 mg mL-1 of PS. It was mixed and allowed to stand overnight at room temperature. Subsequently, it was centrifuged at 7,900 x g for 10 minutes at room temperature. Number of free phages was determined in the supernatant using plaque assay. Phage entrapment efficiency was calculated using the following equation: Entrapment efficiency =
𝐸𝑜−𝐸1 𝐸𝑜
× 100
where E0 = total number of phages, E1 = number of free phages, and (E0-E1) represents the number of phages entrapped in the liposome formulation
8
2.8.
Stability and Leakage studies
The plain liposome formulation and phage loaded liposome (F10, PC/CHOL/SA) was sealed in glass ampoules (in triplicates). The vials were stored at different temperatures viz. 4±2 ºC, 37±2 ºC and room temperature for a period of at least 3 months. Samples were withdrawn periodically after every 7th day and then size and percentage entrapment of phage was determined. 2.9.
Ethics Statement
The animal experiment protocols were duly approved by the Institutional Animal Ethics Committee (Approval ID: IAEC/156), Panjab University, Chandigarh, India. Animals were kept in clear polypropylene cages and fed on a standard antibiotic-free diet (Hindustan Lever Products, Kolkata, India) and water ad libitum. The temperature ranged between 18 and 22 ºC and the relative humidity was between 55 and 65%. Experiments on animals were completed according to the recent guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India which is in agreement with IACUC. All possible efforts were made to decrease the suffering of animals.
2.10
Toxicity testing of phage and liposome loaded phage
BALB/c mice, weighing 20–25 g of around 6–8 weeks age were employed for this study. Phage suspension toxicity was investigated by the method reported by Soothill (1992). Mice were injected with 0.25 mL phage suspension (109 pfu mL-1) by intraperitoneal (i.p.) route in three groups of five mice each. Three un-injected mice were retained as controls for this group. The mice were observed for signs of illness. Rectal temperature was taken hourly
9
during the first 5 h after injection and then daily for the next 4 days. Similarly, the toxicity of liposome entrapped phage was also determined. Plain liposomal suspension, 0.25 mL, was also injected in another group of three mice which acted as control for liposome entrapped phage group. Humane endpoints were not used as none of the animals died.
2.11. Comparison of biodistribution of unentrapped (free) phage and liposome entrapped phage in mice For assessing the in vivo biodistribution of bacteriophage, a group of 18 mice (BALB/c, 20–25 g) was injected intraperitoneally (i.p.) with 0.5mL of phage preparation (109 pfu mL-1). Another group of mice (n=12) was injected with heat inactivated phage suspension that acted as a negative control. Similarly, for checking the in vivo biodistribution of liposome entrapped phage, a group of mice (n=36) was injected i.p. with liposome entrapped phage suspension (109 pfu mL-1). Another group of mice (n=24) was injected with empty liposome that acted as a negative control. After appropriate time intervals, five mice each (3 test and 2 control) from both groups (phage and liposome entrapped phage group), at one time point, were euthanized by cervical dislocation and their organs (lung, liver, spleen, kidney) were removed aseptically into sterile tubes. Phage titer was determined in the supernatant of the homogenized organs and blood. Homogenisation was done in homogenising buffer at room temperature and supernatant was stored in ice. Two mice were injected with sterile normal saline and observed till the duration of experiment.
2.12. Statistical analysis
10
All results were expressed as mean ± S.D. obtained from experiments performed in triplicates. Comparisons between free and liposomal formulations were made by Student ttest, and P-values were considered significant when P < 0.05.
3.
Results
3.1.
Preparation of liposomes
Liposomes were prepared using different ratios of phospholipid and cholesterol. Effect of various product-affecting variables like speed of rotation, vacuum, hydration time and hydration media on the liposome formation were studied. The optimum rotational speed of the flask was noted to be 75 rpm, as it yielded a uniform thin film on the flask and subsequently uniform liposomal dispersion. The film was kept overnight under vacuum to achieve complete drying to avoid the formation of emulsion due to the presence of residual organic solvents. Prepared liposomes were observed under binocular microscope for lamellarity and shape. Light microscopy of the preparations (Fig. 1) showed that uniform liposomes with respect to (w.r.t.) size and lamellarity were obtained with PC:CHOL:T80 ratio of 9:1:2. The effect of PC:CHOL ratio was more pronounced at lower concentration of Tween 80. In general, there was increase in average globule size, as the PC:CHOL ratio was increased. However, at higher concentration of Tween 80, initially there was a milder increase followed by substantial decrease of size. Table 3 shows the average size of various liposomal formulations. The interaction between PC liposomes incorporated with varying quantities of Tween 80 surfactant was
11
examined in order to elucidate the steric stability of empty liposomes. Experimental results showed that the average liposomal size decreased from 921.6 nm to 576.9 nm when the amount of Tween 80 of the liposome composition was increased from 0.1 to 0.2 (T-80: total lipids; molar ratio). When the ratio of T-80: total lipids was further increased from 0.2 to 0.3, liposomes gradually dissolved into mixed micelles. Therefore, the amount of T-80 used for further studies was kept 0.2 as molar ratio of T-80: total lipids. 3.2.
Introduction of charge inducer Sedimentation, observed mainly with neutral liposomes, was the main problem during
storage. Hence, to increase the further stability of liposomes, several charge inducers i.e. dicetyl phosphate (DCP) and stearylamine (SA) at different concentrations were incorporated into the liposomal bilayer membranes which reduced the chances of liposome-liposome contact due to repulsion as it provided electrical like charges on the surface. The charged liposomes thus showing no sedimentation may indicate better physical stability than the neutral liposomes. Light microscopy of all the preparations (Fig. 2) showed that uniform liposomes w.r.t. size and lamellarity were obtained with the PC:CHOL:T80:DCP/SA ratio of 9:1:2:0.5 and 9:1:2:1. Liposomal formulation having PC:CHOL:T80:DCP/SA ratio of 9:1:2:0.5 showed minimum size and polydispersity index (PDI) (Table 4).
3.3.
Selection of hydration temperature
Two sets of formulations were made with selected concentrations of components in which both thin film formation and hydration was done at 50 ºC and 40 ºC. The liposomal dispersion was found to be almost similar at 50 ºC and 40 °C in terms of average size and homogeneity (Fig. 3). Thus, 40 °C was selected for the preparation of liposome in further experiments.
12
3.4.
Entrapment of phage in liposomes
Total lipid (PC, CHOL, T80, SA/DCP) concentration is a crucial factor in deciding the loading efficiency of liposomes (Nallamothu et al., 2006). No decrease in phage titer was observed when free phage was treated with 0.02% w/w of Triton X-100. So, 0.02% w/w of Triton X-100 was safely used with phage to determine total phage. Entrapment of phages increased with increasing total lipid concentration. At 1% total lipid concentration (molar percent), the entrapment efficiencies for negatively charged, positively charged and neutral liposomal formulations were found to be 28, 84 and 43% respectively. For these formulations, the entrapment efficiencies increased to 47, 92 and 71%, respectively when total lipid concentration was increased to the level of 1.5% (molar percent). The F10 (PC/CHOL/SA) formulation was finally selected on account of high physical stability (least aggregation potential), small particle size and relatively higher number of entrapped phages.
3.5.
Electron microscopy
The transmission electron photomicrographs of liposome entrapped phages clearly showed that phages were entrapped within liposome successfully (Fig. 4). Fig. 4(a) shows the morphology of plain liposomes. Fig. 4(b) and 4(c) shows liposomes entrapped phage. Fig 4(c) showed clear structure of KPO1K2 having a short non-contractile tail and icosahedral head with pentagonal nature.
3.6.
Stability study
13
3.6.1. Micromeritics The stability of the two systems was determined by comparing the average size over the various storage time points. Fig. 5 presents the mean size of plain liposomes as well as liposome entrapped phages stored at 4 °C, RT and 37 °C. Plain liposomes and phage loaded liposomes were found to be reasonably stable at 4°C in terms of aggregation and size, over the studied storage period. A polydispersity index of 1 indicates large variation in particle size, a reported value of 0 means that size variation is absent.
3.6.2. Leakage on storage Phage leakage studies were performed to assess the ability of the prepared systems to maintain the loading efficiency over a period of time. The effect of different storage temperatures, i.e., 4 °C, RT and 37 °C on the leakage of entrapped phages has been depicted in Fig. 6. Liposome preparation was found to be most stable at 4 °C as there was insignificant reduction of 0.096 logs in the number of entrapped phages (P > 0.01) during entire period of storage. Preparation was found to be less stable at room temperature as there was significant reduction of 0.406 logs in the number of entrapped phages (P < 0.01) whereas it was highly unstable at 37 °C with 1.16 log reduction as compared to initial number of entrapped phages (P < 0.001).
3.6.3. Toxicity testing of phage and liposome loaded phage The phage KPO1K2 and liposome entrapped phage showed no toxicity in mice on intraperitonial injection. The mean rectal temperature at all time points in all the groups of mice injected with bacteriophage and liposome entrapped bacteriophage was 36.7 °C, which was comparable to the temperature of the control group, 37.1 °C. No symptoms of lethargy or
14
sickness were noted in the test group during the period of observation. These preparations were thus considered safe for in vivo use.
3.6.4. Comparison of in vivo biodistribution of phage and liposome entrapped phage When mice were inoculated i.p. with 109 pfu mL-1 of bacteriophage suspension, blood titers of phage reached 6.35 pfu mL-1 within 1st hour of injection, which increased to 7.72 pfu mL-1 6 h post inoculation. Maximum phage titer in kidney, spleen, liver, lungs and blood was observed at 6 h post inoculation (Fig. 7a). The subsequent concentration of bacteriophage declined gradually to 2.39, 5.32, 3.73, 5.49, 5.28 pfu mL-1 in blood, liver, lungs, kidney and spleen, respectively 12 h post inoculation. Phage became undetectable by 36th h in blood and by 48th h in lungs, kidneys, liver and spleen. In mice inoculated i.p. with 109 pfu mL-1 of liposome loaded bacteriophage, maximum phage titer of 8.23 pfu mL-1 was observed in blood within 6 h of injection (Fig. 7b) whereas maximum titer of 8.06, 7.29, 7.71 and 7.75 in liver, lungs, kidney and spleen respectively was observed between 6 and 12 h post inoculation (Fig. 7b). This is in contrast to results obtained with free phage as titer started to decline at 12 h post injection instead of 6 h post injection. Concentration of phage fell gradually to 1.07, 3.28, 2.31, 2.01, 3.89 pfu mL-1 in blood, liver, lungs, kidney and spleen, respectively by day 3 of inoculation. Phage became undetectable by day 4 in blood and by day 6 in liver, lungs and kidney whereas phage persisted upto 14 days in spleen.
4.
Discussion
The increase in drug resistance among bacterial pathogens has limited the use of conventional therapies, leading to a search for newer therapeutic agents. Lytic bacteriophages
15
with antibacterial property have been recommended as an adjunct to antibiotics. However, certain drawbacks associated with phage therapy have prompted the scientists to look for suitable phage delivery systems. Biocompatible nanomaterials particularly liposomes are preferred now-a-days due to their unique features like size and encapsulation efficiency. In the present study we attempted to adopt a novel approach by incorporating phage particles into liposome by altering physical attributes like lipid composition and surface charge. The bio-distribution of liposome entrapped phages was also evaluated. Preparation and optimization of plain liposomes by using different ratios of phospholipid and cholesterol along with different concentrations of Tween 80 was done. Uniform vesicles were obtained with the PC:CHOL:T-80 ratio of 9:1:2 and their physical stability, including electrostatic and steric stability, was improved by the addition of cholesterol and Tween 80. Though incorporation of cholesterol resulted in enhanced circulation time yet polysorbate 80 (T-80) was incorporated to increase the stability of the system. Sterically stabilized nano-liposomes have been reported to enhance their stability due to their slow clearance and sequestration on in vivo application. The average liposomal size decreased when the amount of T-80 was increased from 0.1 to 0.2. This change might be due to a steric repulsion among the T-80 surfactant molecules, which are exposed from the outer and inner leaflets of the liposomal bilayer membrane (Tasi et al., 2003). On increasing the ratio of T-80: total lipids from 0.2 to 0.3 gradual dissolution of liposome into mixed micelles were observed. This interaction between surfactant and liposomal membrane may be explained via Lichtenberg’s three-step model (Alino et al., 1991). Hence, in the final preparation, the amount of T-80 used for further optimization was 0.2 as molar ratio of T-80: total lipids. Liposomes are thermodynamically unstable and hence are likely to aggregate, fuse, flocculate and precipitate during storage. Increasing interparticle repulsion by modifying the
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surface charges of liposomes is likely to prevent aggregation. So, different charge inducers (SA and DCP) at different concentrations were incorporated into the liposomal bilayer membranes. Earlier researchers have reported that SA at less than 5% concentration (w/w) did not cause hemolysis (Yoshihara and Nakae, 1986). Hence, the formulation having low concentrations of SA and DCP i.e. PC:CHOL:T80:DCP/SA ratio of 9:1:2:0.5 was used for further experiments. Bacteriophage was entrapped within the characterized liposome formulation ratios using 1 % as well as 1.5 % total lipid concentration (molar percent). The results showed that entrapment efficiency of all formulations (neutral, negatively and positively charged) was increased following increase in lipid concentration. This increase can be attributed to increase in number of liposome mL-1 of the formulation. Maximum entrapment was found in positively charged liposome which may be due to the preferential electrostatic adsorption and encasement of negatively charged phage (zeta potential of -15.3 mV) (Anany et al., 2011). The transmission electron photomicrographs of liposome entrapped phage confirmed that phage was entrapped in liposome successfully. Ideal liposomal drug/phage carriers should release the contents only at target site and if such leakage is observed during the shelf life or during in vivo transit before the target site, the purpose of liposomal encapsulation is not achieved in totality (Nallamothu et al., 2006). Thus the ability of vesicles to retain the phage was assessed by keeping the liposomal suspensions at three different temperature conditions, i.e. 4 °C, 25±2 °C (Room temperature; RT) and 37±2 °C for a period of 8 weeks (Tiwari et al., 2000). Liposome preparation was found to be most stable at 4 °C as there was insignificant reduction in the number of entrapped phage during the entire period of storage. The results are in consonance with the previous findings of leakage from liposomes at higher temperatures (Parmar et al., 2010).
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These results indicate that in vitro release testing can be useful for quickly assessing batch-tobatch variation. Biodistribution pattern of phage and liposome entrapped phage was compared to assess whether entrapment increased the retentivity of phage or not. Maximum titer of free phage was retained in vivo only for short duration and became undetectable by 36th hour in blood as well as lungs and by 48th hour in other organs i.e. liver, spleen and kidney. Compared to this, when mice were inoculated with liposome entrapped phage, maximum titer of phage was achieved by 6 h and decreased level of titer persisted till day 4 in blood and day 6 in liver, lungs and kidney and upto 14 days in spleen. The macrophage phagocytic system (MPS) is reported to be the major site of liposome accumulation after systemic administration (Poste and Papahadjopoulos, 1976). Primary organs associated with MPS are liver, spleen, and lungs. The liver exhibited the largest capacity for uptake, whereas the spleen accumulated liposomes for longer duration as its tissue concentration was 10-fold higher than that of other organs. Enhanced biodistribution and bioretention of liposome-entrapped phage may be attributed to biocompatibility and biomembrane mimicking potential of liposome. In addition, the charge on liposomes affects the overall distribution of liposomal carriers as charged liposomes are reported to accumulate preferentially in the spleen, liver and lungs than neutral vesicles (Beaulac et al., 1997). Its longer bioretentivity in lungs and kidneys makes it a suitable candidate for the treatment of K. pneumoniae associated respiratory tract and urinary tract infections. In summary, the results of this study show liposome as potential carriers of phage and have the ability to not only overcomes the hurdles associated with phage therapy but it also increases their availabilty in vivo over a longer period of time. Hence, we speculate that liposome entrapped phage will act as an excellent antibacterial agent that will increase in concentration on interaction with its host. Their prolonged release kinetics in vitro coupled
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with their enhanced survival in blood and various organs on injection in mice proves their utility for human application.
Conflict of Interest The authors declare that they have no conflict of interest.
Acknowledgements We thank Ms. Shikha of the Institute of Microbial Technology (IMTECH), Chandigarh, India for the assistance in the operation of Delsa Nano C. Lead author also acknowledges Department of Science and Technology (DST), New Delhi for awarding her DST-INSPIRE Fellowship.
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References
Alino, S.F., Iruarrizaga, A., Alfaro, J., Pajean, M., Herbage, D., 1991. Stabilization of liposomes with collagen. Int. J. Pharm. 77, 33–40. Anany, H., Chen, W., Pelton, R., Griffiths, M.W., 2011. Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in Meat by Using Phages Immobilized on Modified Cellulose Membranes. Appl. Environ. Microbiol. 77, 6379-6387. Beaulac, C., Clement-Major, S., Hawari, J., Lagace, J., 1997. In vitro kinetics of drug release and pulmonary retention of microencapsulated antibiotic in liposomal formulations in relation to the lipid composition. J. Microencapsul. 14, 335-348. Bhatia, A., Kumar, R., Katare, O.P., 2004. Tamoxifen-entrapped topical liposomes: development, characterization and in vitro evaluation. J. Pharm. Pharma. Sci. 7, 252259. Carlton, R.M., Noordman, W.H., Biswas, B., de Meester, E.D., Loessner, M.J., 2005. Bacteriophage P100 for control of Listeria monocytogenes in foods: Genome sequence, bioinformatic analyses, oral toxicity study, and application. Regul. Toxicol. Pharmacol. 43, 301-312. Gulati, M., Grover, M., Singh, M., Singh, S., 1998. Study of azathioprine encapsulation into liposomes. J. Microencapsulation. 5, 485-494. Hanlon, G.W., 2007. Bacteriophages: an appraisal of their role in the treatment of bacterial infections. Int. J. Antimicrob. Agents. 30, 118–128. Mathur, M.D., Bidhani, S., Mehndiratta, P.L., 2003. Bacteriophage therapy: an alternative to conventional antibiotics. J. Assoc. Physicians. India. 51, 593–596.
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Mudshinge, S.R., Deore, A.B., Patil, S., Bhalgat, C.M., 2011. Nanoparticles: Emerging carriers for drug delivery. Saudi Pharm. J. 19, 129–141. Nallamothu, R., Wood, G.C., Kiani, M.F., Moore, B.M., Horton, F.P., Thoma, L.A., 2006. A Targeted
Liposome
Delivery System for Combretastatin A4: Formulation
Optimization through Drug Loading and In Vitro Release Studies. J. Pharm. Sci. Technol. 60, 144-155. Parmar, J.J., Singh, D.J., Hegde, D.D., Lohade, A.A., Soni, P.S., Samad, A., Menon, M.D., 2010. Development and Evaluation of Inhalational Liposomal System of Budesonide for Better Management of Asthma. Indian J. Pharm. Sci. 72, 442–448. Poste, G., Papahadjopoulos, P., 1976. Lipid Vesicles as Carriers for Introducing Materials into Cultured Cells: Influence of Vesicle Lipid Composition on Mechanism(s) of Vesicle Incorporation into Cells. Proc. Natl. Acad. Sci. (USA). 73, 1603–1607. Raza, K., Kumar, M., Kumar, P., Malik, R., Sharma, G., Kaur, M., Katare, O.P., 2014. Topical delivery of aceclofenac: challenges and promises of novel drug delivery systems. Biomed. Res. Int. 2014, 1-11. Sambrook, J., Russell, D.W., 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Soothill, J.S., 1992. Treatment of experimental infections of mice with bacteriophages. J. Med. Microbiol. 37, 258-261. Sulakvelidze, A., Alavidze, Z., Morris, J.G., 2001. Bacteriophage therapy. Antimicrob. Agents. Chemother. 45, 649–659. Tasi, L.M., Liu, D.Z., Chen, W.Y. 2003. Microcalorimetric investigation of the interaction of polysorbate surfactants with unilamellar phosphatidylcholines liposomes. Coll. Surf. A. 213, 7–14.
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Thiel, K., 2004. Old dogma, new tricks—21st century phage therapy. Nat. Biotechnol. 22, 31–36. Tiwari, S.B., Udupa, N., Rao, B.S.S., Puma, D., 2000. Thermosensiitive liposomes and localized hyperthermia– an effective bimodality approach for tumour management. Int. J. Pharm. 32, 214-220. Uchegbu, I.F., Vyas, S.P. 1998. Non-ionic surfactant based vesicles (niosomes) in drug delivery. Int. J. Pharm. 172, 33-70. Verma, V., Harjai, K., Chhibber, S., 2009. Characterization of a T7-Like Lytic Bacteriophage of Klebsiella pneumoniae B5055: A Potential Therapeutic Agent. Curr. Microbiol. 9, 274–281. Yoshihara, E., Nakae, T., 1986. Cytolytic activity of liposomes containing stearylamine. Biochimica. et. Biophysica. Acta. 854, 93-101.
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LEGENDS
Figures Fig. 1 Photomicrograph (at 100 X) of different liposomal formulations prepared using different ratios of Phosphatidylcholine: Cholesterol: Tween-80 (PC:CHOL:T80). Bar = 10 µm Fig. 2 Photomicrograph of different liposomal formulations (at 100 X) having different concentrations of charge inducers. Bar = 10 µm Fig. 3 Average size and PDI of liposomal formulations prepared at two hydration temperatures. All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions Fig. 4 Electron micrograph of the liposomal formulation F10 (a) plain liposomes (b) white arrow represents the liposome entrapped phage (300000X). (c) White arrow represents the liposome entrapped phage (120000X) with clear pentagonal structure of phage Fig. 5 Average size of (a) plain liposomal formulation (liposomal formulation F10) (b) liposome entrapped phage (liposomal formulation F10)
at three different storage
temperatures i.e. 4°, RT and 37 °C. All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions Fig. 6 Number of liposome entrapped phages (Log10 pfu mL-1) on storage at different temperatures. All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions Fig. 7 Biodistribution of (a) KPO1K2 in various organs of BALB/c mice (n = 3) (b) liposome entrapped KPO1K2 (formulation F10) in various organs of BALB/c mice (n = 3). All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions
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25
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27
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29
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Tables Table 1 Different ratios of lipid, cholesterol and surfactant used for various liposomal formulations F1
F2
F3
F4
F5
F6
7:3:1
8:2:1
9:1:1
7:3:2
8:2:2
9:1:2
Formulation (PC/Chol) Mass ratio of PC:CHOL:T-80
Table 2 Composition of formulations containing different amount of charge inducer Formulation
Mass ratio of PC:CHOL:T-80:DCP/SA
F7 (PC/Chol/DCP)
9:1:0:1
F8 (PC/Chol/SA)
9:1:0:1
F9 (PC/Chol/DCP)
9:1:2:0.5
F10 (PC/Chol/SA)
9:1:2:0.5
F11 (PC/Chol/DCP)
9:1:2:1
F12 (PC/Chol/SA)
9:1:2:1
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Table 3 Average size of various liposomal formulations prepared using different ratios of PC:CHOL:T80. All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions Formulation
Formulation ratio
Size (in nm)
(PC:CHOL:T80)
Zeta Potential (mV)
F1
7:3:1
706.4 ± 6.3
-4.31 ± 0.12
F2
8:2:1
892.5 ± 8.5
0.19 ± 0.01
F3
9:1:1
921.6 ± 7.1
-2.11 ± 0.02
F4
7:3:2
746.5 ± 6.7
-0.98 ± 0.03
F5
8:2:2
797.5 ± 3.5
0.07 ± 0.00
F6
9:1:2
576.9 ± 4.1
-0.11 ± 0.01
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Table 4 Average size and PDI of various liposomal formulations prepared using different ratios of PC:CHOL:T80:DCP/SA. All values represent the mean ± SEM, calculated from two independent experiments, each performed in duplicate on different occasions Formulation
Formulation ratio
Size (in nm)
PDI
(PC:CHOL:T-
Zeta Potential (mV)
80:DCP/SA) F7 (PC/Chol/DCP)
9:1:0:1(DCP)
1006 ± 11.2
0.45 ± 0.12
-53.22 ± 2.17
9:1:0:1(SA)
1052.1 ± 13.7
0.367 ± 0.09
47.91 ± 1.97
F9 (PC/Chol/DCP)
9:1:2:0.5(DCP)
175.7 ± 4.9
0.238 ± 0.23
-31.65 ± 2.32
F10 (PC/Chol/SA)
9:1:2:0.5(SA)
120.7 ± 2.7
0.306 ± 0.17
39.04 ± 1.63
F11 (PC/Chol/DCP)
9:1:2:1(DCP)
426.9 ± 7.4
0.334 ± 0.28
-38.03 ± 2.81
9:1:2:1(SA)
540.8 ± 6.8
0.373 ± 0.14
29.76 ± 1.11
F8 (PC/Chol/SA)
F12 (PC/Chol/SA)
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