An in vitro evaluation of antitumor activity of sirolimus-encapsulated liposomes in breast cancer cells
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Journal of Pharmacy and Pharmacology, 2021, Vol 73, 300–309
doi:10.1093/jpp/rgaa061
Research Paper
Advance Access publication 25 January 2021
Research Paper
An in vitro evaluation of antitumor activity of
sirolimus-encapsulated liposomes in breast
cancer cells
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Uttom Nandi, Ichioma Onyesom and Dennis Douroumis*
Medway School of Science, Faculty of Engineering and Science, University of Greenwich, Chatham Maritime, Kent, UK
*Correspondence: Dennis Douroumis, Faculty of Engineering and Science, University of Greenwich, Chatham Maritime
ME4 4TB, Kent, UK. Email: D.Douroumis@greenwich.ac.uk
Received July 30, 2020; Accepted December 28, 2020.
Abstract
Objectives Design and examine the effect of sirolimus-PEGylated (Stealth) liposomes for breast
cancer treatment. In this study, we developed conventional and Stealth liposome nanoparticles
comprising of distearoylphosphatidylcholine (DSPC) or dipalmitoyl-phosphatidylcholine (DPPC)
and DSPE-MPEG-2000 lipids loaded with sirolimus as an anticancer agent. The effect of lipid grade,
drug loading and incubation times were evaluated.
Methods Particle size distribution, encapsulation efficiency of conventional and Stealth liposomes
were studied followed by cytotoxicity evaluation. The cellular uptake and internal localisation of
liposome formulations were investigated using confocal microscopy.
Key findings The designed Stealth liposome formulations loaded with sirolimus demonstrated an
effective in vitro anticancer therapy compared with conventional liposomes while the length of the
acyl chain affected the cell viability. Anticancer activity was found to be related on the drug loading
amounts and incubation times. Cell internalization was observed after 5 h while significant cellular
uptake of liposome was detected after 24 h with liposome particles been located in the cytoplasm
round the cell nucleus. Sirolimus Stealth liposomes induced cell apoptosis
Conclusions The design and evaluation of sirolimus-loaded PEGylated liposome nanoparticles
demonstrated their capacity as drug delivery carrier for the treatment of breast cancer tumours.
Keywords: sirolimus; stealth; liposomes; breast; cancer; BT-474 cells
Introduction agent.[11] In another study, lipid–polyethylene glycol (PEG)–polymer
hybrid nanoparticles loaded with farnesylthiosalicylic acid demon-
The use of drug delivery carriers to enhance the therapeutic efficacy
strated high affinity for glioblastoma tumours.[12] The development
of anticancer drugs and reduce systemic toxicity has been extensively
of poly (lactic-co-glycolic acid) – methoxy (PEG)-2000 nanoparticles
examined.[1] Liposome drug delivery systems[2–5] presently function
with or without 1,2-dioleoyl-3-trimethylammoniumpropane
as a useful tool for the delivery of anticancer drug substances, such
showed significant tumour reduction for both in vitro and in vivo
as doxorubicin, paclitaxel[6, 7] or topotecan for skin cancers[8] and
studies. In addition, alpha-tocopheryl polyethylene glycol 1000
ginsenoside for lung cancer.[9] Liposomes are also widely used as
succinate (TPGS)-coated docetaxel-loaded liposomes were also
nanocarriers for both passive and active targeting where the later has
developed to reverse multidrug resistance compared with DSPE-
shown a steep rise in preclinical research and demonstrated signifi-
mPEG-coated liposomes (Stealth liposomes) and marketed known
cant clinical advances.[10] Recent studies of targeted liposomes such
as Taxotere.[13] Generally, the advantages of PEGylated liposomes
as PEGylated, binding with oestrogen receptor have demonstrated
have been presented extensively in several studies.[14] The developed
benefits for leukaemia and the delivery of mitoxantrone anticancer
TPGS-conjugated liposomes showed significant in vitro advantages
300
© Crown copyright 2021.
This article contains public sector information licensed under the Open Government Licence v3.0 (http://www.nationalarchives.gov.uk/doc/open-government-licence/
version/3/).
Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 3 301
in human breast cancer MCF-7 and resistant MCF-7/ADR cells sug- cells. Although, sirolimus is not considered a potent anticancer drug
gesting that they could reverse Multidrug resistance and effectively substance, there are strong indications that it could be effectively
treat breast cancer. Another, recent study was reported by Park B. H. used for clinical studies in PTEN-deficient glioblastoma in human
et al. where they have developed negatively charged 1,2-dimyristoyl- breast cancer, showing antcancer activity.[24, 31] The preliminary find-
sn-glycero-3-phosphoglycerol (DMPG)-based liposomes for drug ings of this study provide promising results against tumour progres-
administration. Their novel DMPG-POPC liposomes, combined with sion in breast cancer to develop sirolimus liposomal formulations
the neutral lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholin with improved stability and better.
(POPC), can particularly bind to MCF-7 breast cancer cells and in-
crease cellular uptake in comparison to the CHOL-POPC liposome
resulting in enhanced cytotoxic and anti-colony activity compared Materials and Methods
with free drugs.[15] In this study, distearoylphosphatidylcholine Materials
(DSPC) and dipalmitoyl-phosphatidylcholine (DPPC) were used Sirolimus was kindly donated by the LC laboratories (Massachusetts,
for the liposome preparation as they are known to produce stable USA) for this study and has been used as received. All three GMP
formulations due to the steric hindrance created by larger head grade lipids namely, Distearoyl-phosphatidylethanolamine-methyl-
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groups.[16] DPPC and DSPC, a C16 and C18 long chain has a transi- polyethyleneglycol (DSPE-MPEG-2000), distearoylphosphatidylcholine
tion temperature of 41.5 and 54.5°C, respectively. Theoretically, this (DSPC) and dipalmitoyl-phosphatidylcholine (DPPC) were purchased
variation in their molecular structure will also allow us to advance from Lipoid GmbH (Ludwigshafen, Germany). Both 3T3 and BT-474
liposomes with varius release kinetics that may be drug specific. cell lines were purchased from the American Type Culture Collection
About 20–30% of human breast cancers associated with poor (ATTC: Manassa, Virginia, USA). Cholesterol for the liposome prepar-
clinical prognosis have been reported to have amplification and/ ation and curcumin were purchased from Sigma Aldrich (Dorset, UK).
or overexpression of the HER2/ErbB2 oncogene.[17–19] ErbB2 also Also, Dulbecco's modified Eagle's medium (DMEM) – for cell culture,
known as HER2/Neu belongs to a sub-class of the tyrosine kinase thiazolyl blue tetrazolium bromide for cytotoxicity assay, L-glutamine,
epidermal growth factor (EGF) receptor family.[20] HER2/ErbB2 sig- penicillin, streptomycin, fetal bovin serum (FBS) and trypsin were all
nals via the Akt/PI3-K pathway and leads to the activation of mTOR, purchased from Sigma-Aldrich (Dorset, UK).
a critical mRNA translation regulator that controls cell growth via
translational control of an array of proteins. Several research studies
Preparation of liposome formulations
have demonstrated that amplification and/or overexpression of
The liposome nanoparticles were prepared using the method de-
HER2 in breast cancers resulting in sensitivity to rapamycin; there-
scribed by Bangham et al.[32]
fore evaluation of overexpressed HER2 in breast cancer patient
Firstly, all the lipid mixtures, cholesterol or no drug were weighed
could serve as a prediction of rapamycin sensitivity in breast cancer
according to the formulation design (Table 1) and dissolved in the
patients.[17–19] Mosley et al. reported that rapamycin inhibits multiple
chloroform. The mixture was then evaporated using a rotary evapor-
stages of c-Neu/ErbB2 tumour progression in a transgenic mouse
ator to obtain lipid films. This was then again hydrated using 1000 µl
model of HER2 positive breast cancer.[21] In their study, treatment of
of double deionised water at around 5°C using an Eppendorf vial.
MMTV-c-Neu transgenic mice with rapamycin caused growth arrest
The solution was vigorously agitated for 10 min and then extruded
and regression of primary tumours with no evidence of toxicity or
through Lipex extruder by Northern lipids INC. The solution was
weight loss. The observed effect was proposed to be due to decreased
passed through a 400 nm, 200 nm and then 100 nm polycarbonate
proliferation associated with reduced cyclin D1 expression (an es-
filter (nucleopore), for 20 times respectively at a set temperature of
sential regulator of proliferation in HER/ErbB2 cells) and increased
5°C (above the lipid transition temperature). Only the drug-loaded
cell death in primary tumours. The data from this preclinical study
formulations were passed through a Sephadex G50 column to re-
of ErbB2/Neu induced breast cancer models suggest that HER2/
move any unencapsulated drugs remaining in the solution. The solu-
ErbB2 positive breast cancer may be particularly sensitive to the ef-
tion was then analysed using a particle size analyser from Malvern
fects of rapamycin analog/sirolimus.
instrument, Zetasizer Nanoseries, (Malvern, UK).
Sirolimus is a macrocyclic lactone immunosuppressive agent
Also, rhodamine (10 µg/10 mg of lipid) was dissolved into the
that inhibits the cell division cycle and cellular proliferation by
chloroform to prepare a stained blank liposome formulation for the
facilitating kinase activation and stopping the cellular growth
cellular uptake study. The solution was also passed through a PD-10
phase.[22, 23] Researchers have shown sirolimus as an active anticancer
column to remove any free dye from the formulation.
agent[24] apart from being used in drug eluting stents for treatments
in percutaneous coronary intervention.[25–28] The inhibitors of
mTOR as anticancer agents, such as sirolimus, are undergoing ac- Encapsulation efficiency of liposome formulations
tive evaluation in various malignancies.[29] A recent study showed As unbound drug was removed during the preparation stage,
that sirolimus-loaded PLGA nanoparticle which were also coated drug-loaded liposomes were considered to have sirolimus en-
with polysorbate 80 exhibited enhanced anti-glioma activities using capsulated inside the shell only. To analyse their capacity of
in-vitro models.[30] However, literature review suggests sirolimus-
loaded liposomes are yet to be studied using human breast cancer
Table 1 Composition of studied liposome formulation
cell lines.
In this study, novel formulations containing sirolimus-loaded Formulations Molar ratio
conventional and Stealth liposomes were prepared and their
antitumor efficacy was studied using in-vitro cancer cell line models. DPPC : Cholesterol (DPPC conventional) 18.6 : 9.0
The cytotoxicity, cell uptake and apoptosis of sirolimus encapuslated DPPC : DSPE-MPEG2000 : Cholesterol (DPPC Stealth) 12.6 : 1.14 : 8.0
DSPC : Cholesterol (DSPC conventional) 18.6 : 9.0
liposome nanoparticles were investigated using BT-474 cancer cells
DSPC : DSPE-MPEG2000 : Cholesterol (DSPC Stealth) 12.6 : 1.14 : 8.0
including the cytotoxicity of empty liposomes in 3T3 endothelial
302 Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 3
encapsulation, they were dissolved using 50% acetonitrile solu- liposomes formulations were also incubated at liposome concentra-
tion and then eluted using high performance liquid chromatog- tions of 20, 100, 200, 400 and 1000 µg/ml. Same method was then
raphy. The amount of drug encapsulation was calculated using also used to study cytotoxicity in 3T3 cell lines.
the following equation:
Å ã Cellular uptake
Sirolimus dissolved in Acetonitrile
EE(%) = × 100% Cellular uptake of liposomes was determined using Nikon fluores-
Amount of Siro lim us used during preparation
cent microscope. First, 2 × 103 cells were seeded on a glass coverslip
and incubated in six-well plate for 24 h. Then rhodamine-loaded
Formulation stability evaluations liposomes were added into the cell and again incubated for 24 h. Cell
Prepared liposomal formulations were left to equilibrate at the room medium were discarded after incubation and washed three times
temperature, visually observed for any precipitation and then ana- with PBS. Then cells were fixed to the glass coverslips by adding
lysed using Zetasizer Nanoseries, (Malvern, UK). After evaluation, 1 ml of 4% paraformaldehyde and left in the dark for 15 min. The
the formulations were stored at 4°C, in the refrigerator during the paraformaldehyde solution was then discarded, and cells were again
preparation of their in-vitro release profile study. washed three times with PBS to wash off any remaining solution. The
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glass coverslip was then mounted on a glass slide using vectashiled
Release profile studies mounting medium containing DAPI to stain the cell neucleus. Images
Release study was done using phosphate buffer saline, pH 6.8 as the were acquired using 40× oil immersion mode to analyse the localisa-
method described by Hao et al.[33] First, 1 ml of liposome solution tion of liposomes in the cell.
was placed into a cellulose dialysis bag, with a molecular weight
cut-off 10 kDa. The solution containing bag was then suspended Apoptosis study
in a conical flask containing 20 ml of pH 6.8 solution at 37 ± 2°C. Liposome formulations were conjugated with Alexa Fluor 488 to
Samples were collected at predetermined time intervals and ana- carry out apoptosis study. Drug-loaded DSPC Stealth liposomes
lysed using high-performance liquid chromatography using the were studied using BT-474 cells. First cells were seeded using glass
following method. coverslips at a 2 × 104 cell density for 24 h. Then 20 µl of drug-
HPLC analysis of sirolimus content in liposome formulations loaded Stealth liposomes were incubated with cells for 24 h where
were performed using a HPLC-UV system (Agilent technologies, non-treated cells were used as a control for the experiment. Cells
United Kingdom). Chromatographic separation was obtained using were washed using PBS after incubation and 1 ml of annexin binding
a Hichrome C18 column (150 mm × 4.6 mm, 5 µm). The mobile phase buffer was added to the cells on the coverslip followed by 10 µl of
consisted of 60% acetonitrile and 40% double deionised water. The annexin V conjugate and 5 µl of propidium iodide. After this treat-
temperature of the column was set at 50°C and the wavelength was ment, cells were again incubated for 15 min at room temperature.
set at 278 nm and pump rate of 1 ml/min. The injection volume for Then, 2 µl of mounting medium containing DAPI was added and
all samples was 50 µl and elution time was 6 minutes (min). images were acquired using Nikon fluorescent microscope.
Analysis was done using an Agilent 1200 series instrument
which was equipped with a quaternary pump or gradient elution
system along with a Hicrome C18 column (150 mm × 4.6 mm, 5 µm). Results and Discussion
A 50 µl volume of sample was eluted using a mobile phase consisted
Particle size and drug encapsulation analysis
of acetonitrile and double deionised water at a 60 : 40 ratio. The mo-
The particle size and zeta potential of liposomal formulations were
bile phase was pumped at 1 ml/min flow rate at a column tempera-
measured and results in Figure 1 demonstrate an average particle
ture of 50°C, at a wavelength of 278 nm where the relative retention
size of 170–200 nm for empty liposomes with monomodal distribu-
time for sirolimus was set to 6 min.
tion with a polydispersity index of less than 0.2 indicating a homo-
genous distribution. As shown in Figure 2, the liposome formulations
Cytotoxicity studies demonstrated great encapsulation efficiency (EE) that varied from
For the cytotoxicity study, both 3T3 (endothelial cells) and BT-474 90 to 95%. More specifically, Stealth liposomes displayed consid-
breast cancer cell lines were cultured in an incubator at 37 ± 1°C erably greater EE (up to 3% more, P < 0.001) than conventional
and 5 ± 0.2% CO2, using DMEM culture medium which was sup- liposome which was attributed to the formulation composition i.e.
plemented with 10% FBS, 1% L-glutamine and 1% penicillin and Stealth liposome comprising of MPEG 2000. Furthermore, Figure 1
streptomycin). The culture medium was replaced with a fresh me- shows that for the sirolimus-loaded nanoparticles a reduction in the
dium every three days to ensure the continuous growth of cells. particle size was observed compared to empty. This uncommon be-
Cytotoxicity study of drug-loaded formulation and drug dissolved haviour has been previously studied and is associated to the drug–
in ethanol was initially determined using BT-474 breast cancer cell lipid interactions.[25] Published reports on liposomal drug delivery
line using MTT assay. For the assay, 1 × 105 cells were seeded in each systems also attributed excessive EE due to a number of features
well of a 24-well flat bottom plate for 24 h. The prepared liposome such as the composition of the formulation (e.g. quantity of chol-
formulations were then added into all the wells at specific concen- esterol and MPEG2000), preparation process and the solubility of
trations and left 2 h for incubation. After incubation culture me- the drug.[34–36] Nii and Ishii (2005) estimated the EE of lipophilic
dium was discarded using a micropipette followed by an addition of and amphiphilic drugs in three grades of egg lecithin with various
200 µl of isopropanol. Then 100 µl of the dissolved MTT formazan degrees of saturation. A similar work presented by Rouf et al., re-
crystal was added into each well and absorbance was read at a wave- sults in unstable liposome compositions during stability that pre-
length of 492 nm using an ELISA microplate reader. The study was sented a significant particle size increase after 6 months.[37] Our
done using five different sirolimus concentrations i.e. 20, 60, 100, liposome compositions appeared to be highly stable after 6 months
200 and 500 µg/ml. Blank and drug-loaded (1, 2 and 5 mg sirolimus) stability.[25]
Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 3 303
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Figure 1 Particle size distribution of Stealth liposome nanoparticles (A) DPPC Stealth liposomes blank (red) and loaded (green); (B) DSPC Stealth liposomes
blank (red) and loaded (loaded).
The studies attributed high encapsulation compared to lipid com-
position and drug solubility in relation to the logP value and preparation
process. The liposomal formulation containing the higher saturated egg
lecithin presented higher EE compared to the other formulations. This
observation was attributed to the differences in the packing geometry
of the hydrophobic carbon chains on the liposomal membrane. It was
also stated that the logP value of a drug can affect EE of liposome for-
mulations. Drugs presenting higher lipophilicity demonstrated higher en-
capsulation capacity and vice versa. It was also proved that amphiphilic
drugs resulted in better encapsulation when dissolved in aqueous phase
than chloroform. On the other hand, Ramana et al. (2010) also showed
the effect of egg phospholipid to cholesterol ratios and the drug to total
lipid. The study demonstrated egg phospholipid liposome formulation
to be optimised with high encapsulation capacity of 80% Niverapine Figure 2 Encapsulation of sirolimus in DPPC and DSPC conventional and
at phospholipid to cholesterol ratio of 9 : 1. Furthermore, a significant Stealth liposome formulations (n = 3).
increase in the encapsulated drug amount was reported with increasing
drug to lipid ratios up to 1 : 5, but not further these ratios. Nevertheless,
cumulative release from loaded liposome formulations. The ini-
in this study the increased drug encapsulation attained for the liposome
tial burst release that was observed is attributed to the presence of
nanoparticles could be associated with the preparation process and the
free drug on the liposome surface.[38] As anticipated for a hydro-
sirolimus high lipophilicity (log Po/w of 5.77).
phobic molecule, the drug release rate was comparatively slow.
Approximately 13% of the encapsulated drug was released after 24 h
Sirolimus release profile in liposome formulations from the nanodispersions. A noteworthy difference was obtained
The drug release study was carried out over a period of 72 h at a in the release profile between conventional and Stealth liposomes
controlled temperature of 37 ± 2°C. Figure 3 illustrates the sirolimus where conventional liposomes (DSPC, DPPC) presented higher
304 Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 3
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Figure 3 Sirolimus release profile from DPPC and DSPC conventional and Stealth liposome nanodispersions (n = 3).
drug release (13%) in comparison to Stealth liposomes (DSPC, 5%; Antiproliferative effect of sirolimus versus curcumin
DPPC, 10%;) in 24 h. Similar findings were previously reported by The antiproliferative effects of blank and sirolimus encapsulated
Panwar et al. (2010) and Hioki et al. (2010) where Pegylated lipo- liposomes were investigated against an alcohol solution of sirolimus
somes (Stealth) were found to impede drug release compared with and the results are presented in Figure 4. The antiproliferative effect
the non-pegylated equivalent (conventional).[39, 40] of sirolimus on HER-2 overexpressing BT-474 cells was also com-
Panwar et al. (2010) observed that the albendazole release pared to that of pure curcumin a highly potent anticancer drug
profile from nanosized liposomes decreased in declining order of (control).[42] Curcumin and sirolimus were dissolved in ethanol and
free drug, drug-loaded conventional liposomes and slightest with various concentrations were incubated in the cells. In addition, the
drug-loaded Stealth liposomes. On the other hand, Hioki et al. tolerability of ethanol was assessed by incubating various amounts
(2010) reported the influence of temperature and serum on the re- of solvents in breast cancer cells. The experimental findings revealed
lease profile of both conventional and Stealth liposomes. The study that the highest tolerable ethanol amount was 2% v/v. Consequently,
demonstrated high drug release rates for conventional liposomes both drugs were dissolved and incubated with cells at ethanol con-
with temperature increase and the presence of serum. In Figure 3 centrations below 2%.
release of sirolimus from liposome nanoparticles showed a max- The cytotoxicity study of pure sirolimus (Figure 4) showed ad-
imum release rate after 72 h of 16% (DPPC) and 14% (DSPC) for equate antiproliferative activity at concentrations above 40 µg/ml
conventional liposome 12% (DPPC) and 10% (DSPC), for Stealth without, however, being able to suppress cell viability less than 20%
liposomes, respectively. when the concentration surpassed 500 µg/ml. On the other hand,
For Stealth liposome nanoparticles, the effect of lipids with dif- curcumin, a known potent anticancer agent, showed a pronounced
ferent phase transition temperatures on sirolimus release was also reduction in viability of BT-474 cells at much lower concentration
investigated in vitro. DSPC-Stealth liposomes presented roughly of 40 µg/ml. A further increment of curcumin concentration during
5% release after 24 h while DPPC-Stealth liposomes showed 10% the study reduced the cell concentration to almost 0%, as a reason
sirolimus release. The results are anticipated due to the higher phase curcumin concentration was limited to 100 µg/ml during the study.
transition temperature (Tm) of DSPC compared with DPPC. This is The findings often are regarded advantageous in cancer treatment as
also an indication that the liposome formulations are stable with several reports have emphasised the drawbacks of increased systemic
rigid membranes, which is particularly important for in-vivo ad- toxicity for other highly potent anticancer drugs.
ministration. The rigidity of liposomal membrane should inhibit
or lower any early drug release and increase the circulation time.
Chen et al. (2012) investigated the effect of Stealth formulations on Antiproliferative effect of liposome formulations
drug release using lipids with various phase transition temperatures. Preliminary cytotoxicity studies of unloaded (blank) conventional
Brucine was encapsulated in four different PC-Stealth liposomes and Stealth liposome nanoprticles were assessed using fibroblast
(DPPC, DSPC, SPC and HSPC).[41] In vitro release studies showed endothelial cells (3T3) while sirolimus-loaded formulations were as-
that the brucine release rate increased with decreasing phase tran- sessed on BT-474 cancer cells using MTT assay. In-vitro cytotoxicity
sition temperatures of the PC (DSPC: 6.4%, HSPC: 6.1%, SPC: analysis of blank DPPC conventional and Stealth liposomes carried
13.2%, DPPC: 10.5%), particularly when incubated in rat plasma out on 3T3 endothelial cells in order to estimate their cytotoxicity.
with drug release of 80.9% for SPC-Stealth and 15.5% for HSPC- As shown in Figure 5, the MTT assay of the endothelial 3T3 cells
Stealth liposomes after 10 h. incubated with DPPC conventional and Stealth liposomes displayed
Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 3 305
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Figure 4 Antiproliferative effect of pure curcumins and sirolimus incubated
Figure 6 Cytotoxicity of sirolimus (1.09 mm) loaded conventional DPPC :
in BT-474 cancer cells using MTT assay for 24 h (standard deviation, n = 3).
cholesterol (18.6 : 9, molar ratio) and Stealth DPPC : DSPE-MPEG2000 : chol-
esterol (12.6 : 1.14 : 8.0), molar ratio on BT-474 cell lines (24 h, n = 3).
in breast cancer cells is more profound to sirolimus when compared
to other aberrations.[45]
The findings on sirolimus sensitivity for different breast cancer
cells (such as MDA-MB-231, BT-474 and MCF-7) demonstrated
that BT-474 was the most sensitive to sirolimus and hence improved
the S6K1 association with sirolimus. Figure 6 shows the cytotoxic
of sirolimus-loaded DPPC conventional liposomes on BT-474
cancer cells over 24 h. The drug-loaded DPPC conventional lipo-
somes (1.09 mm) showed reduced cell viability by 25% after 24 h
when compared to the empty DPPC liposomes (8.0%) respectively.
The DPPC conventional liposome nanoparticle presented notably
higher antiproliferative activity (68% cell viability) in a dose de-
pendent manner when compared to their Stealth counterparts (75%,
Figure 5 Cytotoxicity of empty DPPC : cholesterol (18.6 : 9, molar ratio) and
P < 0.05). A, increase is cell cytotoxicity was observed for both con-
Stealth DPPC : DSPE-MPEG2000 : cholesterol (12.6 : 1.14 : 8.0 molar ratio),
ventional and Stealth and liposomes at concentrations of 20 µg/ml,
liposomes on 3T3 endothelial cells (24 h, n = 3).
leading to cell viability of 80%. This cytotoxicity difference between
DPPC conventional and Stealth liposomes is directly associated to
92% (Stealth) cell viability and 90% (conventional) at maximum the lipid composition. As anticipated, MPEG2000 addition to the
concentrations of 1000 µg/ml. liposome composition forms steric hindrance out the outer surface
The findings confirmed that both liposome nanodispersions are of Stealth liposomes resulting in decrease interactions with the serum
non-toxic even at increased lipid concentrations and consequently proteins and hence increased circulation times. In-vitro studies re-
suggest systemic biocompatibility in non-cancerous locations when vealed that due to the steric hindrance formed by Pegylation, Stealth
administered. Previous studies have reported the in vitro biocompati- liposomes show lower antiproliferative action when compared with
bility of Stealth and conventional liposomes. Pitrubhakta et al. (2012) conventional liposomes. These outcomes have been associated with
investigated gematasine hydrochloride liposome nanoparticles and slow drug release rates and less interactions of Stealth liposomes
reported a 78% and 87% cell toxicity of empty conventional and with cancer cells compared with conventional ones (membrane
Stealth liposomes in human lung carcinoma cells.[43] is less dense and thus faster drug release rate). Righeschi et al. re-
Ahmad and Allen (1992) investigated the delivery of doxorubicin ported high cytotoxicity for conventional liposomes compared with
liposomes in lung cancer cells to demonstrate that conventional Stealth's l when studied in vitro.[46] In the study, dihydroartemisinin
liposomes provoked no toxic effects; but, a reduction in cell pro- was encapsulated in Egg-PC conventional and Stealth nanoparticles
liferation of empty Stealth liposomes (IC50 = 68 µm) in comparison and their efficiency was evaluated in MCF-7 cancer cells. The cyto-
to the free drug (IC50 = 8 µm) was observed.[44] Furthermore they toxic effect of the empty liposome nanoparticles showed negligible
also stated that earlier studies of empty Stealth liposomes in bone cell mortality with cell viability above 92%. Nevertheless, loaded
marrow macrophages presented negligible cytotoxicity. Therefore, conventional liposome nanoparticles presented, a significant cyto-
they conlcuded that cytotoxicity of empty liposomes nanoparticles toxic effect (IC50= 48 µm, P < 0.05) with 1.6-fold increase in toxicity
could differ from cells to cells. compared with Stealth nanoparticles (IC50 = 77 µm).
One of the objectives of the study was to estimate sirolimus The influence of several factors including particle size, drug
on BT-474 HER-2 overexpressing breast cancer cells. Although loading and lipid composition, was also studied for the produced
sirolimus presents antiproliferative activity on varius breast cancer liposome compositions. For the investigation of the drug loading
cells the sensitivity has been correlated with the level of PTEN effect, two sirolimus concentrations (1.09 mm and 2.18 mm) were
(Phosphatase and tensin homolog) and phospho-S6K1 or phospho- loaded into the liposome nanoparticles and the antiproliferative
AKT aberrations. Noh et al. (2004) stated that S6K1 overexpression effectiveness was evaluated. As shown in Figure 7, the increase of
306 Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 3
Figure 9 Antiproliferative activity of sirolimus (5.46 mm) loaded conven-
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tional compositions of DPPC : cholesterol (18.6 : 9, molar ratio) and Stealth
on BT-474 cancer cells (72 h, n = 3).
Figure 7 Cytotoxicity of sirolimus (2.18 mm) loaded conventional DPPC :
cholesterol (18.6 : 9, molar ratio) and Stealth DPPC : DSPE-MPEG2000 : chol-
esterol (12.6 : 1.14 : 8.0, molar ratio) on BT-474 cancer cells (24 h, n = 3).
Figure 10 Antiproliferative activity of sirolimus (2.18 mm) loaded DPPC and
DSPC–Stealth compositions (molar ratio 12.6 : 1.14 : 8.0) on BT-474 cell lines
(72 h, n = 3).
unaffected even when sirolimus concentrations increased from 2.18
to 5.46 mm. Comparable results were reported by Zeng et al., where
Figure 8 Cytotoxicity of sirolimus (2.18 mm) loaded DPPC conventional and
sirolimus doses of 5 mg/ml presented similar antiproliferative ac-
Stealth liposomes on BT-474 cell line (72 h, n = 3).
tivity compared with low doses of 1.5 mg/ml.[47]
Figure 10 illustrates the cytotoxic effect (MTT assay) of DSPC
sirolimus encapsulated amounts resulted in further cell viability – Stealth with 58% cell viability compared with 50% of the DPPC
reduction for both compositions. Stealth liposome nanoparticles Stealth liposomes. Liu et al. also proposed that the acyl chain length
showed an additional 10% reduction in cell viability with the in- of phospholipids affects the membrane rigidity of liposomal com-
crease in drug amount and thus the obtained cell viabilities were positions to a great extent.[48] The DSPC acyl chain length consists of
reduced to 75% and 66%, respectively (P ≤ 0.01). 18 carbons in contrast to the 16 of DPPC lipid.[49] When lipid moi-
Figure 8 illustrates the antiproliferative effect of liposome com- eties with long acyl chain length are incorporated in liposome com-
positions, at 2.18 mM sirolimus concentrations, on BT-474 cells positions form membranes with rigid membrane and high transition
where the incubation time was further increased to 72 h. A further temperatures in comparison to lipids with short acyl chain lengths.
reduction in cell viability was observed for both DPPC conventional
and Stealth liposomes with similar cell viability at of 48 and 50%, Cell internalisation and apoptosis of liposome
respectively at liposome concentration of 800 µg/ml. The mechanism compositions
of this behaviour is not fully explained and it could be attributed In order to investigate the liposome internalization, empty DPPC–
to the slower sirolimus release rates for Stealth nanoparticles when Stealth nanoparticles (800 µg) were incubated in BT-474 cancer cells
compared with conventional ones. at various time intervals of 2, 5 and 24 h. The qualitative determin-
Nevertheless, high doses of liposome compositions and extended ation of cell uptake was investigated using fluorescent microscopy and
in vitro incubation times can lead to membrane destabilization for the cell nucleus was stained with DAPI while liposomes were labelled
Stealth liposomal nanoparticles and present antiproliferative ac- with rhodamine. As shown in Figure 11, the internalization of DPPC–
tivity, comparable to that of the conventional liposome. As shown in Stealth liposomes was observed after 5 h while substantial cellular
Figure 9, additional increase the encapsulated sirolimus did not pre- uptake was obtained after 24 h. As seen in Figure 11, DPPC–Stealth
sent significant change in the antiproliferative effect of the liposome nanoparticles are confined in the cytoplasm nearby the cell nucleus.
compositions and cell viability of Stealth liposomes was reduced only The process of apoptosis is considered as a physiologically pre-
3% (P = 0.05). The cytotoxicity of liposome compositions remained determined death of the cells death and is mediated during cell
Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 3 307
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Figure 11 Fluorescent microscopy images illustrating cell uptake of empty DPPC–Stealth liposomes and cytoplasm localization (A- 10× magnification, 5 h; B- 60×
magnification) after e4 h. The cell nucleus was stained with DAPI (blue) and the liposomal nanoparticles were labelled with rhodamine (red).
Figure 12 Fluorescent microscopy images (A1 – 3: after 5 h and B, C: after 24 h) illustrating apoptotic cells of sirolimus encapsulated DPPC–Stealth liposomes
(staining with Annexin V conjugate). Nucleus is stained with DAPI (blue) and apoptosis induced signal (green).
308 Journal of Pharmacy and Pharmacology, 2021, Vol. 73, No. 3
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