Encapsulation of methylene blue in nucleoside-lipid-based liposomes : physicochemical characterization and evaluation for photodynamic therapy of ovarian cancer

Methylene blue liposomes (MB-LPs) intended for photodynamic therapy (PDT) of ovarian cancer were formulated with a negatively charged nucleolipid and phosphatidylcholine using the lipidic film technique followed by an extrusion step. MB-LPs were characterized using dynamic light scattering (DLS), zeta potential (ZP), transmission electron microscopy (TEM), colloidal stability, Fourier-transform infra-red spectroscopy (FT-IR), UV-Visible spectroscopy and cellular studies. MB-LPs were spherical nano-objects with sizes obtained by DLS and TEM between 120 and 130 nm. MB-LPs showed a PDI near 0.10, a negative zeta potential near -42 mV and an encapsulation efficiency around 57 %. Characteristic peaks from ionic interactions of nucle-olipids encapsulation of methylene blue were confirmed by FT-IR. Colloidal stability of MB-LPs was slightly affected in complex ionic media, sizes, PDI and ZP remained stable for 4 weeks at all storage temperature tested, allowing a potential use for in vitro and in vivo assays. The use of MB-LPs allowed slightly higher PDT activity based on the production of ROS against SKOV-3 ovarian cancer as compared to MB alone, a reduction of MB dark toxicity and a better protection against oxidation-reduction reactions.


Introduction
Ovarian cancer is the deadliest gynecological cancer in women [1,2].In the early phases, the ovarian cancer is mostly asymptomatic, which is why in most cases it is diagnosed at an advanced stage, conferring it a poor prognosis [3,4].Cytoreductive surgery is the first option for the treatment of ovarian cancer, and chemotherapy alone or combined with surgery is generally used in order to eradicate the non-removable foci [5,6].Chemotherapy presents various side effects due to the lack of site-specific release, and this can represent a drawback to its usage [7].Photodynamic therapy (PDT) is a therapeutic approach based on a selective activation of chemical substances called photosensitizers (PS) [8].This selective activation is based on an absorption of energy from a light source with an appropriate wavelength and power [9].In this excited form (after absorption), the PS can then present two possible modes of interactions.In the first case (Type I), the PS generates radicals that react directly with Ocontaining substrates basically by electron transfer and then leads to the production of reactive oxygen species (ROS).In the second case (Type II), the PS can transfer its energy to surrounding oxygen molecules, bringing them to an excited singlet state (1O2).These reactive species (1O2 and other ROS) lead to the production of oxygenated free radicals and thus to the tumor destruction [9,10].
PDT can be used in the imaging and treatment of different types of cancer, such as esophageal cancer, prostate cancer, and lung cancer [10].This can be potentially exploited in the treatment of the ovarian cancer disease, as demonstrated by Brandhonneur et al. [11][12][13].
A PS needs to follow some specifications: chemical purity, tumor selectivity and minimal dark toxicity with a fast clearance, high photochemical reactivity and activation wavelengths allowing tissue penetration [14,15].The first-generation of PS (porphyrin, protoporphyrin, uroporphyrin, and hematoporphyrin derivative) partially met these criteria [14], but they had several disadvantages such as a poor selectivity, in terms of target tissue / healthy tissue ratio, low excitation coefficients, relatively short wavelengths for the activation, accumulation in the skin with adverse dermal effects [10,14].These drawbacks led to the development of second-generation PS, such as phthalocyanine, chlorin, bacteriochlorin, or methylene blue (MB).The latter belongs to the phenothiazine family, and is a positively charged molecule soluble in water and in several organic solvents [16].MB has several advantages such as an excellent penetration in the cellular membrane, photochemical properties that produce a good quantum yield in the therapeutic window (600-900nm), hydrophilicity, low toxicity and phototoxicity toward a variety of tumor cell lines in-vitro [14,16,17].Compared to other second-generation PS, MB is low cost and commercially available.It has also high purity, stable composition, and is Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved for therapeutic applications [14,18,19].One limitation of MB use as PS is its sensitivity to biological environment; reduction in leukomethylene blue (LMB).LMB is an inactivated form with a negligible photodynamic activity [14].
Therefore, MB needs to be integrated into biocompatible delivery systems such as nucleolipid nanocarriers to address these issues with a (i) protection against reduction, (ii) reduction of side effect and toxicity, and (iii) accumulation in cancer cells.Several investigations on the MB encapsulation in matrixes have been done, such as the use of polyacrylamide nanoparticles (NPs), sol-gel silica NPs, organically modified silicate NPs, PLGA NPs, microgel and liposomes (LPs) [9,17,[20][21][22][23].The use of nucleolipid-based formulations as potential Drug Delivery System (DDS) has been demonstrated in several studies [24][25][26].
Very few studies have been conducted to assess MB activity in the treatment of ovarian cancer [27][28][29] and these studies did not assess activity based on PDT.
2.2.Nanoparticle formulation 507 µL of diC16dT stock solution (4 mg.mL -1 in chloroform), 135 μL of Lipoid S100 stock solution (15 mg.mL -1 in chloroform), and 5 mL of chloroform were used to obtain a lipidic film after chloroform evaporation under reduced pressure (200 mbar), stirring (150 rpm) at 40°C by a rotary evaporator Laborata 4000 (Heidolph, Schwabach, Germany) for 20 min.The lipidic film was rehydrated using 6 mL of MB stock solution (0.06 mg.mL -1 in deionized water) for 30 min under stirring at 150 rpm and at 40°C.A part of deionized water was then removed under reduced pressure (30 mbar), stirring (150 rpm) at 40°C by the rotary evaporator for 10 min and a final volume of 1 mL of suspension was obtained.The obtained product was centrifuged (7000g, 25°C, 2 min) and the supernatant was extruded eleven times (mini-extruder; 100 nm polycarbonate membrane; Avanti Polar Lipids, Alabama, USA) to obtain a nanosuspension of MB-LPs.
2.3.Nanoparticle characterization 2.3.1.Particles size, polydispersity index and zeta potential Particle size, polydispersity index (PDI) and zeta potential (ZP) analyses were determined using a Zetasizer Nano ZS (Malvern Panalytical, Orsay, France).Samples were diluted by 20-fold in deionized water, and measurements were performed at 25°C.Mean diameter and PDI were measured in low volume disposable cuvettes whereas ZP were obtained using folded capillary zeta cells.All analyses were done in triplicate on 3 different batches.

Transmission electron microscopy (TEM)
MB-LPs were characterized by a transmission electron microscope Hitachi H-7650 (Tokyo, Japan).Samples were prepared by loading 6 µL of MB-LPs nanosuspensions onto a carbon-coated copper grid for 3 min before drying.The air-dried samples were then directly examined.

Encapsulation efficiency
Encapsulation efficiency (EE) was determined by UV-Visible spectroscopy.This determination was based on a previous paper [26].Samples (0.3 mL) were ultra-centrifuged at 40000g for 20 minutes at 20°C.Precipitates were diluted by 20-fold in EtOH and sonicated in an ultrasound bath for 20 minutes.Then the solutions were analyzed in triplicate using a UV-Visible spectrophotometer (Specord®PC 205 UV VIS spectrophotometer, Analytik Jena AG, Jena, Germany).Acquisitions were done in quartz cuvettes with absorbance measurement at 655 nm for quantification.

Fourier transform infrared spectroscopy (FT-IR)
Spectra of Lipoid S100, diC16dt, free MB, blank-LPs and MB-LPs were acquired with a FT-IR spectrometer (FT-IR; VERTEX 70; Bruker).Samples were placed on the ATR module and crushed to ensure a homogeneous distribution on the crystal.All spectra were recorded by averaging 32 accumulations with 4.0 cm -1 resolution between 4000 and 400 cm -1 .2.4.2.Cellular phototoxicity studies SKOV-3 cells were seeded into 96-well plates at a density of 20,000 cells/well and cultured for 24h.The cells were then washed with phosphate buffer (PBS) and then freshly prepared MB-LPs were assessed for MB concentration by UV assay and diluted with FBS-free medium, at different concentrations of MB (0.5, 1, 5, 10, 15, 20, 30 and 50 µM).Blank liposomes not loaded with BM were also tested under dilution conditions equivalent to those of BM-loaded liposomes.After administration of the treatments, the cells were incubated for 4 h.The cells were then activated with a light at 660 nm for 10 min to release singlet oxygen and other ROS using a deep-red LED irradiation lamp (Philips, France) with a power of 36W and an irradiance of 40mW/cm 2 .In parallel, a control plate was not activated.Afterwards, cells were re-incubated for a 24h period.Untreated cells (negative control) were considered to have 100% remaining cells and 20 % DMSO treated cells (positive control) were considered to have 0% remaining cells.The cellular cytotoxicity was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay.The tetrazolium ring it contains is reduced to formazan by the mitochondrial succinate dehydrogenase of active living cells.Formazan forms a precipitate in the mitochondria that is proportional to the amount of living cells (but also to the metabolic activity of each cell).MTT was added to cells at concentration of 0.2 mg.mL -1 for 3 h, then cell medium was removed and 200 μL DMSO were added to dissolve formazan crystals.For each well, 100 μL of formazan solution was transferred to another 96-well plate and made up to 200 μL using DMSO.Finally, cells cytotoxicity was determined by reading absorbance of formazan at 560 nm using POLARstar Optima microplate reader (BMG labtech, Ortenberg, Germany).Each concentration of MB-LPs was tested in 5 replicates per experiment.All experiments were triplicated on different days.

Semi-quantitative study of ROS production by DCFH-DA assay
To monitor cellular ROS levels, SKOV-3 cells were seeded in 96-well dark plates at a density of 25,000 cells per well.A 24h pre-incubation period allowed the cells to adhere to the bottom of the wells.After washing with PBS, the cells were incubated with 100 mL of a 20 mM solution of DCFH-DA (2',7'-dichlorofluorescein diacetate, Sigma-Aldrich) in PBS for 45 min at 37°C.The cells were then washed with 100 mL of PBS and then incubated with MB and MB-LPs samples at different concentrations of MB in PBS.Blank liposomes not loaded with BM were also tested under dilution conditions equivalent to those of BM-loaded liposomes.After 4h of incubation, a plate was activated at 660 nm for 10 min using a deep-red LED irradiation lamp (Philips, France).Positive controls were treated with 50 mM and 200 mM of hydrogen peroxide.ROS production was measured at 20 min intervals with the POLARstar Optima microplate reader (excitation wavelength: 485 nm, emission wavelength: 520 nm).

Statistical analysis Results were expressed as mean values ± standard deviation (SD)
. A Mann-Whitney test was used for statistical analysis using GraphPad Prism 7.0 software, the level of significance was set at p < 0.05.IC 50 calculation were performed with R software (R foundation, Austria) with the "ic50" package.
3. Results and discussion 3.1.BM-LPs formulation and characterization MB-LPs were prepared by lipidic film method associated with extrusion.The choice of diC16dT for the lipidic film was based on his negative charge, which is the opposite of the MB charge.This difference between MB charge and the nucleolipid charge increased their interaction [26].The use of phosphatidylcholine allowed to soften the formed film, because a rigid lipidic film was obtained with phosphatidylcholine-free formulation tests.MB-LPs presented a size of 122.13 ± 5.85 nm by DLS which was confirmed by TEM where particle diameter was around 130 nm, and spherical nano-objects were observed (Fig. 2).MB-LPs size insured the possible use of this DDS by parenteral route [32,33] and the optimal uptake of these particles by tumor cells [34].MB-LPs showed a PDI of 0.11 ± 0.02 with a negative zeta potential of -41.93 ± 2.37 mV.Blank liposomes (without MB) were also formulated in the same conditions.They showed a size of 125.13 ± 4.06 nm, PDI of 0.06 ± 0.01, and a zeta potential of -57.37 ± 2.70 mV.Both formulations (MB-LPs and blank liposomes) PDI values were under 0.25, indicating that formulations were monodispersed.Zeta potential values were negative due to the diC16dT [24,26], and higher than 30 mV absolute values which is generally related to better nanoparticle physical stabilities due to higher electrostatic repulsion between them [35,36].
The ZP value of blank liposomes was higher than that of MB-LPs.This observation can be explained by the presence of MB (positively charged) on the surface of MB-LPs.To further explore the organization of the three compounds (MB, diC16dT and Lipoid S100) in the DDS, spectroscopic analyses were performed (Fig. 3).MB (1593 and 1337 cm -1 in blue dotted line on the spectrum), diC16dT and Lipoid S100 (2919, 2853, 1691, 1165 and 1121 cm -1 for both compound, these bands are shown as green dotted lines on the spectrum) characteristic bands were identified confirming MB-LPs composition, already presented in previous article [26].The EE was estimated at 57.48 ± 3.60 %, lower than the result obtained by Kowouvi et al. [26] with the formulation of MB nanoparticles by precipitation.This difference may be linked to the addition of phosphatidylcholine (zwitterion molecule) which decreased the MB loading.

BM-LPs colloidal stability
The colloidal stability of MB-LPs was assessed in ionic media (NaCl and DMEM) and in different storage conditions (room temperature, 4°C and 37°C).In ionic media, the dilution of MB-LPs did not affect their size in DMEM (123 ± 9.35 nm), but a slight increase was observed in NaCl (167.67 ± 52.11 nm).In both cases an increase in the PDI was observed but lower than 0.25 (PDI of 0.23 ± 0.08 and 0.22 ± 0.04 in NaCl and DMEM media respectively).Colloidal stability (size, PDI and potential zeta) of MB-LPs was evaluated at room temperature, 4°C and 37°C (Fig. 4).LPs size and PDI remained stable, respectively under 200 nm and 0.3 for all conditions allowing a potential use for in vitro and in vivo assays (Fig. 4a and 4b).ZP also remained stable for all conditions (Fig. 4c).3.3.Bio-reduction assessment MB is sensitive to oxidation-reduction reactions.The reduced form of MB, known as LMB, is a photo inactive molecule [37].MB must be protected to have a good PDT activity.This protection can be obtained by its encapsulation.To evaluate the impact of the latter as MB-LPs, a bio-reduction assessment was performed.Ascorbic acid was used as reducing agent and was added to MB and MB-LPs solutions.The reduction kinetics was monitored during 15 min (Fig. 5).The MB reduction was 1.76-fold slower in the case of MB-LPs (24.85 ± 0.63 % preserved) compared to MB solution (8.34 ± 1.68 % preserved).In previous studies a reduction slowdown factor of 1.4 was obtained [26].This result may be linked to the ionic interaction MB/nucleolipids [26], and the MB loading in the DDS stabilized by phosphatidylcholine [23].This result also demonstrates the benefit of this new formulation compared to the previous formulation.

Cellular phototoxicity studies
The cellular cytotoxicity of SKOV-3 (ovarian cancer cell line) was determined for free MB solution and MB-LPs at several concentrations (0.5-50 µM of MB) to select the concentration of MB-LPs to be used in stimulated conditions.Without UV activation, MB solution was non-toxic for concentrations of 0.5 and 1µM (98.05 and 98.93% respectively; Fig. 6).The cytotoxicity then increased progressively with a calculated IC 50 of 14.62 ± 1.21 µM.On the other hand, the MB-LPs presented no cytotoxicity up to 20µM (percentage of remaining cells higher than 80%) and a calculated IC 50 of 48.62 ± 2.10 µM.However, this theoretical value was at the limit of the concentration range used and was considered non-significant.The significant difference between the two calculated IC 50 (p-value <0.001), confirmed that without light activation, the liposomal formulation was significantly less toxic.In order to evaluate the cellular toxic effect of photo-activation, free MB and MB-LPs were incubated for 4 h (Fig. 7).Even if the theorical IC 50 were close, 7.72 ± 0.92 µM for the free MB and 6.49 ± 0.47 µM for the MB-LPs, the difference was statistically significant (p-value <0.1), allowing us to conclude on a slight highest efficiency for the liposomal formulation.MB activity was thus preserved by the liposomal DDS with a slightly increased toxicity by photo-activation and a lowest toxicity without it, decreasing the potential side effects.To find out whether oxidative stress is involved in the biological activity of the compounds, we measured their ability to generate ROS by the fluorometric DFCH-DA.Free MB and MB-LPs at different concentrations were applied.The results showed a significant production of ROS for concentrations of MB (free and encapsulated) between 0,5μM and 50μM (Fig. 8).ROS-induced damage results in cytotoxicity through mitochondrial damage and promotion of apoptotic death [38].Liposomes not loaded with BM, on the other hand, exhibit no cytotoxicity, maintaining cell viability greater than 80% even at the highest equivalent concentration tested, both with and without UV activation.Additionally, these unloaded liposomes do not generate any reactive oxygen species following UV activation, indicating their stability and safety in cellular environments.

Conclusion
In this study, a novel nanoformulation using MB as PS has been developed.This novel nanoformulation was based on the association of MB with a nucleolipid (diC16dT) and a phosphatidylcholine (Lipoid S100).This assembly led to the formation of stable colloidal liposomes (MB-LPs) even in complex media.The MB encapsulation protected it from redox phenomena due to chemical compounds, ie.ascorbic acid.The study of MB-LPs nanoformulation with a PDT activity against ovarian cancer cell SKOV-3 demonstrated an activity by oxidative mechanism (ROS) and a strong reduction of MB dark toxicity.MB-LPs could be an adjunct to surgery as a surgical dye and as a local PDT agent to lower local and distant recurrence rates.We believe that these findings add a valuable contribution to the investigation of ovarian cancer treatment based on photodynamic therapy.

Figure 1 :
Figure 1 : Chemical structures of methylene blue (A), anionic nucleolipid diC16dT (thymidine 30-(1,2-dipalmitoyl-sn-glycero-3-phosphate)) (B), phosphatidylcholine Lipoid S100 (C) Colloidal stability in NaCl and DMEM.To assess MB-LPs stability in in vitro culture medium, a colloidal stability in NaCl and DMEM was performed.MB-LPs were diluted by 20-fold in a NaCl solution (0.9% of NaCl) and in DMEM media.The colloidal stability was assessed based on the size and the PDI.https://doi.org/10.57220/jatpb.v3i2.185Kowouvi et al. 4Colloidal stability at room temperature, 4°C and 37°C.MB-LPs were stored at room temperature and 4°C for 28 days, and at 37°C for two days.Particle size, PDI and ZP modifications were monitored as previously described in triplicate.Bio-reduction assessment.MB-LPs and MB solutions (at the same concentration as MB-LPs) were diluted by 10-fold in deionized water.100 µL of ascorbic acid solution (100 mg.mL -1 ) was added to 1.9 mL of diluted MB-LPs or MB solutions.Bio-reduction kinetics were monitored at 665 nm for 15 min by a UV-Vis spectrophotometer.The percentage of remaining MB was normalized on initial absorbance.All measurements were done in triplicate.2.4.Cellular studies 2.4.1.Cell culture Human ovarian cell carcinoma line SKOV-3 was purchased from Sigma (Sigma Aldrich, France).Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) and supplemented with 10% fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin under a humidified atmosphere (5% CO 2 and 95% air) at 37°C.Cells were cultured in 75 cm2 Nunclon EasYFlask (Thermo Fisher Scientific, Waltham, USA).

Figure 3 :
Figure 3: Colloidal stability of MB-LPs over time with particle size (A), PDI (B) and ZP (C) at 37°C (yellow), 4°C (blue) and room temperature (red).Results are the mean ± s.d. of three experiments.3.3.Bio-reduction assessment MB is sensitive to oxidation-reduction reactions.The reduced form of MB, known as LMB, is a photo inactive molecule[37].MB must be protected to have a good PDT activity.This protection can be obtained by its encapsulation.To evaluate the impact of the latter as MB-LPs, a bio-reduction assessment was performed.Ascorbic acid was used as reducing agent and was added to MB and MB-LPs solutions.The reduction kinetics was monitored during 15 min (Fig.5).The MB reduction was 1.76-fold slower in the case of MB-LPs (24.85 ± 0.63 % preserved) compared to MB solution (8.34 ± 1.68 % preserved).In previous studies a reduction slowdown factor of 1.4 was obtained[26].This result may be linked to the ionic interaction MB/nucleolipids[26], and the MB loading in the DDS stabilized by phosphatidylcholine[23].This result also demonstrates the benefit of this new formulation compared to the previous formulation.

Figure 4 :
Figure 4: Reduction kinetics of MB solution (blue) and MB-LPs (red) in presence of ascorbic acid based on visible absorption at 665 nm.The widths of the curve represent standard deviation of three independent experiments.3.4.Cellular phototoxicity studiesThe cellular cytotoxicity of SKOV-3 (ovarian cancer cell line) was determined for free MB solution and MB-LPs at several concentrations (0.5-50 µM of MB) to select the concentration of MB-LPs to be used in stimulated conditions.Without UV activation, MB solution was non-toxic for concentrations of 0.5 and 1µM (98.05 and 98.93% respectively; Fig.6).The cytotoxicity then increased progressively with a calculated IC 50 of 14.62 ± 1.21 µM.On the other hand, the MB-LPs presented no cytotoxicity up to 20µM (percentage of remaining cells higher than 80%) and a calculated IC 50 of 48.62 ± 2.10 µM.However, this theoretical value was at the limit of the concentration range used and was considered non-significant.The significant difference between the two calculated IC 50 (p-value <0.001), confirmed that without light activation, the liposomal formulation was significantly less toxic.In order to evaluate the cellular toxic effect of photo-activation, free MB and MB-LPs were incubated for 4 h (Fig.7).Even if the theorical IC 50 were close, 7.72 ± 0.92 µM for the free MB and 6.49 ± 0.47 µM for the MB-LPs, the difference was statistically significant (p-value <0.1), allowing us to conclude on a slight highest efficiency for the liposomal /doi.org/10.57220/jatpb.v3i2.185Kowouvi et al. 7

Figure 6 :
Figure 6: SKOV-3 ovarian cancer cell cytotoxicity after 4h of incubation with Blank LPs (black), MB solution (blue) and MB-LPs (red).Results are the mean ± s.d. of three experiments.

Figure 7 :
Figure 7: SKOV-3 ovarian cancer cell cytotoxicity after 4h of incubation with Blank LPs (black), MB solution (blue) and MB-LPs (red) with 10 min photoactivation at 660 nm.Results are the mean ± s.d. of three experiments.

Figure 8 :
Figure 8: Results for semi-quantitative study of ROS by DCFH-DA with Blank LPs (black), MB solution (blue) and MB-LPs (red), following photoactivation (gridded bars) for 10 min at 660 nm and without photoactivation (full bars).