Enhanced Cytotoxic Activity of Novel Synthesized Sulfadiazine Derivatives Bound Liposomes Towards Breast Cancer Cell Line: Comparative Studies with Different Cell Lines

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Enhanced Cytotoxic Activity of Novel Synthesized Sulfadiazine Derivatives Bound Liposomes Towards Breast Cancer Cell Line: Comparative Studies with Different Cell Lines

1 Tarek Said Abd El-Rashed*, Marina Nabil Habib, Marina Mamdouh Makram

B. Sc. Biotechnology Department, Faculty of Science, Helwan University,

Cairo, Egypt

2 Medhat W. Shafaa

Physics Department, Medical Biophysics Division,

Faculty of Science, Helwan University, Cairo, Egypt

Abstract : The present study aims to evaluate the cytotoxic efficacy of sulfadiazine derivative N-(4,6-bis (4- methoxyphenyl) pyrimidin-2-yl) benzenesulfonamide (G16) and its conjugation with L– Phosphatidylethanolamine (Cephalin) liposomes through detecting the possible effects of these compounds on the DNA damage of a human colon (CaCo2), breast (MCF-7) and pancreatic (PANC-1) cell lines carcinoma. The IC50 value for G16-doped liposomes in cytotoxic assay with MCF-7 treated cells was 24 g/ml, while for MCF-7 treated cells with free G16 was 450 g/ml. Using of liposomes increased the anticancer activity of G16 by 20 times than free G16. The IC50value for G16-doped liposomes in cytotoxic assay with CaCo2 treated cells was 62 g/ml, while for PANC-1treated cells was 58 g/ml. IC50value for CaCo2, MCF-7 and PANC-1cell lines treated with free G16 was 296, 450 and 420 µg/ml, respectively. The results indicated that all comet assay parameters for the G16-loaded liposomes treated cells were significantly increased (P < 0.05) compared to free G16 and control values. The results reveal a higher intensity of comet tail in MCF-7 cells treated with G16- loaded liposomes than those treated with free G16. Such high intensity relative to head indicates the presence of several double-strand breaks. The molecular combination between G16 and liposomes was characterized. The encapsulated G16 is probably associated with the lipid bilayers which results in the broadening and shift to higher temperature 143.7 C of the main peak of pure liposomes that exists at 141.2 C. FTIR study revealed structure alterations in vesicles after the encapsulation of G16 into liposomes.

Keywords: G16; Liposomes; DLS; DSC; FTIR; Comet assay; Cytotoxicity

  1. INTRODUCTION

    Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells that have the ability to invade or spread to other parts of the body. If

    the spread is not controlled, it can result in death. It is considered the second most common cause of death in the US, exceeded only by heart disease, and accounts for nearly 1 of every 4 deaths. Treatments include surgery, radiation, chemotherapy, hormone therapy, immune therapy, and targeted therapy (drugs that interfere specifically with cancer cell growth)[47].

    To date, the cancer problem and the failure of conventional chemotherapy to achieve a reduction in the mortality rates for common epithelial malignancies such as carcinomas of the lung, colon, breast, prostate and pancreas, indicates a critical need for new approaches to control cancer development. One of these approaches is chemoprevention, which is a pharmacological approach to intervention with the objective of arresting or reversing the process of multi-step carcinogenesis.

    Common cancer treatment techniques, such as chemotherapy, take advantage of apoptosis, to eliminate malignant cells within tumors [12]. Although this approach is effective on a wide variety of tumors, it not highly selective and thus cause its adverse effects on healthy tissue. An improved strategy for cancer treatment would be to use small molecules to selectively differentiate cancerous cells into normal cells [36].

    Nanocarriers have larger surface area as compared to bigger particles, which can be easily modified to encapsulate large amount of drug, to increase the blood circulation time and to enhance the accumulation of drugs in solid tumors via the enhanced permeability and retention (EPR) effect as well as selective targeting of tumor cells [6].

    Liposomes, the vesicles of phospholipid bilayers, can encapsulate both hydrophilic and lipophilic drugs and protect them from degradation. Liposomes have long been receiving much attention because of their biocompatibility and reduce drug-related nonspecific toxicity. The resemblance between the liposomes and membrane bilayer core makes liposomes a very useful tool to investigate the significance of anticancer drugs-membrane interactions.

    Liposomes have been used to improve the therapeutic index of new or established drugs by modifyingdrug absorption, reducing metabolism, prolonging biological half- life or reducing toxicity. Drug distributionis then controlled primarily by properties of the carrier and no longer by physico-chemical characteristics of the drug substance only [2],[1],[35].

    Clinical applications of liposomes are a vast area of research where cancer therapy is the area of highest impact. A vast of literature describes the feasibility of encapsulation of a wide range of drugs, including anticancer and antimicrobial agents, peptide hormones, enzymes, other proteins, vaccines and genetic materials, in the aqueous or lipid phases of liposomes which showed enhanced therapeutic activity and/orreduced toxicity in preclinical models and in humans when compared to their non- liposomal formulations [9],[16].

    Sulfonamide-substituted heterocyclic compounds exhibit remarkable activities against both Gram-positive and Gram-negative bacteria[39]. Undoubtedly, the efficacy of the sulfonamide groups of this type of compounds, the ease of their oral administration, and its moderately low toxicity distinguish this class of compounds as one of the important antibacterial chemotherapeutic agents. Sulfapyrimidine [38] sulfapyridine [48] and sulfathiazole [13] were some of the early representatives of this group of sulfonamides. The unique antimicrobial properties of sulfapyrimidine inspired the further synthesis of a large number of pyrimidine derivatives of the sulfanilamide compounds, many of which with surprising activity and low toxicity characteristics [48]. Similar to other sulfonamides, sulfapyrimidine derivatives were identified as an inhibitor to dihydropteroate synthase enzyme, which some bacteria use to synthesize folic acid [28].

    L–Phosphatidylethanolamine (Cephalin) derived from sheep brain in powder form and of purity 99% is presented in (Figure 2) with molecular weight of 691.515, Tris base (Hydroxymethyl) in powder form, molecular weight of 121.1, was purchased from CDH, New Delhi, India. Solutions were prepared in distilled ultra-pure water. All other reagents and solvents used in this work were of research grade.

    Fig.1. The chemical structure of G16

    The present study aims to evaluate the cytotoxic efficacy of N-(4,6-bis (4-methoxyphenyl) pyrimidin-2-yl) benzenesulfonamide (G16) and its conjugation with liposomes through detecting the possible effects of these compounds on the DNA damage of a human colon (CaCo2), breast (MCF-7) and pancreatic (PANC-1) cell lines carcinoma (in-vitro study).The work also investigates how G16 modulate the physical structural properties of model lipid membranes and to approximate the subtle perturbation of the lipid bilayer structure using Zeta potential, Dynamic light scattering (DLS), as well as differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy.

  2. MATERIALS AND METHODS

    Chemicals

    N-(4,6-bis(4-methoxyphenyl)pyrimidin-2-yl) benzenesulfonamide (G16) was newly synthesized. It was purified and checked for purity by HPLC. The structure of G16 was confirmed from pectral data and elemental analysis. The molecular weight of G16 is 447.51. The molecular structure of G16 used is shown in Figure (1). Ethanol alcohol, absolute of purity 99.9% was purchased fromDaeJung Chemicals (Seohaean-ro,Gyeonggi-do,Korea).

    Fig.2. Schematic chemical structure of L–Phosphatidylethanolamine (Cephalin).

    Liposome preparation

    Liposome preparation

    Liposomes were prepared by the thin film hydration method as described previously [11]. Briefly, L– Phosphatidylethanolamine (Cephalin) derived from sheep brain was dissolved in ethanol in a round bottom flask. The solution was shacked well for a few minutes then vigorous vortexing took place to assure complete solvation. The organic solved was removed gradually using a rotary evaporator (Re-2010, Lanphan Zhengzhou, Henan, China) under vacuum produced by circulating water aspiration vacuum pump (SHB-III, USA Lab Equipment, USA) in a warm water bath (50°C) at 60 rpm to produce a uniform thin film of lipid on the inner wall of the flask. The flask was then left under vacuum for 12 hours to ensure the evaporation of all traces of ethanol. The lipid film was hydrated with Tris buffer (pH 7.4 in 37) in a water bath at 50°C for 15 min at 60 rpm to form multilamellar vesicles (MLV) as a control

    (empty) liposomes. The flask was mechanically shaken for 1 h at 50 °C. Then the flask was flashed through with nitrogen stream and immediately closed. Parallel to the control L– Phosphatidylethanolamine (Cephalin), G16-loaded liposomes were prepared following the same method as described using only aliquots of mass G16 at molar ratios to lipid 2:7. The liposomal dispersion was stored at a constant temperature of 4°C till use.

    Dynamic light scattering

    The mean particle size, size distribution and zeta potential of freshly prepared emptyand G16-encapsulated liposomes were determined by the dynamic light scattering using a particle sizing system (Nanotrac Wave II, Microtrac, USA) at 25°C in Tris buffer (pH 7.4). The results were an average of three separate measurements.

    DSC measurements

    The thermal behavior of lyophilized samples of empty liposomes and G16 combined with liposomes was investigated using Differential scanning calorimetry (DSC) (model DSC-50, Shimadzu, Japan) calibrated with indium. Analyses are performed on 5-mg samples sealed in standard aluminum pans. The thermogram of each sample covers the 25- 200C temperature range at a scanning rate of 3C/min.

    FTIR Spectroscopy

    FTIR spectra of lyophilized samples of empty L– Phosphatidylethanolamine (Cephalin) liposomes and those liposomes encapsulated with G16 deposited in KBr disks are recorded on a Jasco FT/IR-4100 spectrometer (Tokyo, Japan). Scanning is carried out at room temperature, in the range 4004,000 cm-1 at a speed of 2 mm/s and a resolution of 4 cm-1.

    In-Vitro cytotoxicity assay

    Colon (CaCo2), breast (MCF-7) and pancreatic (PANC-1) cancer cells were cultured in RPMI 1640 media supplemented with decomplemented fetal bovine serum (10%, v/v), penicillin (100 IU/mL), and streptomycin (100g/mL) in a humidified incubator (Akhaton engineering calibration-Thermo scientific – water- USA) under 5% CO2 and 95% air at 37 °C.

    In 96 well culture plates (growth assays), drug treatments were performed. Drugs were added to the culture medium at a calculated final concentration for triplicate experiments. The extent of the cell viability and proliferation (cell numbers) was measured by SRB (Sulfo-RhodamineB- stain) assay.

    Cytotoxicity activity of empty liposomes, freeG16 and G16-loaded liposomes was tested separately using the method of [44]. CaCo2, MCF-7 and PANC-1 cells were plated in 96-multi well plate (104 cells / well) for 24 h before treatment with the applied drugs to permit adhesion of cell to the wall of the plate. Different concentrations of empty liposomes, free G16 and G16-loaded liposomes under test

    (100, 250, 500, 750 and 1000 µg/ml) were added to the cell monolayer triplicate wells were prepared for each individual dose (in Laminar flow cabinet for more fertilization). Monolayer cells were incubated empty liposomes, free G16 and G16-loaded liposomes separately for 48 h at 37C and in an atmosphere of 5% CO2. After 48 h, cells were fixed, washed and stained with Sulfo-Rhodamine – B stain. Excess stain was washed with acetic acid and attached stain was recovered with Tris- EDTA buffer. Color intensity was evaluated in an ELISA reader (ELISA- TECAN-SUNRISE, Germany). The relation between cell viability percentage (surviving fraction) and drug concentration is plotted to find the cell viability curve for CaCo2, MCF-7 and PANC-1 cancer cell line after the treatment of empty liposomes, freeG16 and G16-loaded liposomes.

    The percentage of cell survival was calculated as follows:

    Survival fraction = O.D. (treated cells) / O.D. (control cells).

    The IC50 values (the concentrations of drug required to produce 50 % inhibition of cell growth). The experiment was repeated 3 times for each cell line.

    Single cell gel electrophoresis (comet assay)

    Comet assay is considered as a rapid, simple, visual, and sensitive technique to assess DNA fragmentation typical for toxic DNA damage, including single-and double strand breaks, DNA adducts, cross-links, and early stage of apoptosis.

    The cancer cells of the control and post-treatment groups were homogenized in a chilled homogenizer buffer, pH 7.5, containing 75 mM NaCl and 24 mM Na2 EDTA (ethylene di amine tetra acetic acid) to obtain a 10% tissue solution. A potter-type homogenizer was used, and samples were kept on ice during and after homogenization. Six microliters of cell homogenate were suspended on a 0.5% low-melting agarose and sandwiched between a bottom layer of 0.6% normal-melting agarose and a top layer of 0.5% low- melting agarose on fully-frosted slides. The slides were kept on ice during the polymerization of each gel layer. After the solidification of the 0.6% agarose layer, the slides were immersed in a lysis solution (1% sodium sarcosinate, 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl, 1% triton X- 100, and 10% DMSO) at 4 °C. After 1 hour, the slides were placed in an electrophoresis buffer (0.3 M NaOH, 1 mM Na2 EDTA, pH 13) for 10 minutes at 0 °C to allow the DNA to unwind. Electrophoresis was performed for 10 minutes at 300 mA and 1.0 V/cm. The slides were neutralized with a Tris-HCl buffer, pH 7.5 and stained with 20 µg/mL ethidium-bromide for10 minutes.

    Each slide was analyzed using a fluorescence microscope (with excitation filter of 420490 nm [issue 510 nm]). One-hundred cells were analyzed on each slide. Komet 5 image analysis software developed by Kinetic Imaging, Ltd. (Liverpool, UK) linked to a charge-coupled device (CCD) camera to assess the quantitative and qualitative

    extent of DNA damage in the cells was used. Tail length (µm) is the distance of DNA migration from the center of the body of the nuclear core and is used to evaluate the extent of DNA damage. The tail moment is defined as the product of the tail length and the fraction of total DNA in the tail (tail moment = tail length x % of DNA in the tail). Finally, the program calculated tail moment. Generally, 50 to 100 randomly selected cells were analyzed per sample [29].

  3. RESULTS AND DISCUSSION

    Polydispersity index (PDI), is a number fit to correction data. PDI is a dimensionless and scaled such that values smaller than 0.05 are rarely seen other than with monodisperse standards. PDI effectively accounts for particle homogeneity of colloidal suspension. Values greater than 0.7 designate that the sample has a very broad size distribution and is probably not stable for the DLS technique.

    Figure 1 represents a typical size distribution graphs for both empty and G16-encapsulated liposomes. Figure1A shows the size distribution of empty liposomal sample was concentrated around 330.71±454 nm mean size diameter with 0.216PDI. The incorporation of G16 into liposomes resulted in an increase in the calculated mean sie diameter of blank liposomes from 330.71±454 nm to 976±1701 nm with 1.105 PDI Figure1 B.

    .

    Upon the encapsulation of G16 into liposomes, the mean vesicle sizes were significantly increased to be in the range of 976±1701 nm with 1.397 PDI Figure1B. These results indicate that the liposomes may be physically associated with G16 at the surface and the molecule of G16 tends to be buried in lipid bilayer which could explain why the size is increased.

    The magnitude of the zeta potential gives an indication of the potential stability of the colloidal system. As the zeta potential increases, repulsion between particles will be greater, leading to a more stable colloidal dispersion. If all particles in suspension have a large negative or positive zeta potential, then they will tend to repel each other and there will be no tendency for the particles to come together [30].

    Empty liposomes showed negative zeta potential (-

    67.05 mV), in agreement with the observation of others [32],[17],[22],[26]. It is clear that G16-loaded liposomes had lower negatively zeta potential (-50.82 mV) than empty liposomes due to the incorporation of g16 into the liposomal membranes. Generally, particles with zeta potentials more positive than +30 mV or more negative than -30 mV are normally considered stable.

    Fig.1. Liposomes size distribution measured by dynamic light scattering (DLS) for (A) empty liposomes (B) g16-loaded liposomes.

    DSC characterization was used to study changes in the phase transition of the lipid bilayer due to altered interactions that might occur between the encapsulated drugs and liposomes.

    The temperature at which a transition from the gel phase to the rippled phase takes place is called the pre- transition temperature (Tp) and it is mainly related to the polar region of phospholipids. Subsequently, the melting of the bilayer from the rippled phase to the liquid phase occurs at the main transition temperature (Tm). The melting point (Tm) represents the peak temperature of the endotherm for the lipid gel-to-fluid phase transition recorded during the heating scan. All of the above phases are strongly affected by changes in the lipid structure [18],[37].

    The temperature of fully dehydrated pure L– Phosphatidylethanolamine (Cephalin) liposomes, in the absence of any additives, undergo a sharp main-transition corresponds to the conversion of the rippled gel phase (P) to the lamellar liquid-crystal (L) phase. The smaller transition, called pre-transition (lamellar-to-undulled- lamellar phase transition).

    Liposomes vesicles made of pure L–Cephalin were used as model membranes since this phospholipid can mimic many aspects of biological membranes. Empty liposomes vesicles upon dehydration when submitted to DSC analysis, showed a major endothermic peak (Tm) at 141.2C (Figure 2), in accordance with [19], [45],[42],[41]. The pre-transition temperature (Tp) was around 98C for pure liposomes.

    Fig. 2. DSC diagrams of liposomes made of pure L–Cephalin, liposomes doped with g16.

    The thermotropic parameters of the vesicle transition could be influenced by the presence of a compound in the L-

    – Cephalin membranes. The incorporation of G16 into L– Cephalin liposomes exhibited significant broadening and shift to higher temperature at 143.7C (Figure 2) in a comparison to the main endothermic peak (Tm) of empty L-

    – Cephalin that exists at 141.2C which suggests that G16 had a significant effect on the acyl chains of L– Cephalin bilayers creating a conformational ordering within the acyl chains of phospholipids and increases the transition cooperatively of lipid acyl chains [31],[33].

    The increased temperature of the main L– Cephalin transition process indicated that the incorporation of G16 is more favorable to the formation of acyl chains in an ordered and cooperatively state. The increase in L– Cephalin phase transition and hence, the decrease of the fluidity of the lipid bilayer membrane as a consequence of drug entrapment. The pre-transition temperature (Tp) peak for G16 liposomes was shifted to higher temperature from 98C to 103C, which revealed that it prefers a transformation from a tilted to a rippled chain gel phase. It has been observed using DSC that the mixtures of L– Cephalin and G16 show a single peak, which indicates that they are miscible.

    Such modifications that were observed in the present DSC work can be confirmed by FTIR, which was used to detect any changes in the liposomal membrane structure by analyzing the frequency of different vibrational modes.

    FTIR spectroscopy was used to analyze possible changes in the structure of L– Cephalin by analyzing the frequency of different functional groups investigating the acyl chains and head group region of the lipid molecule in presence or absence of the foreign molecules.

    FTIR spectra of empty lyophilized L– Cephalin liposomes compared with liposomal G16 in the region of 4000400 cm1. The spectrum of the DSPC liposomes displayed the main characteristic bands, the symmetric and

    antisymmetric stretching vibrations of the CH2 in the acyl chain around 2850 and 2920 cm-1, respectively, the OH stretching and bending vibrations at 3,470 and 1,640 cm-1, respectively, the CH2 bending vibration near 1470 cm-1, the carbonyl stretching vibration C=O approximately at 1734 cm-1, and the symmetric and antisymmetric PO2- stretching vibrations approximately at 1090 and 1220 cm-1, respectively. These findings are in good accordance with the data reported in the literature [24].

    The detailed spectral analyses are performed in three distinct wave number regions, namely 35002800 cm1 (Figure 3A), 18001500 cm1 (Figure 3B) and 1800800 cm1 (Figure 3C), since identifiable Raman bands are observed mainly in these regions only.

    Fig. 3A. FTIR spectra (2800-3500 cm-1) of empty and G16 liposomal samples.

    Entrapment of G16 into the L– Cephalin liposomes induced a change in the frequency of the both symmetric and antisymmetric CH2 stretching bands in the acyl chain appeared in Figure 3, suggesting that G16 create a conformational ordering within the acyl chains of phospholipids. In other words, they influenced the order of the lipid bilayer. The frequency peak of the symmetric CH2 stretching bands in the acyl chain which is located at 2851.24 cm-1 for the pure L– Cephalin is disappeared for G16 liposomes. This might indicate a decrease in the number of gauche conformers which implies an increase in the order of the bilayer [40]. Interestingly the signal intensity became more depressed for G16 loaded liposomes. Upon the incorporation of G16 into L– Cephalin, the frequency peak of the antisymmetric CH2 stretching bands in the acyl chain at 2922.592 cm-1 for the pure L– Cephalin has been vanished which implies an increase in the membrane rigidity and thereby stabilization of the system (Figure 3A).

    The frequencies of the CH2 stretching bands of acyl chains depend on the degree of conformational disorder and hence the frequency values can be used to monitor the average trans/gauche isomerization in the systems. The shifts to higher wavenumbers correspond to an increase in the number of gauche conformers. [24].

    Meanwhile, the peaks of the symmetric and asymmetric stretching vibrations of CH2 have been used as a sensitive indicator of the ordering of the alkyl chains. There are significant changes in the frequency of the CH2 stretching bands, revealing that G16 decreased the number of gauche conformers and this implies a decrease in the conformational disorder (trans-gauche isomerization) of the bilayer. [21].

    Fig. 3B. FTIR spectra (1500-1800 cm-1) of empty and G16 liposomal samples.

    The interaction of G16 with the glycerol backbone near the head group of phospholipids in the interfacial region, the carbonyl C=O stretching band is analyzed. The wavenumber variation of this band is shown in (Figure 3B). The wavenumber value of C=O group at 1737.55 cm-1 was shifted to higher frequencies at 1741.41 cm1 for the lposomal sample containing G16, without any evidence of hydrogen bonding formation. This can be accounted for by the presence of free carbonyl groups in the system.

    The absorption bands of ester C=O are sensitive to changes in the polarity of their local environments and are influenced by hydrogen bonding and other interactions. Therefore, any change in the spectra in this region can be attributed to an interaction between G16 and the polar/apolar interfacial region of the membrane [15].

    2

    2

    The interaction between G16 and the head group of L– Cephalin liposomes was investigated by means of the PO2 antisymmetric stretching band, which is located at 1220.86 cm1. Figure 3 C shows the PO antisymmetric stretching band for L– Cephalin liposomes formulations in the absence and presence of G16. As can be seen from Figure 3C, the wavenumber was vanished after the addition of G16 into L– Cephalin liposomes.

    This implied the absence of hydrogen bonding between the liposome head group and G16. The decrease in the frequency value indicates a strengthening of existing hydrogen bonds or even a formation of new hydrogen bonding between the components [40].

    Fig. 3C. FTIR spectra (800-1800 cm-1) of empty and G16 liposomal samples.

    The CH2 scissoring vibration modes which is located at 1463.71 cm1 is affected by the incorporation of G16. The wavenumber was shifted towards higher values at 1461.83 cm1 after the encapsulation of G16 into L– Cephalin liposomes. This might assume that the molecules of G16 act as small spacers of the polar head group which would lead to a slight disorder in the hydrocarbon chains.

    Also, the N (CH3)3 + symmetric deformation band which is located at 1400.07 cm-1 was changed to higher values upon the encapsulation of G16 into L– Cephalin liposomes Figure 3C. This may be attributed to the presence of new intermolecular hydrogen bond formation between G16 and N (CH3)3 +.

    Symmetric stretching band of choline CN-(CH3)3 at

    907.344 cm1 was shifted to higher wave number 909.272 cm1 for G16 doped with L– Cephalin liposomal sample. This could be attributed to the presence of new intermolecular hydrogen bond formation between G16 and choline CN-(CH3)3.

    Table 1 The chemical shifts observed for G16 after the incorporation into L– Cephalin liposomes.

    Peak assignment

    Wavenumber (cm1)

    Wavenumber (cm1)

    Control Liposomes

    Liposoma l G-16

    Symmetric stretching vibration of the CH2 in the acyl chain

    (28002855)

    2851.24

    ————

    anti-Symmetric stretching vibration of the CH2 in the acyl chain

    (29202924)

    2921.63

    ————

    Carbonyl stretching vibrations (C=O)

    (17241740)

    1737.55

    1741.41

    CH2 scissoring vibration

    (1457-1466)

    1463.71

    1461.83

    C-O ester symmetric stretching

    (1150-1350)

    1298.82

    1232.29

    1298.82

    Anti-symmetric PO2 stretching vibration

    (1215-1228)

    1220.86

    ————

    +N-CH3 symmetric deformation

    (1396-1405)

    1400.07

    1402.96

    Symmetric stretching of Choline CN-(CH3)3

    (904-930)

    907.344

    909.272

    In-Vitro cytotoxicity of G16 or its liposomal form against CaCo2, MCF-7 and PANC-1 cell lines

    Cell viability was performed using different concentrations of G16 or its liposomal form and empty liposomes, against colon carcinoma (CaCo2), breast carcinoma (MCF-7) and pancreatic carcinoma (PANC-1) cell lines [44]. Untreated cells served as controls at zero concentration of G16 or either its liposomal form or empty liposomes. The assay was terminated at 48hr and measurements of cell viability were performed. The three cancer cell lines CaCo2, MCF-7and PANC-1were incubated separately with the same series of different drug concentrations 100, 250, 500, 750 and 1000 µg/ml, for 48 h. The potential cytotoxicity of G16 or its liposomal form and empty liposomes, against CaCo2, MCF-7 and PANC-1 cell lines were summarized in (Figure 4).

    Fig.4. In vitro Cytotoxicity of free G16, G16-loaded liposomes and empty liposomes against colon carcinoma (CaCo2), breast carcinoma (MCF-7) and pancreatic carcinoma (PANC-1) cell lines; incubated for 48 h at the same series of different concentrations (100, 250, 500, 750 and 1000 µg/ml). The cell viability was determined using the SRB assay. The data represent mean

    ± standard error of triplicate experiments.

    According to the cell viability measurements, G16- loaded liposomes exhibited the highest cytotoxicity against MCF-7cell lines treated with the same series of different concentrations which applicable for the other drugs. MCF-7 treated cells showed cell viability of approximately 5 %, 48 hours post incubation at the highest G16-doped liposomes concentration (1000 µg/ml). While, free G16 treated cells, the cell viability was approximately 57%. For empty liposomes, the cell viability was approximately 60% at the same concentration (1000 µg/ml).

    CaCo2 treated cells showed cell viability of approximately 20 %, 48 hours post incubation at the highest G16-doped liposomes concentration (1000 µg/ml). While, PANC-1treated cells, the cell viability was approximately 36% at the same concentration.

    CaCo2 treated cells with free G16 showed cell viability of approximately 25 %, 48hours post incubation at the highest free G16 concentration (1000 µg/ml). While, PANC-1treated cells, the cell viability was approximately 36% at the same concentration.

    For G16-loaded liposomes treated cells, the significant decrease in cell viability compared to the free G16 could be attributed to the sustain release of G16 from liposomes. So, as the concentration of the encapsulated drug increases, the amounts of released drug also increase and consequently the cell viability decreases when the encapsulated drug concentration increases.

    Interestingly, empty liposomes indicated noticeable reduction in the cell viability against the three cancer cell lines CaCo2, MCF-7 and PANC-1as treated with same concentration (1000 µg/ml). The cell viability was approximately 60%. This shows that the high concentration of liposomes (lipids) was toxic to the tested cells. Since there is no obvious rule concerning toxicity, different mechanisms might be behind the interferences with cell proliferation that we observed in conjunction with some phosphatidylcholines. So far, these underlying mechanisms are a matter of speculation, and more detailed investigation will be the focus of further work.

    Cytotoxic activity among various drug formulations against CaCo2, MCF-7 and PANC-1cell lines as treated with same concentration (1000 µg/ml) displayed an order of G16- doped liposomes G16 empty liposomes according to (Figure 4).

    At the lower concentration approximately at 100 µg

    /ml, MCF-7 treated cells with G16-doped liposomes showed the lowest cell viability among the tested cell lines of about 30% compared to its free form of about 89%, while 100% of the cell remained viable for empty liposomes (Figure 4).

    CaCo2 treated cells with G16-loaded liposomes showed cell viability of approximately 50 %, 48 hours post incubation at the lowest G16-loaded liposomes concentration (100 µg/ml). While, PANC-1 treated cells, the cell viability was approximately 88% at the same concentration.

    Based on the above results, G16-doped liposomes showed the highest therapeutic efficacy against MCF- 7compared to CaCo2 or PANC-1cell lines. Thus, the cytotoxicity of G16-loaded liposomes depends on th type of cancer cells. G16-loaded liposomes showed selective toxicity towards various cancer cells that might be due to the fact that G16-loaded liposomes targets many signaling molecules, scavenging of free radicals and quenching effect, which cancer cells highly rely on. This may be attributed to the ability of G16-loaded liposomes to be internalized more rapidly into cancer cells (high stability of G16-loaded liposomes and its cellular permeability resulting in enhancement of drug accumulation in cancer cells.

    The IC50 value for G16-doped liposomes in cytotoxic assay with MCF-7 treated cells was 24 g/ml, while for MCF-7 treated cells with free G16 was 450 g/ml. It is worth noticed that the use of liposomes increased the efficacy of G16 by 20 times than unloaded G16 Figure 5. Liposomal encapsulation of chemotherapeutic drugs increases their activity and reduce the needed concentration to give its desired actions. This enhanced efficacy may be ascribed to the lipo-solublized state of the drug, owing to its entrapment within multiple lipoidal domains of vesicles. Furthermore, the phospholipid lamella in the periphery of the vesicles may integrate with cell membrane facilitating internalization of the vesicular contents.

    IC50value for MCF-7 treated cells with empty liposomes was 750 g/ml. Based on above results, the encapsulation of G16 into liposomes showed the highest therapeutic efficacy against MCF-7cell line, depending on the cancer cells type (Figure 5).

    The IC50value for G16-doped liposomes in cytotoxic assay with CaCo2 treated cells was 62 g/ml, while for PANC-1treated cells was 58 g/ml. IC50value for CaCo2, MCF-7 and PANC-1cell lines treated with free G16 was 296, 450 and 420 µg/ml, respectively (Figure 5).

    All tested cancer lines that treated with empty liposomes showed the highest IC50value of about 750 g/ml.

    Fig.5. IC50 curve with significant values for free G16, G16-loaded liposomes and empty liposomes against CaCo2, MCF-7 and PANC-1 cell lines by using SRB assay, 48 h post-treatment.

    (Figure 6) reveals higher intensity of comet tail in all cells treated with G16-loaded liposomes than those treated with g16 alone. Such high intensity of comet tail relative to head indicates the presence of large number of double strand breaks. MCF-7, CaCo2, and PANC-1 cells treated with G16- loaded liposomes were exhibited increased percentage of mortality than those treated with plain G16 only. These findings support the assumption that nano-sized delivery system can more competently pierce into cancerous cells at a faster rate than that of their largesized counterparts.

    On the other hand, all cells treated with free g16 show a highly significant (p < 0.05) DNA damage compared

    to control cells and cells treated with empty liposomes. As a result of the tail length and density reflect the number of single-strand breaks in the DNA, the percentage of DNA in the tail provides a quantitative measure of the damaged DNA. Also, the elevated mean tail moment is indicative of DNA injury.

    (Figure 7) summarizes the comet assay parameters (percentage tailed cells, tail length, percentage tailed DNA, and tail moment) for the control CaCo2, MCF-7 and PANC-

    1 cell lines and post-treatment separately with free G16, G16-loaded liposomes and empty liposomes, and the differences between the control and the post-treatment groups. The results indicated that all comet assay parameters for the G16-loaded liposomes treated cells were significantly increased (P < 0.05) compared to the control values.

    Fig.6. Comet assay images of MCF-7, PANC-1 and CaCo2 cell line (evaluation of DNA damage induced by free G16, G16-loaded liposomes and empty liposomes)

    Fig.7.Comet assay parameters of CaCo2, MCF-7 and PANC-1 cell lines (evaluation of DNA damage induced by free G16, G16-loaded liposomes and empty liposomes).

  4. CONCLUSION

    Our study revealed the highest therapeutic efficacy of G16-loaded liposomes against MCF-7 compared to CaCo2 or PANC-1 cell lines. Thus, the cytotoxicity of G16-loaded liposomes depends on the type of cancer cells. The current data proposes a new treatment routine in which G16 is replaced by liposomal G16 to raise its anticancer activity against MCF-7cancer cell line. The use of liposomes increased the efficacy of G16 by 20 times than unloaded G16. Liposomal encapsulation of chemotherapeutic drugs increases their activity and reduce the needed concentration to give its desired actions.

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