Comparative cytotoxic activity of carboplatin and β-cryptoxanthin in free and liposomal forms against breast cancer cell line

The study of the effectiveness of the synthetic and natural anticarcinogenic compounds in liposomal form is urgent for their possible use in therapy. In this work, the alkylating agent carboplatin and the representative of carotenoids β-cryptoxanthin were used. The aim of the research was to study the toxicity of these compounds in free and liposomal forms against breast cancer MCf-7 cell line. according to DSC and fTIR data, when carboplatin or β-cryptoxanthin were added to liposomal bilayers, a single peak was observed indicating their mutual mixing. Integration of β-cryptoxanthin into bilayer was found to be more proper for the creation of PE acyl chains ordered and cooperative state. It was found that MCf-7 cells sensitivity was much higher to the free β-cryptoxanthin than to the free carboplatin with IC50 42 and 235 μg/ml, respectively. The IC50 values for β-cryptoxanthin loaded into liposomes and for free carboplatin were similar. At the same time, no cytotoxic effect of carboplatin-loaded liposomes was observed. The data obtained allow proposing a possible antitumor treatment regimen where carboplatin is replaced by free β-cryptoxanthin or its liposomal form to increase the effectiveness of breast cancer therapy.

C ancer is characterized by the uncontrolled growth and proliferation of abnormal cells that can invade or spread to other parts of the body. If the spread is not managed, then it can result in death. Treatments include surgery, radiation, chemotherapy, hormone therapy, immune and targeted therapy [1] Breast cancer is the second leading carcinogenic disease as the most common non-cutaneous malignancy among women. Options for treating breast cancer are limited and linked to toxicity. Emerging nanotechnologies have exhibited the potential to treat or target breast cancer. Over the years, different lipidnanoparticlesforbreastcancertherapy have been created, namely liposomes, solid lipid nanoparticles, nanostructured lipid carriers, and lipid polymer hybrid nanoparticles [2,3].
All hydrophilic and lipophilic drugs can be encapsulated and protected from degradation by liposomes, the vesicles of phospholipid bilayers. The liposomal similarity to the membrane bilayer core makes liposomes a very useful tool for examining the importance of drug-membrane interactions with anticancer.
By prolonging biological half-life or reducing toxicity, liposomes have been used to enhance the therapeutic index of new or established drugs [4,5]. Liposomal clinical applications are a large area of research where cancer therapy is the area of greatest impact [6,7].
Carboplatin is one of the platinum anti-cancer compounds and its parent compound is cisplatin with a carboxy-cyclobutane moiety instead of the chloride atoms which makes it more stable and perhaps less doi: https://doi.org/10.15407/ubj93.03.049 toxic than cisplatin. Carboplatin and cisplatin act as alkylating agents causing cross-linkage between and within DNA strands, resulting in inhibition of DNA, RNA, and protein synthesis and triggering programmed cell death, often in rapidly dividing cells. It causes the development of free radicals or reactive oxygen species. Free radicals cause cellular damage in many ways due to their high chemical reactivity. DNA damage, which can lead to a number of pathological conditions, including cancer, is amongthemostdamagingeffectsoffreeradicals. [8].Cisplatinandcarboplatinareeffectivechemotherapy drugs commonly used to treat solid tumors such as cancer of the testicles, ovaries, cervix, lungs, head and neck, esophagus, and bladder [9]. Cisplatin and carboplatin have been investigated in metastatic breast cancer as monotherapies and in combination with other chemotherapeutic agents. [10].
Carotenoids are among the bioactive substances that have a potential impact on cancer risk and progression and have been the focus of numerous investigations [11,12]. Carotenoids are naturally occurring organic compounds in plants and photosynthetic organisms such as algae [13,14]. Present re-portssuggesttheyhaveidentified>600carotenoids. Howe ver, as a result of selective digestive tract uptake, only 14 carotenoids with their metabolites were found in human plasma and peripheral tissues [15].
Several studies have shown that diets rich in vegetables and fruit can reduce the risk of multiple chronic diseases, including cancer, cardiovascular diseases, and diabetes [16]. In addition to β-cryptoxanthin, various carotenoids show more potent activity to suppress the carcinogenic process. Evidenceshowsthatβ-cryptoxanthinhasapotent antioxidant ability in vitro and can defend human cellsfromoxidativedamage,contributingtoinflammation suppression, even serving as a scavenger of free radicals, and preventing biomolecules such as lipids, proteins, and nucleic acids from oxidative dama ge. [17][18][19]. Carotenoids have numerous pathways to exhibit anticancer and cancer prevention mechanisms, including antioxidant and direct antiproliferativeeffects(inductionofapoptosis)and inhibition of oncogene expression and angiogenesis [20].
To our knowledge, no previous studies on the interactionofβ-cryptoxanthinorcarboplatinwith phospholipids were performed from the perspective of the thermotropic phase behavior of phospholipids and to detect the changes of acyl chain conformations and characteristic PO 2 − bands in the polar heads of phospholipids. To date, the anti-proliferative activityofβ-cryptoxanthininfreeandnanoliposomal formstowardsMCF-7(Breastcancercellline)has not yet been studied.
The objective of this study was to determine whether free or integrated β-cryptoxanthin in liposomes could inhibit the proliferation of human MCF-7 cell breast cancer by detecting the possible effectsofthesecompoundsonthecelldeathofhuman MCF-7 cell line carcinoma compared to free carboplatin and its conjugation with liposomes. The formulatedβ-cryptoxanthinorcarboplatin/liposomal conjugate was characterized by analytical techniques to evaluate the size, size distribution, thermotropic and conformation changes of the formulated β-cryptoxanthinorcarboplatin/liposomalconjugate along with in vitro possible cytotoxicity against MCF-7(breastcancercellline).

material and methods
Chemicals. β-cryptoxanthin was purified from natural sources and verified for purity by TLCandHPLC.Thestructureofβ-cryptoxanthin was confirmed from spectral data and elemental analysis.Themolecularweightofβ-cryptoxanthin is 552.85. Carboplatin with a molecular weight of 371.25, was purchased from Asta (Germany). The molecular structure of β-cryptoxanthin and Carboplatin are shown in Fig. 1 In a round bottom flask of capacity 50 ml, 20 mg of L-α-Phosphatidylethanolamine and 3.06 mg carboplatin (LipoCarbo) or 4.57 mg β-cryptoxanthin(LipoCrypto)ofthedrugpowder were mixed. Then 20 ml of ethanol (EtOH) was added,andtheflaskwasshakenuntilalllipidsdissolved in the EtOH. For a few minutes, the solution was well shacked then intense vortexing took place to ensure full solvation. The organic solvent was phased out using a rotary evaporator under vacuum provided by a circulating water aspiration vacuum pumpinawarmwaterbath(50°C)at60rpmtocreateauniformthinfilmoflipidontheinnerwallof theflask.WithTrisbuffer(pH7.4in37℃)inawater bathat50°C.Thelipidfilmwashydratedinawater bathat50°Cfor15minat60rpmtoformmultila-mellarvesicles(MLV)ofliposomes.Followingthe same method as described above using only aliquots ofL-α-Phosphatidylethanolamine(Cephalin),control empty liposomes were prepared.
Size distribution and Zeta potential measurements. InTrisbuffer(pH7.4)at25°Cusingaparticle sizingsystem(NanotracWaveII,Microtrac,USA) for dynamic light scattering, the mean particle size, size distribution, and zeta potential of freshly prepared empty liposomes, LipoCarbo and LipoCrypto were calculated. The results were an average of three individual measurements.
DSC measurements. Using indium-calibrated differential scanning calorimetry (DSC) (model DSC-50,Shimadzu,Japan),thethermalproperties ofdifferentlyophilizedliposomalformulationswere studied. On 5-mg samples sealed in standard aluminum pans the analyses are performed. The ther- fTIR Spectroscopy. On a Jasco FT/ IR-4100 spectrometer (Tokyo, Japan), FTIR spectra of lyophilized samples of empty L-α-Phosphatidylethanolamine (Cephalin) liposomes and those carboplatin (LipoCarbo) liposomes or β-cryptoxanthin (LipoCrypto) deposited in KBr discs are registered. Scanning was performed at room temperature, at a speed of 2 mm/s and a resolution of 4 cm-1 in the range 400-4,000 cm -1 .
The percentage of cell survival was calculated as follows: Survival The IC 50 values(drugconcentrationsneededto inhibitcellgrowthby50%).Theexperimentwasrepeated 3 times for each cell line.
Statistical analysis. All results are recorded as the mean ± SD. Statistical analysis was performed by one-way variance analysis (ANOVA), where a commerciallyavailablesoftwarepackage(SPSS-17 for windows, SPSS Inc., Chicago, Illinois, USA), wasusedandthesignificancelevelwasconsidered at P < 0.05.

results and discussion
Dynamiclightscattering(DLS)isatechnique that is used in particle size measurement. Nano drug delivery systems improve the bioavailability of the entrapped drug according to their size which matches cell dimensions. Table 1 shows the particle size for each formulationofcarboplatinorβ-cryptoxanthin-loaded liposomes as compared to empty liposomes. As These results indicate that the inclusion of β-cryptoxanthin into liposomes decreased the spacing between the adjacent bilayers resulting in the formation of liposomes smaller in size compared with the control ones. The reduction of particle size may be due to stronger β-cryptoxanthin interactions via hydrogen bonding with the lipid bilayer of liposomes. Within a smaller size range, liposomes favor the accumulation of drugs into certain target tissues. Besides, they also have good stability, thus displaying predictable drug-release rates.

T a b l e 1. Particle size distribution measured by dynamic light scattering (DlS) for various liposomal formulations
The possible stability of the colloidal system is indicated by the magnitude of the zeta potential. If the zeta potential increases, there will be an increased repulsion between particles, leading to a more stable dispersion of colloids. If all suspended particles have a strong zeta potential that is negative or positive, they seem to repel each other, and the particles are not likely to join together. [23].

fig. 3. DSC diagrams of liposomes are made of pure PE and liposomes doped with Carboplatin or β-cryptoxanthin
to the integration into the liposomal membranes. Within liposomal membranes, the incorporation of β-cryptoxanthin(LipoCrypto)appearstoincrease the density of negative charge and hence made the zeta potential negative.
The concept statement of DSC is that phospholipid vesicles undergo a reversible phase transition, undertheeffectofincreasingtemperature,froma 'gel' state to a 'liquid crystal' state. The pre-transitiontemperature(T p )atwhichatransitionfromthe gel phase to the rippled phase takes place is primarily related to the phospholipid polar region. Subsequently, the melting of the bilayer from the rippled phase to the liquid phase occurs at the main transitiontemperature(T m ).Changesinthelipidstructure severelyaffectalltheabovephases.
The introduction of carboplatin into PE liposomes showed a slight shift to a lower temperature at 140°C compared to the main endothermic peak(Tm)ofemptyPEthatexistsat143°C,indicating that carboplatin had a significant effect on PE bilayers' acyl chains, creating a conformational disorder within the phospholipid acyl chains and decreasing the transition cooperatively.
The lowered temperature of the main PE transition process suggested that carboplatin incorporation is more favored for the creation of a disordered and loose state of acyl chains. The pre-transition tempera ture(T p )peakforcarboplatinliposomeshas beenmovedtohighertemperaturesfrom100°Cto 103°C, suggesting that it prefers a change from a tilted to a rippled chain gel phase.
The increased temperature of the main PE transition process indicated that the integration ofβ-cryptoxanthinismoreproperforthecreation of an ordered and cooperative state of acyl chains. The increase in PE phase transition, subsequentlythereductionofthefluidityofthelipidbilayer membrane as a consequence of drug trapping. It has been found that the pre-transition temperature for β-cryptoxanthin liposomes was changed from 100°Cto127°C,whichhasconfirmedthatitprefers a transformation from a tilted to a rippled chain gel phase. Using DSC, it was observed that the PE and carboplatinorβ-cryptoxanthinmixturesdisplayeda single peak, which indicates that they are miscible.
Such changes observed in the current DSC workcanbeconfirmedbyFTIR,whichwasusedto detect any vesicle structural alterations in the liposomal membrane structure by analyzing the wavenumberofdifferentvibrationalmodes.
FTIR spectra of empty lyophilized PE liposomes compared with carboplatin or β-cryptoxanthin liposomal samples in the region of 4000-400 cm -1 asshownin (Fig.4).Twopeaksarerelated to the symmetric and antisymmetric stretching vibrations of the CH 2 in the acyl chain around 2850 and 2920 cm -1 , respectively were apparent. The peak noticed near 1470 cm -1 is due to the CH 2 bending vibration and that at 1734 cm -1 is due to the carbonyl stretching vibration C=O. Two peaks corresponding to the symmetric and antisymmetric PO 2 stretching vibrations approximately at 1090 and 1220 cm -1 , respectivelywereobserved.Thesefindingswerein agreement with the data reported in the literature [29].
Encapsulationofcarboplatinorβ-cryptoxanthin into the PE liposomes caused a shift in the wavenumber of the symmetric CH 2 stretching bands in the acyl chain that appeared in Fig. 5, indicating that carboplatinorβ-cryptoxanthincreateaconformational disorder within the acyl chains of phospholipids.
The peak at 2844.489 cm -1 for the pure PE is moved towards higher wavenumber at 2847.381 cm -1 for carboplatin liposomes. This could imply an increase in the number of gauche conformers that indicates an increase in bilayer disorder [30]. The signal intensity became more intensive for carboplatin loadedliposomes.Onceβ-cryptoxanthinisintegrated into PE, the peak for pure PE at 2844.489 cm -1 is moved to a higher wavenumber at 2845.453 cm -1 , whichsuggestsanincreaseinmembranefluidityand thus destabilization of the system in the gel phase (Fig.5).Theshiftstohigherwavenumberscorrespond to an increase in the number of gauche conformers [31]. The wavenumber of the CH 2 stretching bandsissignificantlychanged,showingthatcarboplatinorβ-cryptoxanthinincreasedthenumberof gauche conformers and this suggests an increase in theconformationaldisorder(trans-gaucheisomerization)ofthebilayer.
The C=O stretching band was analyzed for the interaction of carboplatin or β-cryptoxanthin with the backbone of glycerol near the head group of phospholipids in the interfacial region. The wavenumbervariationofthisbandisshownin (Fig.6).As canbeseenfrom (Fig.6),fortheliposomalsample containingcarboplatinorβ-cryptoxanthin,thewavenumber value of the C=O group at 1629.555 cm -1 was moved to higher wavenumbers at 1630.519 cm -1 for the liposomal sample containing carboplatin or β-cryptoxanthinwithoutanyevidenceofhydrogen bonding formation. The absorption bands of ester C=O are sensitive to changes in the polari ty of their local environments and are affected by hydrogen Control bonding and other interactions. Any variations in the spectra in this region may, therefore, be due to an interactionbetweencarboplatinorβ-cryptoxanthin and the membrane's polar/apolar interfacial region. Utilizing the PO 2 antisymmetric stretching band, which is located at 1216.863 cm -1 , the interaction betweencarboplatinorβ-cryptoxanthin and the head group of PE liposomes was investigated. Fig. 7 shows the PO 2 antisymmetric stretching band for PE liposomes formulations in the absence and presence of carboplatin or β-cryptoxanthin. As can be seen from Fig. 7, the wavenumber was shifted to lower values after the addition of carboplatin(1211.077cm -1 )orβ-cryptoxanthin(1213.97cm -1 ) Control into PE liposomes. This implied the presence of hydrogen bonding between the liposome head group andcarboplatinorβ-cryptoxanthin.Thedecreasein the wavenumber value indicates a strengthening of existing hydrogen bonds or even a formation of new hydrogen bonding between the components [30]. The CH 2 scissoring vibration mode which is located at 1461.778 cm -1 isinfluencedbytheinclusionofβ-cryptoxanthinintoPEliposomalpreparation.Followingtheencapsulationofβ-cryptoxanthin into PE liposomes, the wavenumber was shifted towards higher values at 1462.742 cm -1 . This could presumethattheβ-cryptoxanthinmoleculesactas small spacers of the polar head group, resulting in a slight disorder in the hydrocarbon chains. The wavenumber was shifted to a lower value at 1460.814 cm -1 after the encapsulation of carboplatin into PE liposomes.
In a cytotoxic assay with MCF-7 treated cells, the IC 50 valueforfreeβ-cryptoxanthinwas23.43μg/ ml(P<0.05),whilecarboplatinwascounted235μg/ ml similarly in value with IC 50 of MCF-7 treated cells with β-cryptoxanthin-loaded liposomes. It can be noticed that the IC 50 of carboplatin-loaded liposomes (Lipocarbo)wasnotapplicableincytotoxicassay with MCF-7 Fig. 9. Natural medicine may be the perfect alternative for cancer treatment, eliminating themultiplephysicalsideeffectsthatchemotherapy can cause.