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Diffusion of fluorescently labeled bacteriocin from edible nanomaterials and embedded nano-bioactive coatings Muhammad Imran, Anne-Marie Revol-Junelles, Grégory Francius, and Stéphane Desobry ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04621 • Publication Date (Web): 28 Jul 2016 Downloaded from http://pubs.acs.org on July 29, 2016

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ACS Applied Materials & Interfaces

Diffusion of fluorescently labeled bacteriocin from edible nanomaterials and embedded nano-bioactive coatings Authors: Muhammad Imran‡Ͼ*, Anne-Marie Revol-Junelles‡, Grégory Francius†, Stéphane Desobry‡

‡

Laboratoire d'Ingénierie des Biomolécules (LIBio), ENSAIA–INPL, Université de Lorraine, 2

avenue de la Forêt de Haye, 54505 Vandoeuvre-lès-Nancy Cedex, France Ͼ

Department of Biosciences, COMSATS Institute of Information Technology, Park road,

Islamabad, Pakistan †

Laboratoire de Chimie Physique et Microbiologie pour l'Environnement (LCPME), 405 rue de

Vandoeuvre F-54600 Villers-lès-Nancy, France

*

Corresponding Author: Dr. Muhammad Imran

Laboratoire d'Ingénierie des Biomolécules, Université de Lorraine, Nancy, France Assistant Professor, Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan. Email: [email protected] Tel.:

+92.322.6017784

1 ACS Paragon Plus Environment


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Abstract Application of nanobiotechnology to improve the controlled release of drugs or functional agents is widely anticipated to transform the biomedical, pharmaceutical and food safety trends. The purpose of the current study was to assess and compare the release-rates of fluorescently-labeled antimicrobial peptide nisin (lantibiotic/biopreservative) from liposomal nano-carriers. The elevated temperature, high electrostatic attraction between anionic bilayers and cationic nisin, larger size and higher encapsulation efficiency resulted in rapid and elevated release through pore-formation. However, acidic pH and optimal ethanol concentration in food simulating liquid (FSL) improved the stability and retention capacity of loaded drug. Thus controlling various factors had provided partition coefficient K values from 0.23 to 8.78 indicating variation in nisin affinity towards encapsulating macromolecule or FSL. Interaction between nisin and nano-scale bilayer systems by atomic force (AFM) and transmission electron microscopy (TEM) demonstrated membrane activity of nisin from adsorption, aggregation to pore formation. Novel nano-active films with pre-loaded nanoliposomes embedded in biodegradable polymer revealed improved morphological, topographic and roughness parameters (Ra, Rq, r %) studied by confocal microscopy and AFM. Pre-encapsulated nano-active biopolymer demonstrated excellent retention capacity as drug carriers by decreasing the partition coefficient value from 1.8 to 0.66 (~30%) due to improved stability of nano-liposomes embedded in biopolymer network. Keywords: Sustained release, fluorescence spectroscopy, nanoliposome, nanoactive coatings, partition coefficient


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1. Introduction The application of nanobiotechnology to drug delivery is widely expected to transform the pharmaceutical, biomedical and biotechnology trends1-3. Among the most promising antimicrobial agents that are being investigated as substitutes for current antibiotics are antibacterial peptides, such as the lanthionine-containing peptides (lantibiotics)1. Efficient antimicrobial action of lantibiotic nisin against Gram positive bacteria and its low-toxicity have permitted the application of nisin as a biopreservative in the food industries (12.5 mg pure nisin / Kg food product) 4-6 or as an antibiotic in health care 7-8, though health applications are still at the level of potential utilization9. Due to exceptional double mode of action, either non-specific or lipid-II dependent pore formation in cell membranes7, 10, lantibiotics are effective against food borne microbes including Listeria monocytogenes and Clostridium botulinum11-12. Dual mechanisms of membrane pore-formation have led to the concept of encapsulating nisin in cell membrane model i.e. liposome, to ensure its controlled release. Active agents including nisin can not just be added to the product, since they demonstrate a limited stability against chemical or physical degradation, or undergo an uncontrolled release or bioavailability1314

. Liposomes, spherical and self sealing structures with high drug loading capacity, are

considered as biocompatible, biodegradable and non-toxic15. Such drug delivery systems (DDS) are intended to maintain effective drug concentrations over longer time periods. Moreover, liposomes are unique because they can enclose both hydrophilic and hydrophobic molecules due to amphiphilic nature of phospholipids. As nisin has amphipathic character, it is encapsulated concurrently in the central cavity and bilayers of liposomes, which creates the prospect for enhanced controlled release16. Release of pore-forming peptides from liposomes is described as: (a) the released amount increases with time till it reaches its final extent which is dependent on the lipid/peptide ratio; (b) the liberation of vesicle contents follows an all-or-none mechanism, i.e., some of the vesicles release all of their contents, whereas others retain all their contents; (c) the release depends on the size of the vesicles, pores and encapsulated molecules17. The pore-forming peptide GALA (WEAALAEALAEALAEHLAEALAEALEALAA), a 30 amino acid synthetic peptide with a glutamic acid-alanine-leucine-alanine (EALA) repeat, had demonstrated lesser release with decreasing vesicle diameter due to smaller number of bound peptides with liposomes17. Accordingly with emerging nanotechnology trends, nanoliposomes are fabricated as discrete 3 ACS Paragon Plus Environment


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bilayer particles having dimensions of the order of 100 nm or less. Small size in combination with the chemical composition and surface structure can enable nanoliposomes with huge potential as DDS for the controlled release of antimicrobials18-19. One of the key restriction of liposomes based DDS is their tendency to discharge the loaded contents due to pH and mechanical shock, gradual coalescence, pore formation and instability in food system20. A promising improvement in the controlled release behavior can be envisaged by embedding the nano DDS in biodegradable polymers (antimicrobial peptideliposome-polymer conjugate) with eventual use as nano-active films/biomembranes for food and pharmaceutical applications. The dressing of liposomes with biopolymers (chitosan, polyethylene glycol, arbutin) has been reported for improved vaccine delivery (mucoadhesibility), in vivo circulation time, skin permeability21-23 and beneficial effect on the release of nisin24. However, no literature study has disclosed the influence of embedding DDS in biopolymeric coatings to improve the controlled release attributes. In the present study, biodegradable hydroxypropyl methylcellulose (HPMC) is used as matrice to carry active nanoliposomes due to its application in food packaging25 and pharmaceutics (tablets and capsules)26. In recent years, fluorescence-based methods have been recognized widely to detect and quantify the molecules of interest and remarkable advancement had been achieved in both fluorescence-instrumentation and fabrication of new markers. This approach of selective and rapid integration of a low molecular weight fluorescent marker (MW ≈ 10% of biomolecule) into protein molecules generate a final labeled product with identical characters of migration and mode of action27. In the current work, fluorescein-labeled nisin was developed to analyze its release profile in model food systems by rapid spectrofluorimetry. Prediction of lantibiotic diffusion rates from liposomal nano-carriers to food systems is essential to ensure its efficacy during the shelf-life of the product. Likewise, partition-coefficient (K) values for active agents encapsulated in different DDS give an idea about their bioavailability against target microorganisms. Nisin binds to lipid membranes, and its affinity and permeability is sensitive to phospholipids composition (charge)28. Other than composition, size and charge; the prevailing condition during food conservation (pH, temperature, ionic strength, food simulant nature etc.) may influence the controlled release of nisin from different nanoliposomal systems. Up till now, there is no scientific data which can reveal the interactions between pore-forming


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nisin; phospholipids based nanocarriers and food simulants to predict its release rate and partition coefficient for sustained bioavailability of antimicrobial agent. Thus, the purpose of the current study was to assess the controlled release profiles of nisin in liposomal nano-carriers with distinctive charge and composition in different food simulating liquids (FSL) i.e. PBS - phosphate buffer saline (pH 6.8), AA - 0.3% acetic acid (pH 2.8) and EtOH - 10% ethanol; at different temperatures (4° and 37° C). The size, zeta potential, nisinmembrane affinity (atomic force microscopy), pore formation (transmission electron microscopy), nano-liposome distribution in biopolymer matrice (confocal microscopy) and partition coefficients were analyzed to reveal the interactions between nisin, encapsulation system, embedding biopolymer and food simulants. Based on the release obtained, the potential of neutral or charged nano-DDS and pre-loaded nano-active films as carriers of lantibiotic was investigated. 2. Experimental procedures 2.1. Materials The lipids, 1, 2-dioleoyl-glycero-3-phosphatidylcholine (DOPC) and 1, 2-dioleoylglycero-3phosphatidylglycerol (DOPG), were purchased from Avanti Polar Lipids (Alabaster, AL). HPMC powder with hydroxypropyl content of almost 9% and viscosity around 15 mPa.s (2% in aqueous solution at 25°C), was purchased from Fluka Biochemika, Japan. Glycerol (with more than 97% purity) was employed as a plasticizer (Merck, Darmstadt-Germany). Nisin was procured from Honghao Chemicals (Shanghai-China). Nisin formulation utilized in the current study contained >90% pure nisin (Figure S1) (according to the manufacturer, the product contained 38.4 x 106 IU per gram and 6.88% moisture-content) as reported previously dimethyl

formamide

(DMF),

1-Hydroxy

7-Azabenzotriazole

(HOAt),

29

. N,N

N-(3-dimethyl

aminopropyl)-N-ethyl carbodiimide hydrochloride (EDC), trifluoro aceticacid (TFA), ethanol ( 99.5% purity) and bicinchoninic-acid (BCA) reagents were acquired from Sigma-Chemicals (Germany). The label 5-(amino-acetamido) fluorescein (AAA-flu) and Triton X-100 were obtained from Invitrogen (Oregone-USA) and Sigma (Germany), respectively. 2.2. Fluorescent labeling of nisin



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Preliminary trials had shown that completely purified nisin was a prerequisite for successful labeling process28. Thus commercial nisin-Z was purified by HPLC to eliminate the impurities. The procedure of protein labeling described previously for nisin-A30, was optimized for nisin-Z29. The fluorescent marker AAA-flu was coupled with carboxyl-group of nisin-Z by means of HOAt and EDC assisted coupling in 100 μL DMF. The labeled peptide was purified using reversed-phase (C-18) HPLC column. The concentrations of labeled or unlabeled -nisin were measured by means of bicinchoninic-acid (BCA) assay29. 2.3. Nanoliposomal encapsulation of nisin Nisin containing liposomes were prepared by a modified thin film hydration method31. The adequate quantities of DOPC and DOPG stock solutions required for the production of liposomes (5 mg mL-1) were dried in the 10 mL colored glass bottle with flux of nitrogen gas. Uniformly dried lipid layer was hydrated with food simulating liquids (phosphate buffer saline pH 6.8, PBS; acetic acid 0.3% pH 2.8, AA; 10% ethanol, EtOH) containing nisin (labeled and unlabeled nisin mix 1:9) in the desired concentration of 100 µM. Ultrasonication with a probe sonicator (VCX, 400 W, 20 kHz) was carried out in an ice-bath to obtain a homogeneous emulsion. All batches of liposomes were extruded through two polycarbonate-based membrane filters with 200 and 100 nm pores with a hand-driven Lipofast extruder (Avestin, Germany). Non-encapsulated nisin was separated by using Sephadex G-50 syringe columns (5.18 x 1.27 cm) by loading 0.25 mL of nisin containing liposome solution. Peptide-loaded liposomes were eluted by size-exclusion chromatography by centrifuging at 1000g for 3 min. The centrifugation force and durations were optimized to get the loaded nanoliposomes based fraction only which elute first due to bigger size of liposomes as compared to free nisin. 2.3.1. Particle size determination The average diameter and size-distribution of nanoliposomes were analyzed by using Zeta-sizer NanoZS (Malvern). The samples were diluted (50x) with ultra-pure H2O. All dynamic light scattering (DLS) measurements were performed for five replicated at room temperature with aqueous medium refractive-index of 1.335. 2.3.2. Zeta potential The influence of phospholipids composition and stimulant liquid on the zeta potential of nanoliposomes was tested by Zeta-sizer NanoZS equipment. Different formulations were diluted 6 ACS Paragon Plus Environment

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as stated above to measure the surface charge by a combination of laser-Doppler velocimetry and electrophoresis29. 2.3.3. Encapsulation efficiency (EE%) After size exclusion chromatography to remove free nisin, the quantity of encapsulated nisin in the nanoliposomes fraction was measured by using BCA Kit. Samples along with BCA reagent were incubated at 37°C for half an hour; the OD (absorbance) values at 562 nm were calculated by using a UV/Vis spectrophotometer. The value of unencapsulated nisin was determined by subtracting encapsulated nisin value from known concentration of total nisin. Protein standard (BSA) solution 1 mg mL-1 was diluted to obtain the standard curve. EE% was calculated by means of the equation (1): EE% = (Encapsulated nisin / Total nisin) x 100

(1)

2.4. Atomic force microscopy of peptide – liposome interaction The interactions of antimicrobial peptide with nanoscale lipid membranes were studied to judge the affinity of nisin with charged (DOPG) or neutral lipids (DOPC) as explained for vancomycin earlier32. Mica squares (1.5 cm2) were glued onto a steel disc, cleaned carefully with water before use and cleaved to obtain a flat and uniform surface. Immediately, an aliquot of 50 μL of vesicles (DOPC:DPPC [25:75] or DOPG:DPPC [25:75]; adjusted to 250 μM in PBS (1x, pH 6.8) buffer, was deposited on the mica surface and incubated for 1 h at 60°C. The sample was thereafter washed with buffer to eliminate non-adsorbed vesicles. AFM contact mode images in liquid were obtained using a Nanoscope IV Multimode AFM (Veeco Metrology Group, Santa Barbara, CA) with triangular Si3N4 cantilevers (Microlevers, Veeco Metrology Group, Santa Barbara, CA) with a nominal spring constant of 0.01 N m−1. The instrument was equipped with a “J” scanner (120 μm). To minimize the applied force on the sample, the set point was continuously adjusted during imaging. Images were acquired at 90° scan angle with a scan rate of 2 Hz. All images were processed using the MFP3D software.

2.5. Nisin release from liposomal nano-carriers



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To carry out the quantification of released nisin at specific intervals of time from the different nanoliposomes, nisin retention inside the nano-carriers was measured. Thus liposomes prepared in different food simulating liquids containing nisin (PBS, AA, EtOH) were separated from free/non-encapsulated nisin by size exclusion chromatography using sephadex column as explained above (value at T0). Thus at any given time, the value of nisin entrapped inside liposomes (not yet released) was obtained by complete disruption of liposomes using Triton X100 (0.2% by volume). To prevent bleaching and degradation of labeled peptide by light, colored-vials were used with tightly closed septum. The diffusion experiment was carried out at 4° and 37 °C to evaluate the influence of temperature. The concentration of nisin was determined by Xenius-XM spectro-fluorimeter (Safas-Monaco) by means of 490±10 and 520±10 nm as excitation and emission wavelengths, respectively. Partition coefficient is defined as the ratio between migrant concentrations (at equilibrium) in the food simulant (Cs,∞) to the liposomal nanocarrier (CL,∞). (2)

2.6. Negative stain transmission electron microscopy (TEM) Empty and nisin loaded liposomes were diluted 5-times with ultra-pure H2O. Diluted sample and 2% ammonium molybdate solution were mixed in equal proportions and incubated for 3-5 min at 25°C. Single drop of incubated sample on a copper mesh was observed by using TEM (Philips CM 20) at an operating-voltage of 200 KV. 2.7. Active and pre-loaded nano-active biopolymeric coatings HPMC powder was dissolved in ultra pure H2O (6% w/v) by using a modified protocol25. The concentration of labeled peptide was adjusted to attain a final concentration of 20 µM (66 µg mL-1) nisin for active and nano-active coatings (0.528 mg/dried film, 9.04 µg nisin cm-2 based on the exposed-area of coating). For the nano-active treatments, nano vesicles solution (DOPC based) was added into 2x concentrated FFS of HPMC (1:1). Approximately 8 g FFS was poured in Teflon Petri-dishes for subsequent drying at 25°C for two days. Entire procedure of coating production was carried out in the dark to prevent bleaching of fluorescent marker.


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2.8. Atomic force microscopy of nano-active coatings The surface morphology of the non-active (HPMC), active (HPMC-Nisin) and nano-active (HPMC-Liposomal nisin) coatings was analyzed by using AFM with a Nanoscope IV contact mode. The resulting data set of each sample was transformed into a 3D image and a 2D height or deflection image as explained earlier33. Measurements were taken from several areas of film surface using the contact mode. The statistical parameters related with sample roughness (Ra, Rq, r) were estimated. 2.9. Nisin release from nano-active coatings The method of diffusion assay was adapted from the procedure reported earlier34. Coatings with a defined surface area and average thickness (58.4 cm2, 50.5±1 µm respectively) were cut into 6×1.5 cm pieces, which were immersed vertically into 10 mL water ethanol (10:90) solution. To prevent the bleaching or degradation of labeled peptide by light, colored-vials were employed with firmly closed septum. The initial temperature of water-ethanol solution was set to either 4°C or 37°C before immersing the coatings in each vial (3 replicates). For quantification, a 200 µL sample was withdrawn at pre-determined time interval. The difference in total volume after each withdrawal was taken into account while calculating the peptide concentration. Diffused nisin was analyzed by spectro-fluorimeter as explained above. 2.10. Confocal microscopy of nano-active coatings Nano-active coatings were examined using Olympus Fluoview confocal laser scanner microscope using a 10x lens (excitation λ 490±10 nm, emission λ 500-550 nm), and images were acquired by FV-10i software version 1.2. The pinhole, offset, gain control and all other settings were kept constant during all experiments. 60 frames per Z level were set prior to initiation of the Z series. Images were recorded at intervals of 1 µm in the Z direction. Images were represented as vertical distribution of fluorescence with upper and lower surface of film presented as the bottom and top respectively of film cross-section. 2.11. Antimicrobial potential of nano-active coatings The antimicrobial activity of the nisin-containing active and nano-active coatings against Listeria monocytogenes ATCC 13932 was determined either by :a) qualitative evaluation by a 9 ACS Paragon Plus Environment


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modified agar diffusion assay, or b) quantitative evaluation in Listeria inoculated saline solution suspension. To determine the anti-listerial potential, 1 cm2 discs of different active and nanoactive coatings were tested on inoculated-medium (TSA-YE)35. Petri dishes were incubated at 4°C for 5 h to permit the nisin diffusion in absence of microbes and then placed at 37°C for 24 h. Antagonistic activity was indicated by a halo (clear zone) in the bacterial lawn. HPMC based biopolymeric coatings without active agent and embedded with empty liposomes were employed as negative controls. Subsequently for a quantitative assessment, pieces of nisin loaded coatings (20×20 mm2) were immersed in 10 mL saline (sodium chloride 0.85%) suspensions of Listeria monocytogenes ATCC 13932 (107 CFU/mL) as reported previously36. These suspensions were incubated at 37 °C for 72 h with continuous shaking and bacterial counts were determined, at regular intervals, by spreading serial tenfold dilution(s) on TSA-YE plates. Biopolymeric coatings without active agent and embedded with empty liposomes were also employed as negative controls. 2.12. Statistical analyses KyPlot version 2.0 was used for comparative analyses by using a parametric multiple Tukey test (p ≤ 0.05). 3. Results and discussion 3.1. Nanoliposome physico-chemical attributes: Size, zeta potential and encapsulation efficiency The influence of different food stimulant liquids in relation to phospholipids (PL) composition has not yet studied on the fundamental characteristics of liposomes as active agent carriers. Nano-sizer data revealed liposomes with a polydispersity index of ≤ 0.25 prepared in various food simulating liquids (FSL) i.e. PBS - phosphate buffer saline (pH 6.8), AA - 0.3% acetic acid (pH 2.8) and EtOH - 10% ethanol. Average liposomes sizes varied from 68 ± 3 nm to 88 ± 2 nm in AA depending on lipid composition i.e. for DOPG and DOPC liposomes containing nisin respectively (Table 1). Among FSL having pH close to neutral, PBS and EtOH with pH 6.8 and 7 respectively, the size of liposomes were fairly close. Thus the ionic strength, due to presence of salt in PBS, did not influence the size of liposomes encapsulating nisin. However, acidic pH in AA had affected the liposomal size with regard to phospholipids composition. This could be due to the fact of electrostatic repulsion between encapsulated nisin (+4 charge) and 10 ACS Paragon Plus Environment

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DOPC, which have a net positive charge at acidic pH. The obtained nisin loaded liposomes were relatively small due to dual sheer force applied by mild sonication followed by multiple runs of extrusion31, 37, thus providing small unilamellar vesicles (SUV < 100 nm)16.

Table 1: Influence of phospholipids composition (PC, PG) and food stimulant liquids (Phosphate buffer saline, pH 6.8; Acetic acid 0.3%, pH 2.8; Ethanol 10%, pH 7) on average size, polydispersity index (PI), zeta potential and encapsulation efficiency (EE%) of fluorescently labeled nisin. Composition

DOPC

Food stimulant liquid (FSL) PBS AA 0.3% EtOH 10%

Mean diameter (nm) ± S.D. 76 ± 1 a 88 ± 2 b 70 ± 1 c

PI ± S.D.

0.23 ± 0.03 0.25 ± 0.02 0.24 ± 0.02

EE (%) ± S.D. a a a

44 ± 2 a 28 ± 1 b 43 ± 3 a

Zeta potential ± S.D. (mV) -14.16 ± 0.4 a 21.88 ± 3.2 b 6.90 ± 0.6 c

PBS 77 ± 1 a 0.20 ± 0.02 a 21 ± 1 bc -54.12 ± 6.0 AA 0.3% 68 ± 3 c 0.23 ± 0.01 a 24 ± 1 b -22.86 ± 1.7 a EtOH 10% 76 ± 2 0.23 ± 0.02 a 20 ± 1 bc -59.9 ± 4.8 a to d different superscripts indicate significant difference (p< 0.001) DOPG

d ad d

Zeta potential is usually utilized as an indicator of surface charge. Instability of liposomes is attributed to collisions (random Brownian motion) and eventual merging of multiple liposomes. Repulsive forces can reduce the frequency of interactions. Different studies have suggested the role of charged lipids on liposomal stability and drug interaction38, however no study reveal the influence of liposomal environment (FSL) on zeta potential in relation to neutral (zwitterionic) or anionic phospholipids. The zeta sizer data revealed that zeta potential ranged from -54.12 ± 6 mV to 21.88 ± 3.2 mV for DOPG and DOPC in PBS and AA respectively (Table 1). There existed significant difference of surface charge for PG and PC based nisin loaded liposomes due to anionic and neutral charge of the phospholipids. However, liposomes prepared in FSL at acidic pH had exposed the influence of environmental conditions on surface charge, without taking into account the composition of phospholipids. As anionic PG liposomes possessed less negative charge in AA as compared to PBS or EtOH (neutral pH). Similarly neutral PC liposomes had more positive 11 ACS Paragon Plus Environment


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charge in AA as compared to neutral pH of PBS. This change in the surface charge of liposomes for different compositions may eventually result into variability of EE%, drug-lipid interactions, release profile and liposomal stability. One possible explanation of this variation of surface charge for liposomes is the dissociation constant pKa of DOPC and DOPG vesicles. The pKa of DOPG vesicles were estimated as a lower pKa of ~3 and a higher pKa of ~10. While for DOPC vesicles, a lower pKa of ~3 and a higher pKa of ~11 were estimated. The lower pKa most likely correspond to the protonation of the phosphatic acid groups for the two samples, and the higher is due to the protonation of glycerol and choline, respectively. For DOPG vesicles at neutral pH, each lipid can be assumed to be negatively charged, due to the low pKa of the DOPG lipids 39. However at acidic pH in AA, DOPG based vesicles encapsulating nisin had significantly less negative surface charge due to protonation of phosphatic acid group. Similar protonation in DOPC based liposomes encapsulating nisin contributed for higher positive surface charge. Thus, AA environmental conditions will result in more repulsion for DOPC liposomes as compared to anionic DOPG vesicles encapsulating nisin. If other factors are not taken into account (acidic shock), positive or neutral charge may also result in lower electrostatic interaction between cationic nisin and phospholipidic vesicles. The encapsulation efficiency (EE %) of liposomal nano-carrier system for drugs and active agents depends on multiple factors including size, charge, pH, preparation method, lamellarity, nature and molecular mass of the drug and peptide. In the present study, liposomes encapsulating nisin have diameter size between 70-90 nm indicating unilamellar liposomes due to multiple run (>15) through the extrusion membranes. These nano-scale vesicles are suitable for controlled release as the extent of leakage decreases with a reduction in vesicle diameter. This trend is based on the fact that while the vesicle size distribution is shifted towards smaller vesicles, there would be smaller numbers of bound-peptides per vesicle17. Results of encapsulation efficiency for different liposomal compositions are compiled in Table 1. The data presented the average of triplicate analyses with 100 µM initial concentration of fluorescently labeled nisin, since it is previously observed that the µM concentration of nisin is capable of pore formation in bacterial or model membranes10. The lowest concentration of labeled nisin was incorporated in DOPG liposomes with 20±3 % EE, whereas the highest lantibiotic encapsulation 12 ACS Paragon Plus Environment

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of 44±2 % was realized with DOPC-based liposomes at neutral pH (PBS and EtOH). Were et al., has reported higher encapsulation efficiency (54-71%) of nisin in PC and PG based liposomes with or without addition of cholesterol38. However, the EE was calculated indirectly by addition of calcein. The encapsulation and release behavior rely on the initial concentration of peptide. Furthermore, the cholesterol addition improves the barrier property of liposomal bilayers by rendering the fatty acid tails more compact. The EE of nisin at nano-scale was almost double for DOPC as compared to DOPG, except in acidic environment, indicating the significance of selecting ideal composition for desired active agent. As explained above, nisin has exceptional mode of action against pathogens by pore formation in cell membranes. Thus, the concentration of compounds that can be entrapped is a function of lipid composition of model membranes i.e. liposomes, and may be attributed to electrostatic and hydrophobic interactions between antimicrobials and phospholipids38. As cationic nisin can interact with anionic DOPG membranes by electrostatic interaction due to it higher affinity, it eventually led to higher permeability of nisin by rapid pore-formation. However, Since PC liposomes demonstrate properties of uncharged monolayers at neutral pH values, electrostatic interactions with peptides might be minimal. Hence, the insertion of peptides into PC vesicles could possibly be due to hydrophobic interactions and association with PC bilayer structures38. The significantly lower EE of DOPC liposomes in AA may possibly be due to high positive charge of DOPC at lower pH (pKa ~ 3) that can result in electrostatic repulsion between strongly cationic nisin and phospholipids membrane. Hence, the knowledge on the pKa values of the phospholipids utilized for liposome manufacture is critical for drug loading and release. Secondly, DOPC based liposomes’ higher average size of 88 ± 2 nm in AA as compared to PBS or EtOH could result in higher peptide to lipid ratio in vesicle, which resulted in quick pore formation and thereby induced rapid leakage of encapsulated nisin. At 10% EtOH, the drug loading capacity of liposomes was unharmed. As previous study indicated that the optimal ethanol concentrations ranged from 10% to 15% (v/v) did not significantly affect liposome size, retention of active agent or, most importantly, the stability of the lipid bilayer. The main conclusion drawn from physico-chemical measurements is that zwitterionic phospholipids contribute most to the encapsulation of lantibiotic nisin in liposomes at neutral pH. 3.2. Peptide – liposome interaction: Atomic force microscopy



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To investigate the effect of lantibiotic nisin on supported phospholipids bilayer (SPB) at nano-scale, we tested the nisin induced changes in the lipid membrane organization in domains of DPPC with DOPC or DOPG. Fusion of unilamellar vesicles on freshly cleaved mica resulted in phase-separated SPBs. After careful and extensive rinsing with sterile PBS buffer, clean bilayers were obtained. The small liquid-crystalline domains of DOPC with a packing similar to that of biological membranes can be readily distinguished from the surrounding gel state DPPC bilayer, because DOPC SPBs are 0.79 ± 0.11 nm lower than DPPC and thus show up as darker in the AFM image (Figure 1, Top image and its cross-section). Subsequent to replacement of the PBS buffer covering the SPBs with a 10 µM nisin solution in PBS buffer, a concentration resulting in bactericidal effect in Listeria monocytogenes29, a very slow transformation process of the SPBs was observed (Figure 1). Scans were recorded every 10 min after addition of micro-molar nisin solution. Thus 1-3 µm large gel domains transformed in a time dependent manner (from left to right: at 0, 40 and 120 min respectively). The process started at the boundaries of the domains, which were increasingly pervaded by deposition/erosion. Figure 1 shows the DPPC gel phase domains protruding by 0.79 ± 0.11 nm from the surface of the DOPC fluid-phase. Forty minutes after the addition of nisin most of the DPPC gel-phase that was not yet transformed still protruded by the typical value of 0.8 nm from the surface, while in the section where nisin adsorption occurred, a principal difference of approximately 0.5-0.7 nm in height were observed. Next, 120 minutes after addition of nisin (Figure 1, top right image), an increase in height especially at the boundaries between fluid and gel-phase of supported bilayers was observed with 1 nm addition in DPPC height, which can be linked to the adsorption of nisin on the SPBs. A second possible explanation of this might be damage of DOPC bilayer by nisin prolonged interaction resulting in its erosion and subsequent deposition at borders of gel and fluid phase bilayers. This hypothesis is supported by the cross-section of AFM image after 120 min (Figure 1, top right image and cross-section height profile), revealing 0.4 ± 0.1 nm decrease in the height of DOPC bilayer and increased roughness as compared to the base line of previous images at 0 or 40 minutes of nisin exposure. As bacterial membranes are dominated by negatively charged phospholipids, whereas human-cell-membranes are rich in neutral lipids, electrostatic attraction contribute in antimicrobial peptide-membrane interactions, and common antimicrobial peptides including nisin carry a net positive charge. Thus, negative surface charge is responsible for increased peptide 14 ACS Paragon Plus Environment

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adsorption which eventually results into membrane lysis40. Similarly, the electrostatic attraction or repulsion of anionic and neutral phospholipids based nano-carriers with nisin may result in difference of drug release required for achieving specific dose of active agent in the medium. In the case of DPPC/DOPC mixtures, the gel phase (DPPC) was found to be organized in large, well-defined domains. Replacing the zwitterionic PC head group with the negatively charged PG rendered the fluid and gel phases with no domains distinction. These results are in accordance with previous findings showing similar height profile of 6.0 ± 0.1 nm and 6.1 ± 0.4 nm for DPPC:DPPC and DPPC:DOPG SPBs respectively. When a 10 µM nisin solution was added to the SPBs containing anionic DOPG, the nisin adsorption/agglomeration phenomena could be observed, further the turnover process took place markedly faster (Figure 1, bottom image). After 40 minutes of the peptide addition, distinct patch formation of nisin electrostatically attracted towards negatively DOPG was observed, although this phenomenon was less pronounced for DOPC:DPPC SPBs. After 120 min (Figure 1, bottom right) an increasingly rough bilayer with regularly distributed patches of adsorbed nisin was observed. The section analyses of the bilayers revealed that, as compared to time 0, the differences in height of the domains increased about 2 nm shortly after addition of nisin (Figure 1, bottom cross-section).



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(A)

*

*

*

*

*

*

*

*


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(B)

Figure 1: AFM scans of the time dependant nanoscale membrane activity of nisin on supported phospholipids bilayer (SPB). AFM height images (5µm x 5µm; z-scale 10 nm) after the addition of 10 µM of nisin dissolved in PBS (left to right: 0, 40, 120 min respectively), against SPB composed of DOPC – DPPC (A) and DOPG - DPPC (B), respectively. Vertical cross-sections were taken along the position indicated by the continuous red line. Upper domain of gel phase lipid (DPPC) and lower domain of liquid crystalline lipid (DOPC) are indicated by black and white stars respectively. 17 ACS Paragon Plus Environment


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After 40 min, the roughness increased significantly, and after 2 h, a more irregular surface structure with homogenously distributed small patches was observed. In general, the effects induced by nisin in DOPG:DPPC were significantly elevated to those of DOPC:DPPC as erosion of SPBs resulted in 3 nm decrease in base-line of cross-section height after 2H of exposure to cationic nisin. This process was quicker and somewhat more efficient than PC SPBs mainly due to increased affinity of peptide towards negatively charged PG. As the pore formation in liposomes is a step-wise process of adhesion, agglomeration and then permeability or leakage; present AFM data of peptide-lipid interaction suggested that in similar given set of conditions, DOPC based nano-carriers must be opted for longer retention of nisin or vice-versa. Previous studies had reported similar trend between DOPC and cationic peptides using other analytical techniques38, 41. 3.3. Nisin release from liposomal nano-carriers Fluorescence spectroscopy can detect and quantify the active agents at nM concentration and below, thus fluorescently labeled nisin has enabled us to encapsulate nisin at sufficiently lower concentration which could permit liposomal stability against spontaneous disruption by nisin induced pore-formation. As expected, the increase in temperature resulted in higher release of the nisin from nanocarriers, which was 5-10 % higher in case of DOPC for all FSLs (Figure 2). Similar trends were found for DOPG based liposomes encapsulating nisin, however as the EE% of DOPG liposomes were lower (< 25%), the apparent variation of released nisin was more conspicuous in AA and EtOH (25-30% higher) due to higher temperature. This increase in release was possibly due to the increased Brownian motion of molecules and less physical stability of liposomes at higher temperature. Thus the diffusion of loaded nisin from different liposomes had revealed that elevation in temperature resulted in significant increase in permeability of nisin. Considering the role of neutral or negative surface charge of vesicles, higher electrostatic interaction between anionic lipids and cationic bacteriocin had resulted in rapid release of nisin as a subsequent step to higher adsorption/aggregation of peptide on liposomes as expressed in AFM study explained above.


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The most interesting findings were obtained about the influence of the FSL on the release pattern of pre-encapsulated nisin. In case of DOPC liposome, higher nisin release (%) was observed for PBS as compared to the AA or EtOH (Figure 2 A). The possible explanation of higher retention by AA is the higher net positive charge as compared to PBS, which had resulted in lesser peptide-lipid interaction due to electrostatic repulsion resulting in lesser permeability. Secondly, the net EE% of nisin in DOPC based liposomes in AA was considerably lower than other FSL, resulting in lower peptide: lipid ratio which can lead towards lesser aggregation and pore formation by nisin. Considering the higher retention of EtOH, it may be due to positive effect of ethanol at optimal concentration ( 85% in PBS at 4°C. In acidic pH as is the case of AA, DOPG based SUVs encapsulating nisin had significantly less negative surface charge due to protonation of phosphatic acid group, which could result in lower peptide-lipid affinity. However, at higher temperature, the kinetic energy of moving molecules were so high that it overcome the repulsion between peptides and phospholipids moieties, which had decreased the variation of percent release among the FSLs. The partition coefficient (K) values were useful to establish the interactions of nisin with different encapsulating nano-carriers; as low-values of K involve less permeability of nisin from the encapsulating carriers (Table 2).

Thus the K values ranged from 0.23 to 8.78 for DOPG

based liposomes dispersed in AA and PBS, at 4° and 37°C, respectively. However, different encapsulation efficiencies of nisin in different simulant liquids have significant impact on its release behavior due to concentration gradient, electrostatic repulsion and pores formation kinetics in liposomes. Thus different concentrations or encapsulation efficiencies of nisin in liposomes can influence the obtained K values. In general, PBS has allowed higher permeability from carriers as the observed K values are highest in this FSL. While increase in temperature had increased partition coefficient values, that can explain the lesser retention of peptide during storage at elevated temperatures.



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Figure 2: Controlled release of nisin from nano-carrier systems influenced by phospholipids composition and temperature. Release profiles from DOPC based nanoliposomes carried out at 4°C and 37°C are depicted in A and B, respectively. Nisin release profiles from DOPG based nano-carriers tested at 4°C and 37°C are depicted in C and D, respectively. Whereas, FSL are represented as phosphate buffer saline, pH 6.8 (triangles); acetic acid 0.3%, pH 2.8 (circles); and ethanol 10%, pH 7 (squares). Table 2: Partition coefficient (K) of lantibiotic nisin in different nanoliposomal carriers: Influence of phospholipids composition (PC, PG) and food stimulant liquids (PBS-Phosphate buffer saline, pH 6.8; AA-Acetic acid 0.3%, pH 2.8; EtOH- Ethanol 10%, pH 7) and storage temperature (4°C and 37°C). Composition DOPC

T °C 4°C 37°C 4°C 37°C 4°C 37°C

FSL PBS AA EtOH

K 2,53 ± 0,29 a 4,48 ± 1,13 b 1,68 ± 0,27 a 2,25 ± 0,51 a 1,26 ± 0,18 a 2,33 ± 0,52 a

DOPG 4°C PBS 4,90 ± 1,19 b 37°C 8,78 ± 1,82 c 4°C AA 0,23 ± 0,06 d 37°C 1,52 ± 0,29 a 4°C EtOH 0,59 ± 0,12 d 37°C 2,15 ± 0,51 a a to d different superscripts indicate significant difference (p< 0.001) 21 ACS Paragon Plus Environment


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3.4. Stability of nisin loaded liposomes: Negative stain electron microscopy (TEM) The EE% data had revealed that the pre-encapsulated nisin in different FSL is present at micro-molar concentrations that can destabilize the liposome by pore-formation. Due to amphipathic characteristics of nisin, it is located in the inner hydrophobic inner-domains of the bilayers. Thus nisin interacts with liposomal bilayers and perturb the membrane structure, thus inducing release of active agent. An additional reason is the instability of liposome due to chemical hydrolysis of glycerol-phospholipids to free fatty-acids, which may lead to an increase in permeability through liposomal bilayers37.

A

A

B

C

D

Figure 3: Transmission electron micrographs representing the structure and stability of DOPC bases nanoliposomes at 4°C. Nisin loaded DOPC liposomes at time 0 (A) and after 2 weeks (B, D) (peptide induced pores are clearly visible). However, empty DOPC liposomes (control) were stable after 2 weeks (C) without showing any pore formation. 22 ACS Paragon Plus Environment

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As the maximum EE% values were found for DOPC based liposomes in PBS, these liposomes had highest peptide-lipid ratio that could lead to subsequent pore-formation. An insight of pore-formation by pre-encapsulated nisin in neutral phospholipids based liposomes are presented (Figure 3) at time zero and after 2 week exposure with lantibiotic nisin. As most of the pore-formation revealed openings of in the core and not the disruption of bilayer; the nisin retained in bilayers due to hydrophobic interactions would possibly be released during longer storage.

3.5. Active and pre-loaded nano-active coatings: Surface morphology Surface roughness of the films observed by atomic force microscopy (AFM) shows the marked influence of nisin addition, in free/unencapsulated form or pre-loaded nanocarriers embedded in HPMC coatings (Figure 4). The surface of the native HPMC film is practically smooth without irregularities. However, the active films with free nisin present certain irregularities in the matrix due to partially homogenous distribution of nisin. However, the preencapsulated liposomal nisin was homogenously distributed in film matrix resulting in lower surface roughness (Figure 4C). Previous studies had demonstrated that the greater droplet size of dispersions make them unstable, thus promoting greater accumulation of lipid aggregates on the surface33. However in the present study, reduction of size to nano-scale avoids such kind of surface agglomeration of nano-carriers. On the other hand, the corresponding topographic image (2µm x 2µm) shows the presence of nanocarriers with a size less than 100 nm (Figure 4D). Similar results were obtained for deflection and height images by AFM, thus revealing differences in term of homogenous distribution of active agent in active and nano-active coatings (Figure 5). Different parameters quantify the surface roughness obtained from the data analysis of the height plots. Average roughness (Ra): is the average of the absolute value of the height deviations from a mean surface; root mean square roughness (Rq): is the root mean square average of height deviations taken from the mean data plane and roughness factor (r): is the ratio between the threedimensional and two-dimensional surface area produced by projecting the surface onto the threshold plane. All the parameters indicated a significant increase when free nisin was present in the film as compared to the control film. Average and RMS surface roughness, Ra and Rq values



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indicated the similar trend of higher values when free nisin was present in the HPMC matrice (Table 3).

A

B

C

D

Figure 4: 3D plots obtained from AFM of non-active HPMC film (control) (A), active film with free nisin (B), and nano-active film embedded with nisin nano-carriers (C, D – zoom image).

Table 3: Roughness parameters acquired from height trace of atomic force microscopy images: Ra, Rq and r. (Three images were analyzed in each case) Ra (nm)

Rq (nm)

r (%)

Control

HPMC

2,13 ± 0,3a

2,68 ± 0,4a

0,29 ± 0,1a

Active film

HPMC + Nisin

9,95 ± 2,2b

14,1 ± 3,0b

0,85 ± 0,4b

Nano-active film

HPMC + Liposomal Nisin

2,25 ± 0,8a

2,8 ± 1,1a

0,95 ± 0,2b

a to b

means with different superscripts indicate significant difference (p< 0.001) 24 ACS Paragon Plus Environment

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A

B

C



Figure 5: AFM deflection, height and section analysis of pure, active and nano-active HPMC matrice as biopolymer nisin carrier (from left to right, A to C respectively). However, pre-encapsulation of nisin in liposomal nano-carrier and their subsequent embedding in native films decreased the surface roughness values. But the roughness factor (% r) remained elevated with unencapsulated or encapsulated nisin. By observing topographic and cross-section analysis (Figure 5, images on right side), the surface irregularities ascribed to nanoactive films are minor and better dispersed than those qualified to active films, which are bigger but fewer in number. 3.6. Nisin release from nano-active films The above explained controlled release of fluorescently labeled nisin from different phospholipids based nanocarriers in 3 different FSL has highlighted major limitation of liposomal encapsulation system i.e. its tendency to discharge the loaded molecules over time due to pH and temperature, surface charge, fusion, pore formation and kinetic instability. A promising improvement in the controlled release behavior can be envisaged by embedding the nano DDS in biodegradable polymers (antimicrobial peptide-liposome-polymer conjugate) with eventual use as nano-active films/biomembranes for food and pharmaceutical applications. As HPMC is 43

, the

C

B

biodegradable and its biopolymer network disrupts in the presence of high water activity A

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controlled release of free or pre-encapsulated nisin embedded in films were studied in alcoholic FSL (water: ethanol 10:90 v/v) at 4° and 37°c. At refrigerated temperature, HPMC acted as good carrier of nisin and retained more than 50% of the total nisin after one week (Figure 6). However, increasing the temperature resulted in higher release into FSL. In case of active films, the release and retention of active agents are both vital attributes as nisin necessitates reaching in the FSL to express its antimicrobial action against bacteria. In parallel, adequate amounts of this bacteriocin are required at food surface as well, where the microbial load is maximal. Thus, a balance of release and retention may guarantee protection of food against food borne pathogens during longer conservation periods. Partition coefficient (K) values may suggest the relative affinity of nisin with the coating or FSL. K values lesser than 1 indicate that a low quantity of nisin has migrated from the active coatings. Concerning active film with un-encapsulated nisin, the data revealed that K values were 0.77±0.2


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and 1.8±0.3 at 4°C and 37°c respectively. Thus at elevated temperature, HPMC demonstrated poor capacity to hold free nisin for longer periods.

Figure 6: Controlled release of nisin from HPMC (A, active films) or nanoliposomal carriers embedded in HPMC (B, nano-active films) at 4°C and 37°C (triangles and circles respectively) in 90% aqueous ethanol solution.

Confocal microscopy was employed for observing the interface and exterior defects of coatings44. For understanding the location, distribution of labeled nisin, and structural characteristics of active and nano-active film; CLSM was employed to observe the surface and cross-section (z-scan) distribution of free or pre-encapsulated nisin in biopolymer network 27 ACS Paragon Plus Environment


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(Figure S2). The z-scans along the cross-section of active or nano-active film revealed homogenous distribution of nano-carriers in film matrice. Further, these nano-liposomes are rendering the nano-composite films with hindrance function for nisin release (zigzag path) from biopolymer carriers (Figure S2).

A

B

C

D

Figure 7: Confocal images of the HPMC bio-membranes surface carrying either free labeled nisin or nano-encapsulated nisin (A, C: fluorescence; B, D: combination of fluorescence and phase contrast images) respectively. In addition, combining fluorescence and phase contrast images for film upper surface revealed homogenous distribution without any kind of creaming or coalescence (Figure 7). Relative to active nisin films (higher intensity in middle of film cross-section, the nano-active films provides equally high intensity at surface also. Concerning cumulative release values, promising results were demonstrated by the nanoactive films carrying pre-encapsulated nisin in liposomal nanocarriers (neutral lipids) subsequently embedded in HPMC matrice. Thus, the nisin has to release from liposomal bilayers and further diffuse through the biopolymer network to reach the FSL. The nanoliposomes embedded in the polymer network act as the nanocomposite hurdles which further slow down the passage of nisin towards food, resulting in higher retention of nisin in coatings to ensure longer 28 ACS Paragon Plus Environment

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bioavailability of fresh nisin diffusing from bioactive packaging. The reduction in K values to 0.66 ± 0.03 and 0.45 ± 0.04 by nano-active films at 37° and 4°C in FSL established that biopolymer-coated nanoliposomes carrying pre-encapsulated nisin had superior antimicrobial retention capacity as compared to liposome or biopolymer alone, which can be eventually employed in pharmaceutical applications or biopreservation of food through controlled delivery. 3.7. Antimicrobial potential of nano-active coatings To demonstrate the efficacy of nano-active against Listeria monocytogenes ATCC 13932, diverse coatings (1 cm2 discs) were tested on inoculated agar medium. As anticipated, non-active coatings had not demonstrated any inhibition zone (Figure 8) against Listeria monocytogenes. Similarly, the non-active nano-coatings incorporated with empty nano-liposomes were not effective to reduce the growth of pathogen. Active HPMC coatings containing antimicrobial peptide had generated larger inhibition-zone than disc size owing to discharge and diffusion of peptide in the agar network. The nano-active coatings had exhibited better control of pathogen as compared to non-active controls. The active films had demonstrated larger halo zone due to faster release from active coatings as compared to nano-composite films in 24 hours (Figure 8). Figure 8B reveals the data observed during the quantitative antibacterial evaluation. After 24 h of exposure, the viable counts (CFU mL-1) of Listeria monocytogenes ATCC 13932 in the solutions in contact with the active coatings containing nisin decreased by more than 1.5 log units in comparison with the results observed for non-active and nano-coatings without nisin. In comparison, the nano-active coatings could only reduce the viable counts by about 1 log unit due to controlled release phenomenon after 24 h. Listeria enumeration was further reduced to a difference of 3 to 3.5 log units after 72 h for active and nano-active coatings, respectively. This continued decline of the Listeria counts revealed that there was a progressive release of nisin from active and nano-active coatings. Thus as demonstrated by partition coefficient studies, the controlled release of nisin from nano-composite coatings is advantageous to improve the bioavailability of active nisin to ensure the food safety.



A

III

IV

II

I

8

B

7 6

log CFU/mL

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5 4 3 Control (HPMC coating)

2

Active coating (with nisin) Nano coatings (with liposome)

1

Nano-active coatings (with nisin encapsulated liposome)

0 0

10

20

30

40

50

60

70

80

Time (hours)

Figure 8: Antimicrobial activity of active and nano-active HPMC coatings. (A) Inhibition zones of coatings without nisin (I control), coatings embedded with empty nano-liposomes (II control), active coatings containing nisin (III) and nano-active coatings containing nano-liposomes encapsulated nisin against L. monocytogenes in TSA medium. (B) Quantitative antilisterial activity assessment (log CFU mL-1) of active and nano-active coatings against Listeria monocytogenes ATCC 13932 in sterile saline solution suspension. 30 ACS Paragon Plus Environment

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4. Conclusion We have reported the controlled release of fluorescently labeled nisin, a pore-forming lantibiotic, pre-encapsulated in phospholipids based nano-carriers. The study revealed the influence of encapsulating material composition (charge, head group), temperature (4°C, 37°C), size, zeta potential and pH on diffusion of nisin from nanoliposomes. The elevated temperature, high electrostatic attraction between anionic bilayers and cationic nisin, larger size, higher EE% (peptide-lipid ratio) resulted in superior interaction of nisin with nanocarriers resulting in rapid and elevated release through pore-formation. However, acidic pH and optimal EtOH concentration in FSL improved the stability and retention capacity of loaded drug. Thus controlling various factors had resulted into a range of nisin partition coefficient K values from 0.23 to 8.78 indicating the variation in affinity for encapsulating molecules. Our work has advanced the characterization of interaction between nisin and nano-scale bilayer encapsulating systems by atomic force and transmission electron microscopy demonstrating membrane activity of nisin from adsorption, aggregation to pore formation. Finally, we have developed nano-active films with nisin pre-loaded nanoliposomes embedded in biodegradable polymer HPMC. The morphological, topographic, and roughness parameters studied by CLSM and AFM revealed improved attributes of nano-active biopolymer membranes. This concept has demonstrated excellent retention capacity as drug carriers by decreasing the partition coefficient value from 1.8 to 0.66 (app. 30% less release) due to improved stability of liposomes coated in biopolymer network. The nano-active (pre-encapsulated nano-carriers embedded in biopolymers) carrier system for controlled release may subsequently be of significant interest for the improved delivery of functional agents and drugs to improve preventive or therapeutic notions. Associated Content: Supporting Information: Chromatogram obtained during nisin Z purification on semi-preparative HPLC. Confocal laser scanning microscope based Z-stack images representing cross-section distribution of fluorescently labeled-nisin in free form or encapsulated in liposomal nano-carriers after embedding in HPMC based coatings.



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Author Information: Corresponding Author: Dr. Muhammad Imran Email: [email protected] Tel.:

+92.322.6017784

Notes: The authors declare no competing financial interest.

Acknowledgements: The authors are grateful to Jacquline Chanél and Dr. Christophe Stenger for excellent technical and scientific assistance considering TEM and CLSM. We are thankful to Higher Education Commission, Islamabad for funding the doctoral studies of Muhammad Imran (Grant No. HEC200656).

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