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 Downloaded from https://academic.oup.com/jpp/article/73/3/300/6118582 by guest on 10 August 2021 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- Downloaded from https://academic.oup.com/jpp/article/73/3/300/6118582 by guest on 10 August 2021 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 Downloaded from https://academic.oup.com/jpp/article/73/3/300/6118582 by guest on 10 August 2021 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 Downloaded from https://academic.oup.com/jpp/article/73/3/300/6118582 by guest on 10 August 2021 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 Downloaded from https://academic.oup.com/jpp/article/73/3/300/6118582 by guest on 10 August 2021 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 Downloaded from https://academic.oup.com/jpp/article/73/3/300/6118582 by guest on 10 August 2021 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- Downloaded from https://academic.oup.com/jpp/article/73/3/300/6118582 by guest on 10 August 2021 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 Downloaded from https://academic.oup.com/jpp/article/73/3/300/6118582 by guest on 10 August 2021 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).
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