Examples of Tumor Growth Inhibition Properties of Liposomal

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ACS Biomaterials Science & Engineering

Examples of Tumor Growth Inhibition Properties of Liposomal Formulations of pH-Sensitive Histidinylated Cationic Amphiphiles Arup Garu, †⊥ Gopikrishna Moku, †⊥ ‡ Suresh Kumar Gulla, †‡ Dipankar Pramanik, †ζ Bharat K. Majeti, †¶ Priya P. Karmali, †λ Shaik Haseena , †ρ Bojja Sreedhar, ‡‡ and Arabinda Chaudhuri† ‡* †

Biomaterials Group, CSIR-Indian Institute of Chemical Technology, Hyderabad-500 007, India;

‡

Academy of Scientific and Innovative Research, India; ζ Present Address: Haldia Institute of

Technology, Purba Medinipur, West Bengal 721657, India; ¶ Present address: Molecular Therapeutics Department, Nitto Denko Technical Corporation, Oceanside, San Diego, CA92058, USA; λ Present address: Formulation Development, Regulus Therapeutics, San Diego, CA92121-1121, USA; ρ Present address: Albany Molecular Research Hyderabad Research Centre Pvt. Ltd, Genome Valley, Hyderabad 500 078, India;

‡‡

Inorganic and Physical Chemistry

Division, CSIR-Indian Institute of Chemical Technology, Hyderabad-500 007, India. KEYWORDS : Endosomal pH-sensitive lipids, histidinylated cationic amphiphiles, cancer cell selective cytoxicity, anti-cancer liposomes; mitochondrial membrane depolarization.

ACS Paragon Plus Environment

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ABSTRACT: Herein we report on the unexpected cancer cell selective cytotoxicities of the liposomal formulations of aspartic & glutamic acid backbone-based four novel lipids with endosomal pH-sensitive head-groups and aliphatic n-hexadecyl& n-octadecylhydrophobic tails. Surprisingly, while the formulations killed cancer cells efficiently, they were significantly less cytotoxic in non-cancerous healthy cells. Importantly, intratumoral administration of the liposomal formulations efficiently inhibited growth of melanoma in a syngeneic C57BL/6J mouse tumor model. Western Blotting experiments with the lysates of liposomes treated cancer cells revealed that liposomes of lipids 1-4 induce apoptosis selectively in cancer cells presumably by releasing cytochrome c from depolarized mitochondria and subsequent activation of caspases 3 & 9, upregulation of Bax and down regulation of Bcl-2. In summary, the present report describes for the first time tumor growth inhibition properties of the liposomal formulations of endosomal pH-sensitive histidinylated cationic lipids under both in vitro and systemic settings. INTRODUCTION A number of prior reports demonstrated efficient bioactives delivery properties of pH-sensitive peptides,1,2 pH-tunable endosomolytic oligomers,3 charge-conversional polyion complex micelles,4 cationic polymers,5-8 and cationic liposomes.9,10 During cationic liposome mediated gene delivery, lipoplexes (complex of liposomes and plasmid DNA) enter cells usually by endocytotic pathway and gets initially localized in the endosomal compartment.11 Efficient liposomal gene transfer depends on fast release of endosomally trapped DNA into the cell cytoplasm. To this end, Wolff and co-workers pioneered the use of endosomal pH-sensitive cationic lipids with imidazole head-groups.12 Since the pKa of the weakly basic imidazole headgroups lies within the pH-range of endosomes (5.5-6.5), the imidazole head-groups of cationic


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lipids gets protonated (i.e. act as a proton sponge) once the lipoplexes enter the endosome compartments. Such endosomal buffering actions by the imidazole head-groups leads to osmotic swelling and subsequently to bursting of the endosomes due to entry of lots of hydrated chloride counterions.12,13 Subsequent studies demonstrated the promise of cationic amphiphiles with endosomal pH sensitive histidine head-groups in enhancing gene transfer efficiencies of cationic liposomes.9,14-16 With a view to designing potent transfection enhancing endosome pH-sensitive histidinylated cationic amphiphiles through structure-activity study, in the present investigation we synthesized aspartic & glutamic acid backbone based four new histidinylated cationic amphiphiles with their n-hexadecyl & n-octadecyl hydrocarbon tails covalently tethered to the histidine head-groups via ester linkages (lipids 1-4, Figure 1). NH+ Cl N H - + Cl H 3N

NH+ Cl -

H N

O O

O

n

CH 3

N H - + Cl H 3N

H N O

O O

CH3

O O ( )2

CH 3

O

O

CH3

n

n

Lipid 1; when n=11 Lipid 2; when n=13

n

Lipid 3; when n=11 Lipid 4; when n=13

Figure 1. The structures of the presently described endosome pH-sensitive histidinylated lipids 1-4 and the previously described guanidinylated lipid 5. We envisaged that use of these newly designed potentially endosomal pH-sensitive cationic lipids 1-4 as additional ingredients in the liposomal formulation may further enhance the transfection efficiencies of our previously disclosed17 guanidinylated cationic amphiphile lipid 5 (Figure 1). Findings in the initial formulation optimization experiments (data not shown)


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revealed formation of stable liposomes prepared with equimolar ratios of the histidinylated lipids, lipid 5 and DOPC (di-oleyolphosphatidylcholine, a non-cytotoxic co-lipid). While evaluating the cellular toxicities of these new liposomal formulations by conventional MTT assay, a completely unexpected trend was observed. We measured cytotoxity of the liposomal formulations with 150-170 nm hydrodynamic radii and 3-5 mV surface potentials (Table S1) in multiple cultured animal cells including four cancer cells (A549, B16F10, C26 and ASPC-1) and four non-cancerous cells (RAW264.6, COS1, NIH3T3 and human dermal fibroblast).

The

liposomal formulations were found to be mostly non-cytotoxic in all the four non-cancerous cells. Contrastingly, the liposomal formulations were found to be significantly cytotoxic in all the four cancer cells.

Such unexpected cytotoxicity profiles revealed cancer cell selective

cytotoxic characteristics of the histidinylated lipids 1-4.

Under in vivo settings too, the

liposomal formulations of lipids 1-4 significantly inhibited the growth of melanoma tumors in a syngeneic C57BL/6J mouse tumor model. EXPERIMENTAL SECTION General methods and reagents. Mass spectral data were obtained using a commercial LCQ ion trap mass spectrometer (ThermoFinnigan, SanJose, CA, USA) equipped with an ESI source or micromass Quatro LC triple quadrapole mass spectrometer. 1H NMR spectra were recorded on Varian AV 300, 400 & 500 MHz NMR Spectrometers. Aspartic acid, glutamic acid and histidine were purchased from Spectrochem, India. EDCI and HOBt were procured from SigmaAldrich, USA. Column chromatography was done on silica gel (Acme Synthetic Chemicals, India, 60-120 mesh) and the purities of all the target lipids (1-4) were confirmed to be ≥ 95% by reversed phase HPLC analysis using two mobile phases (A: pure methanol & B: 95:5, v/v, methanol/water). Cell culture lysis reagent was purchased from Promega, USA. Antibodies


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against BAX, Caspase 3 & 9, Cytochrome c, Bcl-2, GAPDH and β-Actin were purchased from Pierce, USA. Antibiotics were purchased from Hi-media, India. All the other reagents were purchased from local suppliers and used without further purification. B16F10, A549, ASPC-1, C26, COS-1, CHO, RAW264.7, NIH3T3 and human dermal fibroblasts were purchased from the National Centre for Cell Sciences, NCCS, Pune, India. Cells were grown at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS in a humidified atomosphere containing 5% CO2. C57BL/6J mice (6-8 weeks old each weighing ~20-22 g) were purchased from National Institute of Nutrition, Hyderabad, India. The in vivo experiments were performed using animal protocols approved by our Institutional Bio-Safety and Ethical Committee Guidelines. Preparation of liposomes. Appropritate equimolar ratios of lipids 1-4, lipid 5 and DOPC were dissolved in chloroform and the solvent was removed with a thin flow of moisture free nitrogen gas. The dried lipid film was then kept under high vacuum for 8 h. Sterile deionised water (1 mL) was added to the vacuum dried lipid film and the mixture was allowed to swell for overnight. The hydrated lipid films upon vortexing for 2-3 min at room temperature produced multilamellar vesicles (MLVs). MLVs were finally sonicated in an ice bath until clarity using a Branson 450 sonifier (using 100% duty cycle and 25 W output power) to produce small unilamellar vesicles (SUVs). The final lipid concentrations used for in vitro and in vivo experiments were 1 mM and 5 mM, respectively. Zeta potential (ξ) and size measurements. The sizes of the liposomal formulations of lipids 1-5 were measured by photon correlation spectroscopy and their surface potentials were measured by electrophoretic mobility using Zeta sizer 3000HSA (Malvern UK), respectively. The sizes were measured by diluting with deionised water and using refractive index of 1.59 and


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a viscosity of 0.89. 200 nm + 5 nm polystyrene beads (Duke Scientific Corps. Palo Alto, CA) were used for system calibration and the hydrodynamic diameters of the liposomes were calculated using the automatic mode. The zeta potentials of the liposomal formulations were measured using the following parameters: viscosity, 0.89 cP; dielectric constant, 79; temperature, 25 °C; F(Ka), 1.50 (Smoluchowski); maximum voltage of the current, V. The system was calibrated by using DTS0050 standard from Malvern, UK. Measurments were performed 10 times with the zero field correction and the surface potentials were calculated by using the Smoluchowski approximation. Transmission electron microscopy. FEI Tecnai 12 TEM apparatus operated at 100 KV was used in carrying out transmission electron microscopy experiments. A 10 µL drop of the liposomal sample was transferred onto an ultrathin-carbon coated copper grid by placing the grid on top for 1 min. After removing the excess fluid from one side, the grid was placed on top of a 20 µL water drop for 30 s. The excess fluid was removed and the grid was placed on top of a 20 µL drop of freshly filtered uranyl acetate (1.33%) for one min. The vesicles were finally imaged with TEM after wicking away the excess fluid and air drying the grid. FRET Assay. The pH-dependent membrane fusion activity of the liposomes (of lipids 1-4) were measured by FRET assay following a previously reported protocol14 using NBD-PE and Rho-PE (Avanti-Polar Lipids, USA) as the donor and acceptor fluorescent lipids, respectively. The biomembrane mimicking liposomes of DOPC/DOPE/DOPS/Chol (45:20:20:15, w/w ratio, with the total lipid concentration of 0.5 mM) were labeled with the donor and acceptor lipids so that the final concentrations of both the donor & acceptor lipids were 0.005 mM. The total lipid concentrations in the liposomes of histidinylated lipid/DOPC (at 1:1 mole ratio) were also kept at 0.5 mM. Equimolar amounts of the liposomes of lipids 1-4 were mixed with labeled model


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biomembrane liposomal formulations in an FLX 800 Microplate Fluoroscence Reader (BioTek Instruments Inc., U.K.) at room temperature, at pH 7.4, 6 and 5 and the fluorescence intensities were recorded as a function of time using 485 nm excitation wavelength and 595 nm emission wavelength. 100% fusion was determined from the Rho-PE fluorescence intensity observed for labeled biomembrane liposomal formulations dissolved with 1% Triton X100. Cell Culture. Mycoplasma free A549, B16F10, ASPC-1, C-26, COS-1, RAW246.7, NIH3T3 and human dermal fibroblasts cells were procured from National Center for Cell Sciences (Pune, India) and cultured at 370C in a humidified atmosphere of 5% CO2 in air in DMEM/RPMI-1640 medium (Sigma) containing 10% fetal bovine serum (South American Origin, Gibco, USA) and 1% penicillin-streptomycin-kanamycin. 85-90% confluent cultures of cells were used in both in vitro and in vivo experiments. MTT Assay. Cells at a density of 10,000 per well were seeded in 96-well plates and allowed to grow for 18-24 h before the treatment. Liposomes of histidinlated lipids 1-4 and lipid 5 in the concentration range 16-40 µM in the serum free medium were added to cells. Medium was replaced with 100 µL fresh complete medium containing 10% FBS to each well after 4 h of incubation, allowed to grow for 24 h. MTT solution (5 mg/ mL in PBS, 10 µL) was added to the cells and incubated for 4 h. MTT is reduced to formazan (purple color) by living cells but not by dead cells. The formazan crystals were dissolved with 50 µL of 1:1 (v/v) DMSO/Methanol. Absorbances of the wells were determined with a microplate reader (ELISA) at 550 nm wavelength. Percent cell viabilities were calculated using the formula percent viability = [A550 (treated cells)-background/A550 (untreated cells)-background] x 100.


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Flow Cytometry. B16F10 and RAW 264.7 cells (~3 x 105 cells/well in 6 well plates) were seeded 18-24 h before treatment. 1 mM stock liposomes of lipids 1-4 (25 µL) were added to the cells, the total volume diluted to 1 mL with plain DMEM, and after 4 h of incubation in a humidified atmosphere containing 5% CO2 at 37 °C, the medium was replaced with fresh complete medium containing 10% FBS. Cells were then trypsinized and resuspended in 500 µL binding buffer having 5 µL of annexin-V FITC and 10 µL of PI. Cells were incubated for 20 min at dark (BD, USA) and were analyzed immediately by a flow cytometer (FACS Canto II, BD). Mitochondrial membrane potential depolarization. B16F10 cells (~106 cells/well in 6 well plates) were seeded for 18-24 h and stock liposomes of lipids 1-4 (25 µL of the 1 mM ) were added to the cells. The total volumes were diluted to 1 mL with plain DMEM and, after 4 h of incubation, the cells were stained with JC-1 for 1/2 h after removing the medium. Cells were trypsinized, washed with PBS (500 µL) and resuspended in PBS (500 µL). Immediately thereafter, cells were analyzed with a flow cytometer (FACS Canto II, BD). Western Blot Analysis. B16F10 cells (~1 x 106cells/T-25 flask) were seeded for 24 h and thereafter treated with liposomes of histidinylated lipids 1-4 in plain DMEM media (3 mL total volume having final 25 µM concentration of each lipid) at 37 °C in a humidified atmosphere containing 5% CO2. After 4 h, the media was replaced with 3 mL of fresh complete DMEM containing 10% FBS and the cells were incubated for 24 h. Cells were detached from the flask with a cell scrapper and lysed at 4 °C with lysis reagent (CCLR, Promega). BCA assay method was used in quantifying the total protein contents of the cell lysates. 60 µg total proteins were dissolved in SDS-PAGE sample buffer and the components were separated using 12% SDSPAGE. Proteins were transferred to PVDF membrane (Millipore, USA) by wet blotting,


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membranes blocked for 2 h at room temperature with 5% non-fat milk in PBS containing 0.05% Tween-20 (PBS-T). The blots were incubated with primary antibodies against BAX, Caspase 3 & 9, Cytochrome-c, Bcl-2, GAPDH or β-Actin raised in rabbit (Pierce, USA) at 1:1000 dilutions for overnight at 4 °C, washed with PBS-T (3 x 10 mL), incubated with goat anti rabbit secondary antibody conjugated to alkaline phosphatase (Calbiochem, USA) at 1:2000 to 1:5000 dilution for 1 h. Protein bands were visualized using with BCIP/NBT (Sigma, USA) following manufacturer’s protocol and quantified by densitometry using image j software. Tumour growth inhibition assay. On day 0, female C57BL/6J mice (6-8 weeks old each weighing 20-22 g) were subcutaneously injected with ~1 x 105 B16F10 cells in 100 µL Hank’s buffer salt solution (HBSS) into their left flanks. Mice were then randomly sorted into total five groups and on day 14, 16, 18, 21 and 24, each group (n = 5) was injected with liposomal formulations of lipids 1-4 and with control liposome of lipid 5 & DOPC. Tumor volumes (V) (calculated using the formula V = 1/2 ab2 where, a = maximum length of the tumour and b = minimum length of the tumour measured perpendicular to each other) were measured for up to 27 days with a slide calipers. The mean tumor volume +/- SD (n = 5) were used in plotting tumor growth inhibition graphs. Statistical analysis. Error bars represent mean values ± SEM. The statistical significances in the tumor growth inhibition studies and ratios of Bcl-2/Bax in western blotting analysis were determined by two-tailed Student’s test. P values less than 0.05 were considered to be statistically significant. RESULTS AND DISCUSSION


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Chemistry. The histidinylated cationic lipids 1-4 were synthesized by covalently grafting Nim, Nα-di-BOC-L-histidine to the appropriate aspartic or glutamic acid di-esters via conventional peptide coupling as shown schematically in Scheme I. Details of the synthetic procedures, 1H NMR & ESI Mass Spectra and the HPLC profiles of the purified lipids 1-4 are provided in Supplementary information (Figures S1-S12, supporting information). Scheme I. Synthesis of lipids 1-4. O

O BocHN

BocHN

OH + HO

( )x

CH3

n

OH

a

O

CH3

I

N

CH3

n

H N

c

O

BOCHN CH3

O

O

O

d,e O ( )x

n

n

CH3

O

O

II

CH3 n

III

NH+ClH N O

b

O

NBoc

N H - + Cl H3N

CH3

n

O

( )x

n

O

O

H2N

O ( )x

O O ( )x

CH3

n

O

O

CH3 n

Lipid 1, when n=11; x=1 Lipid 2, when n=13; x=1 Lipid 3, when n=11; x=2 Lipid 4, when n=13; x=2 Reagents and conditions: a) Dry DCM, EDCI, HOBT, DIMAP, 12 h; b) Dry DCM, TFA (2:1; v/v), 0 °C, 3 h; c) Nα, Nim-di-t-butyloxycarbonyl-L-Histidine, EDCI, HOBt, DIPEA, Dry DCM, 12 h; d) Dry DCM, TFA (2:1; v/v), 0 °C, 3 h; e) Amberlyst A-26 for Cl- ion exchange.


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Physicochemical Characterizations of Liposomes. Dynamic laser light scattering technique (Zetasizer 3000A, Malvern Instruments, U.K.) was used in measuring the sizes and the surface potentials of all the liposomal formulations. The hydrodynamic radii and the surface potentials of the liposomes were found to be in the range of 150-170 nm and 3-5 mV (Table S1). However, the TEM images of the liposomes revealed a populations of heterogenous vesicles with distorted shapes (Figure S13) presumably induced during the drying steps in which the samples are exposed under vacuum. Endosomal pH-sensitivity of lipids 1-4. First, to confirm the endosomal pH-sensitivity of the presently described histidinylated cationic lipids, we measured the biomembrane fusibility of the liposomes of lipids 1-4 and DOPC using the fluoroscence resonance energy transfer (FRET) assay developed by Struck et al.18 In such FRET assay, the liposomes are mixed with a model biomembrane containing dioleyol-phosphatidylcholine, dioleyol-phosphatidylethanolamine, dioleyol phosphatidylserine, and cholesterol (at a ratio of 45:20:20:15, w/w). This model biomembrane was labeled with both the donor and the acceptor fluorophores ( N-(7-nitro-2-oxa1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine Rhodamine

RedTM-x-1,2-dihexadecanoyl-sn-glycero

respectively) as described previously.19

(NBD-PE)

3-phosphoethanolamine

and

(Rho-PE),

Such FRET technique depends upon the interactions

that ensue between the donor and the acceptor fluorophores when they are in close physical proximity (e.g. the above described biomembrane mimetic liposomal formulation where both are present within the lipid bilayer region of the liposomes). Under this condition, overlap happens between the emission band of the energy donor and the exicitation band of the energy acceptor. In consequence, the photon energy absorbed by the energy donor (NBD-PE) is transferred to the physically nearby energy acceptor (Rho-PE) causing the latter to fluoresce. Thus, the efficiency


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of resonance energy transfer should be decreased upon fusion of the double-fluorophore labeled model biomembranes with cationic liposomes of lipids 1-4 & DOPC (devoid of any fluorophore) because of larger physical separation between the donor and the acceptors after fusion. Significantly enhanced fusion of the liposomes of lipids 1-4 with the biomembrane mimetic liposomes were observed when FRET experiments were carried out at pH 5 & 6 (i.e. near the endosomal pH range) compared to degree of fusion observed at pH 7.4 (Figure S14). Importantly, no such pH-dependent fusion properties were observed for liposomes of control lipid 5 and DOPC (Figure S14, last panel). These findings were also consistent with the observed significant increase in liposomal sizes when fusions of the biological model membranes with the liposomes of lipids 1-4 were effected in pH 5 & 6 compared to the extent of liposomal size increase for fusion at higher pH 7.4 (Tables S2-S4). All these findings collectively support the endosomal pH-sensitivities of the presently designed lipids 1-4. Liposomes of lipids 1-4 selectively kill tumor cells by inducing apoptosis and inhibit mouse tumor growth. Next we evaluated the cellular cytotoxicity profiles of the stable liposomal formulations of lipids 1-4 (containing equimolar amounts of lipid 5 and DOPC) by MTT assay protocol.20

Taking the significant transfection efficiencies of our previously reported non-

aspartic acid based simple histidinylated cationic amphiphiles14 and simple guanidinylated cationic amphiphiles (lipid 5)17 in both cancerous and non-cancerous cells into account, we envisaged that the presently disclosed liposomal formulations of the aspartic acid based cationic lipids 1-4 and the guanidinylated cationic lipid 5 to be non-cytotoxic in both cancerous and noncancerous cells.

The liposomal formulations, as expected, were significantly less cytotoxic in

all the four non-cancerous cells including RAW264.7 (mouse macrophage cells), NIH3T3 (mouse fibroblast cells) and COS1 (African Green Monkey Kidney cells), human dermal


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fibroblasts (Figure. 2a-d). Contrastingly, the liposomal formulations were found to be cytotoxic in all the four cancer cells after 24 h including A549 (human lung carcinoma cells), B16F10 (mouse melanoma cells), ASPC1 (human pancreatic cancer cells) and C26 (mouse colon cancer cells) (Figure 2e-h). Since in all our liposomal formulations, we have used two additional lipids as co-lipids (DOPC & lipid 5), toward getting insights into whether these co-lipids contribute in any significant way to the observed cell viabilities, we also performed additional control experiments in all our MTT assays in which we measured the percent cell viabilities for cells treated with liposomes of only DOPC & lipid 5. Importantly, these control liposomes of only DOPC and lipid 5 were found to be least cytotoxic in both cancer and non-cancer cells (Figure 2a-h). Therefore, liposomal ingredients DOPC and lipid 5 are unlikely to contribute to the above-mentioned cancer cell selective cytotoxicities of the liposomal formulations of lipids 1-4, DOPC & lipid 5. We incubated the cells with our liposomal formulations for 24 h. Toward confirming where the observed percent cell viabilities remain similar for cancer cells treated with our formulations of lipids 1-4 for longer than 24 h time periods, we also carried out MTT assay for 48 and 72 h. The cytotoxicity profiles for these longer period incubations were not found to be significantly affected (Figure S15). Thus, the cellular toxicity profiles depicted in Figure 2a-h and Figure S15, taken together, demonstrated cancer cell selective cytotoxicities of lipids 1-4. Consistent with their cancer cell selective cytotoxic nature, the liposomal formulations of lipids 1-4 induced significant late apoptosis in representative B16F10 cancer cells and failed to do so in representative non-cancerous RAW264.7 cells as was revealed in the flow cytometric study using Annexin V/Propidium iodide (PI) (Figure 3 and Figure S16).

Notably, the

contrasting cellular cytotoxicity profiles of the presently described liposomal formulations in cancerous and non-cancerous cells observed in the MTT assay done in absence of added serum


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(Figure 2) were observed to be significantly less contrasting when the MTT assays were repeated in presence of 10% added serum (Figure S17). Based on this finding, we administered the formulations in mice intratumorally, and not intravenously, in the tumor growth inhibition studies. Importantly, intratumoral administration of liposomal formulations of lipids 1-4 in general and lipids 1 & 3 in particular showed significant mouse tumor growth inhibtion properties in syngeneic C57/BL/6J mouse tumor model (Figure 4a- b). Previously we showed that untreated vehicle control group of mice (i.e. mice treated with only 5% aqueous glucose solutions) usually develops large melanoma tumor on day 22 and in consequence the group had to be sacrificed at that points.21

Toward confirming that the liposomal ingredients lipid 5 and

DOPC do not play any significant role behind the observed tumor growth inhibition properties of the presently described liposomes of lipids 1-4, control liposomes containing equimolar amounts of DOPC and lipid 5 (labeled as lipid 5 group in Figure 4) were also injected intratumorally in melanoma tumor bearing mice. Such control liposomal formulations of only co-lipids namely, lipid 5 & DOPC, did not inhibit melanoma tumor growth (Figure 4a-b).


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Figure 2. Cytotoxicity profiles of the liposomal formulations of lipids 1-4 & DOPC (labeled as Lipids 1-4). The absorbance of reduced formazan in untreated cells was taken as 100. The MTT assays were done 24 h after treating the cells with the liposomes of lipids shown in the inset for each figure.


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Figure 3. Measuring efficiencies of the liposomal formulations of histidinylated lipids 1-4 (containing 25 µM lipids) in inducing apoptosis in melanoma (B16F10) cells in 24 h through Annexin-V binding based flow cytometric assay. Degree of apoptosis in both the untreated (control) and the treated B16F10 cells were measured by flow cytometric analysis after staining the cells with FITC-Annexin V and propidium iodide (PI). Cells labeled with FITC-Annexin V and PI are shown in the dot plot by the horizontal and the vertical axes, respectively. The upper right quadrants of the dot plot show the population of late apoptotic cells (positive for both annexin V and PI). The individual shifts in the FITC- and PI-histograms for untreated control cells and cells with liposomal formulations of lipids 1-4 are also shown next to the dot plots.

Figure 4. Tumor growth inhibition studies in syngeneic mouse tumor model by intratumoral administrations of liposomal formulation of lipids 1-4 & DOPC (labeled as Lipids 1-4). a). On day 0, female C57BL/6J mice (6-8 weeks old, each weighing 20-22 g) were subcutaneously injected in their left flanks with ~1 x 105 B16F10 cells in 100 µL Hank’s buffer salt solution


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(HBSS) and the mice were then randomly divided into five groups. Each group (n = 5) was injected with different liposomal formulations namely, liposomes of lipids 1 & 3 (light blue and red, overlaped); liposomes of lipid 2 (green); liposomes of lipid 4 (black) and control liposomes of lipid 5 & DOPC (dark blue) on day 14, 16, 18, 21 and 24. Tumor volumes (V) were measured with a slide caliper for up to 27 days using the formula V = 1/2 ab2 where, a = maximum length of the tumour and b = minimum length of the tumour measured perpendicular to each other. Results represent the means +/- SD (n = 5) (*P < 0.01 vs lipid 2 and **P

Examples of Tumor Growth Inhibition Properties of Liposomal

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