Materials and Methods

Materials and Animals:  Soybean phosphatidylcholine (PC)
was purchased from Lipoid, Inc. (Alabaster, AL, US). Cholesterol, Vancomycin hydrochlorid, 2-Iminothiolane
hydrochloride (Traut’s reagent), Lysostaphin, L-Cystein, Triton
X-100 and Sodium Chloride (NaCl) were purchased from Sigma Aldrich (St Louis, MO, USA). Blood
agar, Tryptic Soy Broth (TSB), Methanol, Chloroform, Glycine and Ammonium
Sulfate were obtained from Merck,
Germany. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-maleimide
(polyethylene glycol)-2000 (DSPE-PEG(2000) Maleimide) and  1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cyanur
PEM1 ) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Sepharose CL-4B was purchased from GE
Healthcare. (—)
and Silver nitrate
solution was purchased from —-. Antibiotic disks were
purchased from MAST (Merseyside, UK).  Male NMRI mice, 6-8 wk old, were purchased
from Center for Comparative
and Experimental Animals Studies. (Iran University). All animal experiments were carried
out according to the protocols
approved by the Ethics Review Committee of Animal Experimentation of Iran University.

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Bacterial StrainM2 : S. aureus was isolated from a
patient with a skin wound infection (Hamadan hospital, Iran) And identi?ed by conventional biochemical tests. Finally S. aureus confirmation was carried out by nuc
gene, as previously describe (?????? ????? ????).
Antimicrobial susceptibility testing was performed
using the disk diffusion method according to the Clinical and Laboratory
Standards Institute (CLSI) guidelines (Table 1) (?).  S. aureus ATCC 25423 was used as a control strain.

Preparation of Vancomycin Liposome: Liposomes were
prepared using the thin-film hydration method in a molar ratio of 9:1 (47 mg of
PC and 3 mg of Cholesterol) (?). The lipids were individually dissolved
in Chloroform and methanol in rate 9:1. And a thin film was formed on the inner
side of the round bottom flask, by evaporating the solvent under vacuum using a
Rotary Evaporators (IKA,
Germany). The lipid film was then hydrated with using 1.5 mL of PBS (PH: 7.4)
containing 15 mg of vancomycin. Following vigorous vortex for 5 min, the obtained MLVs were
sonicated using aprob sonicator for 3 min. the solution was extruded through a
200 nm pore-sized polycarbonate membrane (Whatman, USA)  for 21 times to produce (????? ????) small unilamellar vesicles (SUVs). The un-encapsulated
vancomycin was then separated using Sepharose CL-4B column. Alternatively,  encapsulation
efficiency was determined by using 10% Triton X-100M3 .  Briefly, 90 ?l liposomal suspensions was added
to 200 ?l 10X Triton X-100 and after incubated with water bath at 37 ?C for 5
min, then vortexed for
5 min. The supernatant was
collected and used for HPLCM4 

Conjugating Lysostaphin to
Liposome by Cyanur DPPEM5 :

The coupling of lysostaphin to
liposome was performed by cyanur DPPEM6  ligand. Briefly, PC, cyanur DPPEM7  and cholesterol (8:1:1, 42 mg, 5.6
mg and 2.6 mg, respectively) were prepared using the thin film hydration method
and 1 mL borate buffer (PH: 8.3) was added in lipid film to form liposome. Following by vortexing
and sonicating for 5 min, it was extruded through a 200 nm pore-sized
polycarbonate membrane for 21 times to homogenized the size of the liposome. 500
?g (? mol or n mol?) of lysostaphin (18.5 nmol, in 50 ?l 10 mM sodium acetate solution)
were addedM8  to liposome solution (? ? mol) and
the mixture was shacked and incubated for 16 h at 4 ?C in the darkness.
Uncouple lysostaphin was harvested by 100 kDa MWCO dialysis tubing by centrifugation at 4000 rpm
for 2.5 h. and after that it was concentrated by 3 kDa MWCO dialysis tubing. The coupling efficacy
was determined using HPLC analysis.

Preparation of Vancomycin Liposomal
Conjugated with Lysostaphin:

Lysostaphin conjugation was
performed for liposomes -containing
vancomycin by two methods.

Post Insertion Method: the lipid mixture including PC:
Mal- PEG 2000- DSPE and cholesterol, in a molar ratio of 8.7: 0.3: 1 (42 mg,
5.6 mg and 2.4 mg, respectively), were employed in this method. Thiolated of
lysostaphin were obtained by reacting the protein (18.5 nmol) with 2-Iminothiolane
at a 1:1 molar ratio in 1 mL PBS (PH: 8) for 1 h at Rome Temperature (RT) and condensed by 3 kDa MWCO dialysis tubing to
removed excess 2-Iminothiolane. Dried lipid films containing Mal- PEG 2000-
DSPE were hydrated by the lysostaphin thiolation solution M9 with gentle agitation and
incubated 16 h at RT. Free Maleimide group were quenched with Cystein (50 mM) for 1 h at RT. To
prepared liposomes – containing
vancomycin, small unilamellar liposome composed of PC: cholesterol was
prepared using the thin film hydration method. Hydration was carried out by 1
mL of PBS (PH: 8)
containing 15 mg of vancomycin. Following by sonication and extruder (21 times,
200nm) were performed. Unencapsolation of vancomycin was separated by 100 kDa MWCO
dialysis tubing. The lysostaphin- modified liposome were added to the liposomes – containing vancomycin
and incubated for 2 h at RT. After 10
min bath
sonication, again, unbounded lysostaphin was separated with 100 kDa and 3 kDa MWCO tubing. The
concentration of unencapsulation of vancomycin and unbounded of lysostaphin
were analysis by HPLC.

Lysostaphin Conjugation Liposome
by Ammonium Sulfate gradient:

The liposomal formation was
prepared using the lipids PC: cyanur DPPEM10  and cholesterol, in a molar ratio of 8: 1: 1 (42 mg, 5.6
mg and 2.6 mg, respectively). The lipid film was then hydrated in ammonium
sulfate solution (250 mM, PH: 5.5). After vortexing
and sonication,
the mixture was extruded for 21 times. Unloaded ammonium sulfate was removed by
100 kDa MWCO dialysis tubing by centrifugation at 4000 rpm for 30 min. lysostaphin
(18.5 nmol) was added to solution and incubated for 16 h at 4 ?C in the darkness upon shaking.  Free cyanur group were quenched with Glycine (50 mM) for 1 h at RT.
Un bounded lysostaphin were separated by 100 kDa MWCO dialysis tubing and
analyzed by HPLC. Liposome solution and vancomycin solution (1 mL NACL with PH:
5.5; 302 mM; containing 10 mg of vancomycin), were mixed and then incubated for
2 h at 4 ?C. The mixture was dialyzed by 100 kDa MWCO dialysis tubing to separate
the unloaded vancomycin and the concentration was measured by HPLC.

Antimicrobial Susceptibility

The minimum inhibitory concentration (MIC)
and the minimum bactericidal concentration (MBC) of Vancomycin, Lysostaphin,
Liposome- conjugated lysostaphin and Vancomycin liposomal conjugated with
lysostaphin, were determined using a liquid growth inhibition assay in 96 well
microtiter plates as recommended by CLSI guidelines
(?). The MIC was
considerate as the lowest concentration drugs, that completely inhibited the visible growth
of bacteria were determined after 16-18 h of incubation at 37 ?C. The MBC was
performed after transferring of aliquots from the wells of the broth microliter
dilution plates on to
blood agar. The plates incubated for 16-18 h at 37 ?C.

Size Distribution and Zeta Potential:

The particle
size, PDI as well
as zeta potential were determined for each liposome. Briefly, the liposomes
were diluted in
a ratio M11 in 1:15 with PBS, individually. Then,
after passing through the 0.2 nm filter, size of liposomes were measured by dynamic light
scattering (DLS) analysis using the Brookhavon BI-90 particle size analyzer V7.2 (Brookhaven Instrument,
Holtsville, new York).










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