According and lipids 10% (w/v) S100PC and MPEG2000-DSPE,

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 processing parameters , 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 molar
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 then connected 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)


Functionalized liposomes

Liposomes speak to a versatile medication
carrier system that may be enriched for other properties moving forward their targeting
on towards those tumors. A novel amongst those approaches will be shaping an
stealth liposome.

Paclitaxel encapsulated in pegylated liposomes (long-circulating

Fast freedom of the accepted
liposomes Toward RES speaks to a novel  amongst those significant limitations in the
drug delivery. This issue might have been understood  eventually by utilizing the long-circulating
liposomes. Those grafting about accepted liposomes for an inactive and
biocompatible polymer for example, polyethylene glycol (PEG) prompts those
shaping of a protective and hydrophilic layer on the liposomal surface 46.

the surface change could
limit the abstraction of liposomes by cell of RES and clearly prolongs those
half-life from purporting  liposomes
Throughout coursing period47.

Those long-circulating
liposomes would likewise alluded should similarly as pegylated, sterically
settled alternately stealth liposomes. It might have been showed that the
permeability of  the slim endothelium in
the tumors may be increased as compare to ordinary tissues 48.

the macromolecules are
passively gathered will more excellent degree to more drawn out period in the
tumor over in the non-malignant capillary endothelium. This wonder may be alluded
will similarly as an improved permeability and 
Also provide maintenance (EPR) effect 49.

the zeta possibility of the conventional
liposome might have been Practically unbiased as anticipated since S100PC
Furthermore cholesterol donot have any charge. addition of 3% (v/v) Tween 80 in
the hydration medium, those imply zeta possibility about traditional liposome
scattering might have been that’s only the tip of the iceberg negative which
will be steady with past reports. The reason behind those lower zeta potential
was because of the halfway hydrolysis of Tween 80. Those zeta potential from
claiming PEGylated liposomes might have been more negative over that of conventional
liposomes because of those contrarily charged phosphate aggregation from
claiming MPEG-DSPE, which is likewise about the outcome the reports in
literature. In this case, the impact from claiming Tween 80 for zeta-potential
appears to be unimportant since those negative charge because of those
PEGylation may be to such an extent bigger.(Yang et al., 2007)


long-circulating liposomes (100 nm) showed An higher recurrence for
encountering permeability  in vessels of
the tumor and extra-vasating under those fenestrated tumor tissue. This aggregation
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
introduction  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 circulation   result of PTX was observed after i.v.
administration of 7.5 mg PTX/kg (single dose) of Taxol®, conventional and
pegylated PTX liposomes in mice model having tumor xenograft. Cr-P was rapidly amassed
and cleared by the liver, spleen and lung, while PTX liposomes exhibited a enhanced
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 prominent inhibition
of the tumor growth as compared with the other methods at the end of the
observation period of 60 days. Long circulation time and slow delivery of PTX
from pegylated liposomes gives opportunity 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  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, needle like
precipitates and aggregated liposomes were detected . 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.,


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

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

Inhibition of tumor

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 observed. The study on the control (saline) group
terminated on the 35th day reason is that 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 < 0.05). This better anti-tumor activity of the PEGylated liposomes can be clarified by the increased local concentration of pacltiaxel near the tumor via EPR effect.(Yang et al., 2007)


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