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According thenassembled with a BUCHI R-200 rotary

According to a research
published in an article paclitexal loaded Liposomes were produced with
SPC:CHOL:PEG2000- DSPE:tocopherol:PTX?16.2:3.8:1.3:0.2:1 molar concentration by
thin film hydration method (Umrethia et al. 2007). Briefly, SPC, CHOL, and PTX were
weighed correctly   and then dissolved in organic phase, that is,
chloroform (5 mL) in a 100-mL round bottom flask. This was assembled with a
rotary evaporator and the organic phase was evaporated at 45?2°C, which forms
the film on the wall of the flask. The other processingparameters , such as
rotational speed of evaporating flask (100 rpm) and vacuum (250 mmHg) were
maintained constant. The round bottom flask comprising  thin lipid film was left in vacuum desiccator
overnight to evaporate  the solvent
residuals if any. After that it was hydrated with phosphate-buffered saline
(PBS), pH 7.4, employing vortex mixture for about 2 min to form conventional
liposomes. This liposomal suspension was left at room temperature for about 2 h
to obtain  complete swelling. The resulting
suspension was sonicated for 12 min in probe sonicator (220 W) to get small and
homogenous vesicles and extruded via  polycarbonate membrane of 0.2 mm pore size.  (Xu
and Meng, 2016)

 

According to another
research , Both the conventional liposome consists of S100PC/CH (90:10, molar
concentration) and the PEGylated liposome consisting  of S100PC/CH/MPEG2000-DSPE (90:10:5 as a mola concentration)
were produced by improved thin-film hydration technique .Temporarily , the
hydrophobic excipients, paclitaxel (3.5 mg/mL), CH and lipids 10% (w/v) S100PC
and MPEG2000-DSPE, were dissolved in chloroform and transferred into a appropriate
conical flask. The flask was thenassembled with a BUCHI R-200 rotary evaporator
¨ (Flawil, Switzerland) and water bath (BUCHI B-490) with tem- ¨ perature
maintained at 40 ?C under the aspirate vacuum. The thin-film layer formed was washed
with nitrogen gas for 5 min and maintained overnight under vacuum to evaporate traces
of chloroform. The thin-film was re-suspended in phosphate buffer saline (PBS,
pH 4.0) with or without 3% (v/v) Tween 80 by rotating the flask at
approximately t 300 rpm till the lipid film was entirely hydrated. Then, the
liposome dispersion was passed through 1.2, 0.4 sequencially and finally 0.2 m
pore size filters (IsoporeTM) under nitrogen gas with an extruder (Northern
Lipids, Inc., Canada). Un-entrapped paclitaxel was detached from the liposome
suspensions by centrifuging at 1000 rpm for 10 min, after that  the supernatant liposomal dispersion was
centrifuged at 50,000 rpm for 30 min to precipitate the liposomes. Entire precipitation
of liposomes was revealed  by observing
the absence of particles in the supernatant utilizing a NICOMP 370 Submicron
Particle Sizer. The supernatant was wasted , and the liposome pellet was washed
two times with PBS (pH 7.4). The pellet was then suspended in distilled water havingsucrose
(molar ratio of sugar-to-lipid = 2.3), and freeze-dried (Laboratory Floor Model
Freeze-dryer FD5512, Ilshin, Seoul, Korea). The concluding liposome particles
were kept in tight containers at 4 ?C for additional experiments. (Yang et al., 2007)

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Functionalized liposomes

 Liposomes represent a versatile drug delivery
system that could be endowed with other properties improving their targeting
towards the tumors. One of the  approache
is  forming a stealth liposome .

Paclitaxel encapsulated in pegylated liposomes (long-circulating
liposomes)

Rapid
clearance of the conventional liposomes by RES represents one of the major
disadvantages in the drug delivery. This problem was solved by employing the
long-circulating liposomes. The grafting of conventional liposomes with an
inert and biocompatible polymer such as polyethylene glycol (PEG) leads to the production
 of a shielding and hydrophilic coating  on the liposomal surface 46.

·        
The surface modification
reduces a clearance of liposomes by the cells of RES and apparently prolongs
the half-life of liposomes during circulation 47.

·        
The long-circulating liposomes
are also referred to as pegylated, sterically stabilized or stealth liposomes.
It was explained that the permeability of the capillary endothelium in the
tumors is increased in comparison with normal tissues 48.

·        
The macromolecules are
passively aggregated to greater extent for longer period in the tumor than in
the non-malignant capillary endothelium. This phenomenon is termed as an
enhanced permeability and retention (EPR) effect 49.

·        
The zeta potential of the
conventional liposome was almost neutral as expected since S100PC and
cholesterol do not bear a charge. With the addition of 3% (v/v) Tween 80 in the
hydration medium, the mean zeta potential of conventional liposome dispersion
was more negative which is consistent with previous reports (Lee et al., 2005;
Yang et al., submitted for publication). The reason for the lower zeta
potential could be due to the partial hydrolysis of Tween 80. The zeta
potential of PEGylated liposomes was more negative than that of conventional
liposomes due to the negatively charged phosphate group of MPEG-DSPE, which is
also in accordance with the result reported in literature (Hinrichs et al.,
2006). In this case, the effect of Tween 80 on zeta-potential seems to be
negligible since the negative charge due to the PEGylation is so much larger.(Yang et al., 2007)

 

Small
long-circulating liposomes (100 nm) demonstrated a higher frequency of
encountering permeable capillaries of the tumor and extravasating into the
fenestrated tumor tissue. This accumulation of long-circulating liposomes with
encapsulated drugs by EPR effect represents a passive targeting mechanism
enhancing the drug delivery and drug therapeutic potential
50. Liposomal formulations containing 4 mol% of PTX were prepared either as
conventional ones made up of PC/PG/cholesterol (molar ratio, 9:1:2) or as
pegylated ones composed of PC/PG/cholesterol/ DSPE-PEG (molar ratio,
9:1:2:0.7). However, both types of liposomes were physically stable only for
less than 1 day in the hydrated state at 4 °C and reserved
only 50% of the initial PTX content 51. Conventional and pegylated liposomes were produced by extrusion of
MLVs producing PTX liposomes with a average  size of 120 nm. The addition of cholesterol at
more than 20% caused a PTX precipitation and liposome destabilization. The
conventional PTX liposomes were more stable than previously pegylated ones
52. However, the pegylated PTX liposomes were long-circulating showing a
half-life time of 48.6 h against 9.3 h for the conventional ones. It is a
result of a less clearance of the pegylated PTX liposomes. Their
biodistribution established a                                                                                                                       considerable
decline in PTX uptake in RES-containing organs (liver and spleen) after 0.5 and
3 h in comparison with their conventional complements in Balb/c mice model
52. The biodistribution  result of PTX
was assessed after i.v. administration of 7.5 mg PTX/kg (single dose) of
Taxol®, conventional and pegylated PTX liposomes in mice model bearing tumor
xenograft. Cr-P was rapidly accumulated and cleared by the liver, spleen and
lung, while PTX liposomes exhibited a prolong half-life of 1.6-fold and
7.1-fold for the conventional and pegylated formulation, respectively. In tumor,
after 6 and 24 h the PTX concentration of pegylated liposomes (0.4 and 0.1
?g/g) was expressively higher than that of the conventional liposomes (0.1 and
0.03 ?g/g) and Cr-P (0.05 ?g/g and cleaved). In case of pegylated PTX
liposomes, the drug concentration in tumor after 6 h was more than that in
spleen, lung, heart, kidney and brain. The aggregation of the pegylated PTX
liposomes after i.v. administration of 7.5 mg PTX/kg (3 cumulative doses in
4-day intervals) in tumor resulted in a signifi- cant inhibition of the tumor
growth as compared with the other preparations at the end of the observation
period of 60 days. Long circulation time and slow delivery of PTX from
pegylated liposomes gives a chance for PTX to be retained at tumor through EPR
effect and uphold the effective therapeutic level for a long-time period via a
depot effect. The passive tumor targeting was explained by an application of
pegylated PTX liposomes of an suitable size of b200 nm 53. The arrangement of
lipids comprising EPC, HEPC, cholesterol and DSPEmPEG was optimized to make
better the encapsulation capacity of PTX and prepare stable pegylated
liposomes. The addition of cholesterol allowed a preparation of small-sized
liposomes with high drug incorporation. The presence of pegylated PL gave a
steric stabilization of the liposomes. Increasing portions of HEPC (25 to 82
mol%) have headed towards to an increased average diameter of the liposomes
(113 to 203 nm), in the mean time , the encapsulation efficacy of PTX slowly decreased
(69 to 37%). Established on these results, the liposomal formulation of
EPC/HEPC/ cholesterol/DSPE-mPEG (molar ratio, 15:5:2:1) was found to be optimum
. Liposomes were made by sonication of MLVs followed by extrusion through 0.2
?m filters. The maximum encapsulation capacity of stable liposomes during the
preparation was observed to be 20 mol%.furthermore , PTX accelerated liposome
destabilization, needlelike precipitates and aggregated liposomes weredetected .
Liposomes encapsulating up to 15 mol% of PTX reserved the initial drug content
and the real size (about 140 nm) for 6 months at 4 °C.furthermore , i.v.
administration of liposomal PTX (40 mg/kg) triggered neither acute toxicity nor
mice death, which Taxol® at the consistent dose did 54. For the in vivo
studies employing Colon-26 solid tumor-bearing mice, it was established that
PEG-coated PTX liposomes delivered a significantly higher amount of PTX to
tumor tissue and gave more excellent anti-tumor effect than PEG uncoated PTX
liposomes 55. These results proposes that PEG liposomes would aid as a potent
PTX delivery carrier for the future cancer chemotherapy and signifies a appropriate
platform for the advancement of targeted liposomal PTX systems (Koudelka and Turánek, 2012)

 

In yet one more revision where long circulating and targeted liposomes
of paclitexal for FGF receptors were arranged employing a thin film
evaporation-extrusion method .Provisonally , paclitaxel, egg
phosphatidylcholine, cholesterol, COOH-PEG2000-cholesterol,
and DSPE-PEG2000 (2:60:30:5:3
mol/mol) were dissolved in 4 mL of methanol and chloroform (1:3, v/v) as a mixed
solvent at 37 Celsius
 and dried to a thin film, firstly with
nitrogen gas and after that under vacuum for several hours. The lipid film was
hydrated with 2 mL of 10 mM
2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 5.0) at 40°C for one hour. To obtain
small and homogeneous vesicles, the liposome suspension was sonicated for 10
minutes in a bath-type sonicator (Bransonic 12) along with  three extrusion cycles via  polycarbonate filters with 0.2 ?m pores
(Lipex™ Extruder, Northern Lipids Inc, Vancouver, BC).40 For CL-PTX, paclitaxel, egg phosphatidylcholine, and cholesterol
(molar ratio, 2:60:30) were dissolved in chloroform/methanol (3:1, v/v), and
then ready as for the above description of paclitaxel-loaded targeted PEGylated
liposomes (TL-PTX) to obtain persistent CL-PTX. The resultant liposomes were purified
on a Sephadex G-75 column to remove the non-encapsulated drug particles.(Cai
et al., 2012)

 

Application
of pegylated paclitaxel-containing liposomes in metronomic chemotherapy

 Metronomic chemotherapy or common
administration at doses much less than MTD
represents an alternate method of treatment with respect to common strategy
utilizing  MTD
chemotherapy of the drug. As an advantage , this strategy shows a lower
destructive effect and metronomic regimen could exploit the growth-limiting
effects as well as the anti-angiogenic properties. The pegylated PTX liposomes
and Taxol® formulation were used to predict the influence of metronomic and MTD action on the tumor growth inhibition and
antiangiogenic activity. The uncoated  Balb/c mice bearing MDA-MB-231
cells were in developing stage to be treated after 11th day of xenograft
implantation. PTX formulations were administered at 15 mg PTX/kg on the 11th,
15th, 19th and 23rd day and at 6 mg PTX/kg every day from the 11th to 15th day in
addition to the 22nd to 26th day for MTD
and metronomic chemotherapy, respectively. On the 32nd day, mice were
sacrificed and the tumor volume was measured .Mostly , the tumor growth in the
groups of metronomic and MTD
pegylated PTX liposomes as well as MTD
Taxol® showed the same inhibition effect, while important tumor progression was
observed for the metronomic administration of Taxol®. The metronomic use of pegylated
PTX liposomes was more effective in anti-angiogenic action as determined by
micro-vessel compactness calculation. These results postulates that conventional
administration of pegylated PTX liposomes had an anti-angiogenic effect that disrupts
the blood stream and may be more effective in overcoming tumor growth in vivo
56.(Koudelka and Turánek, 2012)

Tissue distribution study:

·        
In case of Taxol®, plasma absorption of
paclitaxel was nearly negligible at 6 h, and it was readily uptake and clean by
the liver, spleen and lung.Though , when paclitaxel was encapsulated in
liposomes, the plasma concentration was sustained for up to 24 h.

·        
Furthermore , PEGylated liposomes gave  greater plasma level than that of conventional
liposomes, which is consistent with the results from the pharmacokinetic study
in rats. In tumor tissue, paclitaxel concentration in PEGylated liposomes was significantly
higher than that in conventional liposomes and in Taxol® at 6 and 24 h. Also,
in the case of PEGylated liposomes, the paclitaxel
concentration in tumor was higher than that in spleen, lung, heart, kidney and
brain tissues from 6 h. These results proposed that PEGylated liposomes were noticeably
localized in the tumor tissues.

·        
It look like that
long-circulating time and slow discharge of PEGylated liposomes might offer sufficient
chance for paclitaxel to be achieved at the tumor site through the EPR effect
and preserve the effective therapeutic concentration for a long period of time
through the depot effect.

·        
Therefore, these results designate
that our PEGylated liposomal formulation successfully increased the antitumor effectiveness
while declining the potential side-effects.

Inhibition of tumor
growth:

·        
Since the paclitaxel loaded
classical liposomes and PEGylated liposomes were highly stored in the tumor
tissues of MDA-MB-231 human breast cancer xenograft model, the tumor growth
inhibition effect was further estimated. The study on the control (saline)
group ended on the 35th day because the tumor capacity was extremely enlarged
(about 2000 mm3), while other groups lasted until the 60th day.

·        
The PEGylated liposomes inhibited
tumor growth most efficiently, followed by the conventional liposomes and
Taxol® (p

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