Improvement of a liposomal formulation with a native molecule: calcitriol

Abstract

Many studies have been conducted to improve liposome's potency towards cancer treatment. Recently, modifications of liposomal formulations turned towards either dual loading of anticancer agents to minimize the dose of each agent or the use of targeting moieties to target them to or near cancer cells for minimizing side effects with maximum treatment. In this study, a natural molecule, calcitriol, was used to improve the effectiveness of doxorubicin loaded liposomes by co-loading approach. Calcitriol treatment alone was reported to create an anti-proliferative effect on several carcinoma cell lines. However, to create an antiproliferative effect, a high dose of calcitriol needs to be used which might result in hypercalcemia. Therefore, co-loading calcitriol into liposomes will improve its efficiency on cells besides increasing hydrophobic calcitriol's chemical stability in circulation. At first, possible doxorubicin cytotoxicity improvement against Namalwa cells by calcitriol pretreatment was investigated. The enhancing effect created was over 40% after 72 hours of calcitriol pretreatment. Upon co-loading into liposomes, enhanced and early cytotoxicity was observed even after 24 hours. Hence, the synergistic effect of calcitriol and doxorubicin on Namalwa cells was shown both in free and liposomal forms for the first time. A confocal study confirmed that early cytotoxicity was related with cell internalization. This potency ended when liposomes targeted non-internalizing antigen, CD20. Liposome co-loading of calcitriol and doxorubicin increases the effectiveness of their synergistic activity by creating a dual delivery system. Liposomal co-loaded calcitriol delivery would overcome calcitriol dose limitation problems and decrease possible side effects of both therapeutic agents.

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Introduction

Calcitriol interacts with cells by the vitamin D receptor (VDR) which induces several metabolic pathways and gene transcriptions. The biological action of calcitriol includes induction of cell differentiation, inhibition of cell growth, immunomodulation, and control of other hormonal systems.^1,2 However, in vitro and in vivo studies show the antineoplastic activity of calcitriol on several cell types including carcinomas of the breast, prostate and myeloid leukemia cells, and others.^3–6 There are several studies that investigate doxorubicin (DOX) cytotoxicity enhancement by various vitamin derivatives. Danhier et al., used a vitamin E derivative, D-α-tocopherol polyethylene 2000 succinate (TPGS), with DOX in a micelle formulation with the hypothesis that TPGS was able to inhibit the function of P-glycoprotein, an efflux pump that creates resistance to anti-cancer agents like DOX.⁷ In the study of Gruber and Anuszewska, the influence of calcitriol pretreatment on DOX cytotoxicity over several cell lines was tested. The study did not confirm any enhancement of DOX cytotoxicity by calcitriol pretreatment over normal human fibroblasts and human melanoma cells. Growth inhibition was only reported when melanoma cells were incubated with calcitriol for 72 hours.⁸ However, different than Gruber's study, cytotoxicity improvement was reported when calcitriol was used in combination with anti-cancer agents which indicates that different cell lines can respond differently to calcitriol treatment. Synergistic effects of calcitriol with chemotherapy, radiation, and other anticancer drugs have been reported in tumor models and it was shown that the mechanism of additive effect of calcitriol differs with different chemotherapy agents.¹ Synergistic effect of calcitriol and DOX was reported in the study of Ravid et al.,⁴ on breast cancer cell line. The binding of calcitriol to VDR of breast cancer cells inhibits the production of a cytoplasmic anti-oxidant enzyme (Cu/Zn superoxide dismutase). By this aspect it was shown that oxidative damage of DOX can be enhanced with calcitriol pretreatment. However, the side effects of calcitriol create a major problem when it is administered systemically at high doses. Serum level has to be below 50 pg mL⁻¹ (0.12 nM) to avoid hypercalcemia effect. This amount suggests that a dose of 0.25 μg can be taken safely for daily treatment, yet, it is not enough to create a therapeutic activity on cancer cells.⁵ There were efforts to overcome hypercalcemia effect of calcitriol by modifying the molecular structure or applying intermittent high doses during a treatment.⁶ Liposome delivery systems are another way to overcome side effects and dose limitations of therapeutic agents. These spherical vesicles with hydrophilic core and hydrophobic membrane compartments are able to encapsulate both hydrophilic and hydrophobic molecules. Liposomes are preferred drug delivery vesicles owing to their modifiable characteristics as lipid composition, drug release rate and attachment of functional molecules.⁹ Dual drug loaded liposomal systems can be developed to decrease the required dose for individual drugs while creating therapeutic activity.¹⁰

The anti-cancer agent, DOX, has many side effects including skin irritation, fatigue and cardiotoxicity. The clinically approved liposomal formulations, Doxil®, Myocet® and Caelyx™ partially overcome these effects by reducing systemic toxicity. Co-loading of a second therapeutic agent to DOX liposomes could enhance liposomal cytotoxicity towards cancer cells and decrease the amount of DOX needed which will reduce the total DOX side effects. Dual loaded liposomal systems are superior to multi-drug chemotherapeutic treatments in terms of delivery of both agents into the cells at the same time and thereby decreasing needed drug amounts and preventing drug resistance.¹¹

CD20 is an overexpressed, non-internalizing antigen for Namalwa and some other lymphoma disease related cells. Hence, it is distinguished as a target for liposomal drug therapy. Anti-CD20 antibody is currently used in clinics as an anticancer drug (Rituximab®) against lymphoma.^12,13 Anti-CD20 was preferred as a targeting ligand in many studies to treat B cell lymphoma.^14,15 Here, a non-internalizing antibody is chosen to modify liposome groups in order to evaluate the enhanced and early cytotoxicity achieved by co-loading of calcitriol and DOX into liposomes. The possible unspecific fusion of liposomes to cells was prevented by targeting to CD20 and the cytotoxicity was compared to untargeted liposome groups.

A liposomal system that carries both calcitriol and DOX was developed for the first time by our group to create a dual delivery system. The effect of calcitriol co-loading on DOX loaded liposome formulations was investigated. For this purpose, at first, the possible enhancement of DOX cytotoxicity against Namalwa cells upon calcitriol pretreatment was proved with free forms of these agents at different concentrations. Then, DOX, calcitriol or their co-loaded liposome formulations were developed and characterized in terms of calcitriol and DOX encapsulation efficiency, particle size distribution, in vitro release profile and cytotoxicity on Namalwa cells.

Experimental

Materials

Liposome preparation. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), L-α-phosphatidylcholine-hydrogenated (Soy) (HSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000] (18:00 mPEG2000-PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE), mini-extruder set, filter supports, Nucleopore Track-Etch Membranes (800, 400, 100 nm) were purchased from Avanti Polar Lipids, Inc. (Birmingham, Alabama). Methoxypolyethylene glycol p-nitrophenyl carbonate (PEG-(pNP₂)), doxorubicin hydrochloride, cholesterol, dialysis sacks, dialysis tubing, HPLC grade; chloroform, methanol and ethanol were the products of Sigma Aldrich Chem. Co. (St. Louis, Missouri). Sephadex G-75 and PD-10 disposable columns were purchased from GE Healthcare (Piscataway, New Jersey). Calcitriol was obtained from Cayman Chemical Company (Ann Arbor, Michigan).

Cell culture. Namalwa cell line was kindly provided by Gulhane Military Medical Academy, Cancer and Stem Cell Research Center. These cell lines were originally obtained from ATCC (Rockville, Maryland). RPMI 1640 medium was purchased from GIBCO-BRL/Life Technologies (Gaithersburg, Maryland). Fetal bovine serum (FBS) was purchased from Biological Industries (Kibbutz, Beit HaEmek, Israel). L-Glutamine, 1× penicillin–streptomycin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Invitrogen (Carlsbad, California). Cell culture plastic-wares were the products of Orange Scientific (Braine-l'Alleud, Belgium).

Methods

Preparation of DOX loaded liposome. DOX was loaded into liposomes by ammonium sulfate gradient method.¹⁶ Briefly, lipid film was formed by dissolving lipids (HSPC : mPEG-DSPE : cholesterol, 2 : 0.2 : 1 molar ratio) in chloroform solution and removing chloroform by evaporation under N₂ gas followed by overnight incubation in vacuum oven (Nüve EV 018, Ankara, Turkey). Lipid film was hydrated with 120 mM solution of ammonium sulfate by continuous two minute cycles of incubation in water bath (65 °C) and vortex for about an hour. To reduce the size, vesicles were sequentially passed through polycarbonate membranes of decreasing pore sizes (800, 400, 200 and 100 nm) at 65 °C. Liposomes were dialyzed against 0.9% NaCl solution for 20 hours in order to change extra-liposomal solution and create ammonium sulfate gradient. Loading of DOX (500 μg) into liposomes (25 mg total lipid) was performed by incubation at 65 °C in water bath. After 10 minutes, they were quickly dipped into ice bath. Loading procedure and following steps were performed in the dark due to light sensitivity of DOX. Unloaded DOX was removed by using Sephadex G-75 chromatography column. Turbidity measurement of each tube was performed at 410 nm by using UV-Visible spectrophotometer (Hitachi U-2800A, Tokyo, Japan). Liposome fractions were pooled accordingly. Samples were freshly prepared before characterization and cell culture studies. They were stored at 4 °C at most for 1 day before analysis.

Preparation of calcitriol loaded liposome. Calcitriol was loaded into the lipid bilayer of liposome due to its hydrophobic nature. Calcitriol loading was initially achieved through hydration step. Calcitriol (480 μM) was added to lipid mixture that was dissolved in chloroform (1 mL). After obtaining a homogenous mixture, nitrogen gas was applied for 2 hours and formed lipid film was incubated overnight in vacuum oven. Hydration was performed by ammonium sulfate solution and formed vesicles were extruded to obtain liposomes with 100 nm size. Calcitriol loaded liposomes were obtained after the removal of unloaded calcitriol by Sephadex G-75 chromatography column.

Doxorubicin encapsulation into calcitriol loaded liposome. DOX encapsulation into calcitriol loaded liposomes was performed with the same steps described in DOX loading. The only difference was the addition of calcitriol into lipid mixture and obtaining a calcitriol loaded lipid film. Briefly, calcitriol loaded lipid film was hydrated with ammonium sulfate solution (120 mM) and 100 nm size was obtained by extrusion. After dialysis, DOX was loaded into liposomes by incubation with liposomes at 65 °C. Unloaded DOX and calcitriol were separated with Sephadex G-75 chromatography column.

Quantification of doxorubicin. Amount of DOX loaded into liposome was determined by dissolving liposomes in methanol. DOX was quantified by measuring optical density at 480 nm with UV-Visible spectrophotometer. DOX amount was calculated from the calibration curve constructed. Non-interference of lipids at the same wavelength was also checked.

Quantification of calcitriol. High performance liquid chromatography (Shimadzu, Kyoto, Japan) was used to quantify calcitriol. C18 reverse-phase separation column (5 μm, 4.6 × 250 mm) was used with methanol : dH₂O (98 : 02) as the mobile phase. Column was heated up to 35 °C. Flow rate was 1 mL min⁻¹. Optical density measurement was performed at 255 nm. Method was modified from several studies.^17,18

Quantification of phospholipid (HSPC). Phospholipid amount was determined by the Stewart Assay, which is based on formation of a complex between phospholipid and ferrothiocyanate molecules.¹⁹ Liposome samples were dried in vacuum oven and dissolved in 2 mL chloroform solution in a glass tube. Then, 2 mL of ferrothiocyanate solution was added, vortex mixed (1 min) and centrifuged at 2000 rpm (5 min) (Hettich EBA 20, Tuttlingen, Germany). After centrifugation, the chloroform phase was removed and used to measure optical density. Lipid amount was calculated according to the calibration curve constructed with known amounts of HSPC.

Encapsulation efficiency and loading. In order to quantify encapsulated DOX and calcitriol, liposomes were dissolved in methanol solution and a homogeneous mixture was obtained. Then, DOX and calcitriol amounts were quantified by optical density measurements that performed as described previously.

Encapsulation efficiencies of DOX and calcitriol were calculated as follows:

Percent loading was calculated according to the equation:

Preparation of targeted liposome. The antibody-PEG-PE unit was introduced into the structure of readily formed liposomes. Antibody coupling was formed by combining two methods from literature.^20,21 First, pNP-PEG-PE unit was synthesized and conjugated to antibody. Then, antibody conjugated PEG-PE molecules were integrated into liposomes by co-incubation.

In order to synthesize the pNP-PEG-DOPE molecule, PEG-(pNP)₂ at eight fold molar excess over DOPE was dissolved in chloroform. Triethylamine was added at 2 fold molar excess over PEG-(pNP₂) and incubated overnight at RT, under nitrogen gas. Chloroform was removed by evaporation under nitrogen gas followed by 4 h incubation in vacuum oven. Dried film was suspended in 0.01 M HCl, 0.15 M NaCl solution while applying sonication. Unreacted molecules were separated by column chromatography (Sepharose CL-4B). Micelle suspension was kept frozen at −80 °C and freeze-dried. pNP-PEG-PE was extracted with chloroform.

Antibody-PEG-PE conjugate was formed as follows. Briefly, (pNP₂)-PEG-PE solution, 2% mol of total lipids in liposome, was dried under nitrogen gas and hydrated in C₆H₅Na₃O₇ (5 M) and NaCl (0.15 M) solution with octylglucoside (10 μL mL⁻¹). Anti-CD20 solution at a concentration of 1 mg mL⁻¹ was prepared in Tris buffered saline. Two solutions were mixed at equal volumes and incubated at 4 °C overnight to form antibody-PEG-PE molecule. Antibody-PEG-PE molecules were incorporated into the preformed liposomes by post-insertion method. Antibody-PEG-PE solution was mixed with liposome solution and incubated at 4 °C for 5 hours. Free antibody-PEG-PE molecules were separated through Vivaspin filtration (300.000 MW cutoff).

Determination of antibody conjugation efficiency. Fluorescamine, an amino group reactive dye, was used to determine the pNP-PEG-DOPE conjugation efficiency as described by Lukyanov et al.²⁰ Fluorescamine solution in acetone (3 mg mL⁻¹) was added to antibody or antibody-PEG-PE solution at a 3 : 1 volume ratio. The solution was incubated in the dark for 30 minutes at RT. The optical density of fluorescamine was measured at 359 nm using UV-Visible spectrophotometer. Absorbance readings were compared for fluorescamine reacted with antibody alone and reacted with antibody-PEG-PE. The amount of pNP-PEG-PE molecule conjugation to a single antibody molecule was calculated with absorbance decrease ratio.

Determination of antibody to liposome incorporation efficiency. To determine antibody incorporation efficiency, immunoliposomes were prepared with Oregon Green 488 labeled antibodies. Antibody amount on liposomes was calculated by measuring absorbance value of Oregon Green 488 modified antibodies attached to liposomes. Briefly, antibody solution (5 mg mL⁻¹) in 0.1 M sodium bicarbonate buffer (pH 8.3) was added to Oregon Green solution in dimethyl sulfoxide to obtain 15 : 1 final molar ratio and incubated at 25 °C for an hour. Microdialysis against 0.1 M sodium bicarbonate solution was performed for 12 hours in order to remove excess dye. Then, immunoliposomes were formed using Oregon Green labelled antibodies. The average number of antibodies conjugated to a single liposome was calculated by dividing antibody number detected to the number of liposomes present in the solution.

Doxorubicin and calcitriol release. Release profiles of the liposomal DOX and calcitriol were studied for all formulations. Briefly, 1 mL of liposome sample was put inside a dialysis bag (MWCO: 12 kDa). The dialysis bag was transferred into polypropylene tubes containing 10 mL phosphate-buffered saline (PBS) (0.1 M, pH 7.4). Tubes were placed in a shaking water bath (Nüve ST 402, Ankara, TURKEY) set at 37 °C. Samples (1 mL) were taken from the release medium for optical density measurements at predetermined times; 2, 6, 12, 24 and 48 hours and the total release media were changed with fresh PBS at time points.

Cell culture conditions. Namalwa cell line was cultured in RPMI 1640 medium, 10% FBS, 2 mM L-glutamine and 1× penicillin–streptomycin. Cells were incubated at 37 °C under humidified atmosphere of 5% CO₂ and 95% air (Shel lab, Cornelius, Oregon). Cultures were maintained by replacement of fresh medium in every two to three days. Cultures were started at 5 × 10⁵ cells per mL and subcultured at 2 × 10⁶ cells per mL.

In vitro cytotoxicity assay. Cytotoxicity experiments were performed with free and liposomal formulations of DOX and calcitriol on Namalwa cell line using MTT test. Cells were grown in 96 well plate after an initial seeding density of 20 000 cells per well. Control group in all studies were Namalwa cells without any treatment. Calcitriol dose cytotoxicity was investigated by 24, 48 and 72 hours of incubation in order to observe calcitriol's solitary effect on Namalwa cells. Calcitriol and DOX cytotoxicity experiments were performed in three sets; (i) free calcitriol pretreatments followed by DOX IC₅₀ (11.4 ± 0.46 μM) treatment for 24 hours to see the effect of combined treatment and to optimize calcitriol pretreatment time for enhancement effect, (ii) targeted or untargeted liposomal formulations of DOX and calcitriol, (iii) free DOX and calcitriol dose treatment mimicking liposome drug contents. After incubations, MTT test solution (20 μL) was added to each well and plate was left in the dark for 4 hours at 37 °C. The test solution was removed and wells were treated with isopropyl alcohol (100 μL) for 3 hours at 25 °C. Optical densities were measured in microplate spectrophotometer (Biorad, GMI Biotech 3550, Ramsey, Minnesota) at 570 nm. Results were presented as percent relative cytotoxicity of the control cells.

Laser scanning confocal microscopy. For confocal microscopy studies, liposome groups were labeled with 0.1 mol% Rh-PE. Interaction between liposomal formulations and Namalwa cells after 6 hours of incubation was evaluated for cell surface binding and uptake with detection of DOX and Rh-PE fluorescence by Laser Scanning Confocal Microscopy (LSCM). Namalwa cells were suspended in fresh media at an initial density of 1 × 10⁵ cells per mL. Liposomal formulations, each having 400 nmol mL⁻¹ total lipid content, were added to cell suspensions and incubated in dark for 6 hours at 37 °C. After incubation, cells were collected by centrifugation at 800 rpm for 10 minutes and media containing unassociated liposomes were removed. Cells were washed and resuspended in 100 μL of PBS (0.01 M, pH 7.4). One drop of cell solution was put on glass slide and lam-lamella system was immobilized at the edges by sticking.

Cells were imaged with LSCM using Plan-Neofluar 40× Oil DIC objective (Zeiss LSM 510, Middle East Technical University Central Laboratory, Molecular Biology and Biotechnology Research Center). Rh-PE and DOX fluorescence was excited with a He–Ne laser at 543 nm, and the emission was collected through a 585 nm long-pass filter. The pinhole was adjusted to obtain 1.0 μm optical sections and images were taken.

Statistical analysis. One-way Analysis of Variance (ANOVA) test was applied to compare groups for single parameter. Tukey's Multiple Comparison Test for the post hoc pairwise comparisons (SPSS 22 Software Program, USA) was used; p < 0.05 was considered as statistically significant.

Results and discussion

Encapsulation efficiency and loading

Due to hydrophobic nature of calcitriol, it was entrapped in the lipid bilayer during lipid film formation step. Entrapped calcitriol was detected by HPLC with a retention time of 5 minutes. Calcitriol encapsulation efficiency was calculated as 42.84 ± 6.24% and over 95% encapsulation of DOX into liposomes was achieved. DOX loading was 8.27 ± 0.56%. DOX/calcitriol combination groups were also analyzed for a possible effect of calcitriol co-loading on DOX encapsulation efficiency. DOX encapsulation was again over 95%, which showed that membrane incorporated calcitriol had no effect on exchange between DOX and ammonia ions. Lipid recovery ranged between 60–75% percent. Liposomes had an average size around 100 nm. A liposomal delivery system that is co-loaded with DOX and calcitriol was produced for the first time by our study group by encapsulating calcitriol into lipid bilayer and DOX into the aqueous core. The encapsulation efficiency of calcitriol achieved (42.84 ± 6.24%) was in agreement with the literature; similar calcitriol entrapment results were reported (48 ± 7%) by Frankenberger et al.²² and study concluded that such loss could be the effect of liposome formation steps that follows encapsulation. The high DOX encapsulation efficiency achieved was the result of DOX encapsulation to readily formed liposomes by ion exchange method which was carried out after hydration and extrusion steps. With this method, it was suggested that due to high ion gradient force, DOX was encapsulated more than its solubility, even accumulating as aggregates inside the liposome.^23,24 For targeting, anti-CD20 molecules were attached to readily formed DOX loaded liposomes and during this process approximately 30% of the DOX leaked out. Drug leakage has probably occurred due to the disruption of liposome membrane by the detergent, octylglucoside, during antibody incorporation.²⁰ On the other hand, post-insertion sustains separate antibody modification and liposome preparation steps that allow separate optimization conditions for best result. Besides that the drug loss was compensated by the therapeutic activity created by anti-CD20 and increased binding efficiency towards Namalwa cells.

Anti-CD20-PEG-PE modification and liposome surface conjugation

Anti-CD20 modified liposomes were prepared and prior to in vitro experiments, anti-CD20-PEG-PE modification degree and antibody–liposome conjugation were evaluated. Number of PEG-PE units attached to antibodies was estimated by the loss of primary amino groups by anti-CD20 molecules due to formation of urethane (carbamate) bonds. When fluorescamine was reacted with PEG-PE modified antibody molecules, approximately 35% decrease in the absorbance of fluorescamine was recorded compared to its reaction with unmodified antibody molecules. The decrease in fluorescamine absorbance indicates that a lower number of fluorescamine molecules were able to react with modified antibodies due to binding of PEG-PE molecules to fluorescamine binding sites.²⁰ According to O.D. measurement and calculations, an average of 15 of the 49 lysine residues of anti-CD20 molecule were modified with PEG-PE molecules. The number of antibody molecules conjugated to liposome was calculated by labelling antibodies with Oregon Green 488 dye. On the average “92” anti-CD20 molecules were attached to a single 100 nm sized liposome. The number of antibody attached was enough to create a specific targeting, which was in agreement with the literature.²⁰

In vitro release study

Calcitriol release from DOX/calcitriol co-loaded liposomes was very slow as expected from its hydrophobic nature. Calcitriol was retained in the lipid bilayer mostly and only 3.25 ± 0.49% was released after 48 hours. DOX release from DOX/calcitriol co-loaded liposome was presented in (Fig. 1). The presence of calcitriol within the bilayer was affected the release profile of DOX. When liposomes were loaded only with DOX, approximately 26% of the drug was released after 48 hours. However, when DOX was co-loaded with calcitriol, only 3.51 ± 0.58% of the drug, despite the same initial loading amounts, was released after 48 hours. The conjugation of anti-CD20 targeting molecule to liposome surface also affected the release of DOX by slightly decreasing initial burst amount. There were no data present in literature about calcitriol release from a liposome but a slow release rate was expected and rational in accordance with the nature of hydrophobic molecules. As a comparison, Nounou et al. reported a release study of hydrophobic drug, 5-fluorouracil, from liposomes with different phospholipid/cholesterol ratios, and observed a burst release followed by a very slow drug release rate when stability increased.²⁵ The encapsulation efficiency and release profile of calcitriol were suitable for creation of anti-proliferative effect and to the purpose of developed liposomal formulation; to release all or most of its content in a sustained manner.

Fig. 1 DOX release profile of only DOX loaded, DOX/calcitriol co-loaded, CD20 targeted liposomes and * calcitriol release result from DOX/calcitriol co-loaded liposomes.

Liposomal formulation of HSPC, cholesterol and mPEG-DSPE proved its high stability by providing slow release of hydrophilic drug, DOX. The release rate of DOX decreased even further when calcitriol was co-loaded into liposome which might be due to decreased permeability of the bilayer when calcitriol was loaded into liposome membrane. Calcitriol has a chemical structure that is very similar to cholesterol which will affect the membrane fluidity in the same way. Increasing levels of cholesterol in the membrane have been reported to disrupt the packing of lipid molecules in the liposome membrane which increased membrane fluidity.²⁶ That is why, the amount of cholesterol like molecules incorporated to the lipid membrane should be between tolerable rates. Yacoub et al. was reported that when cholesterol was added to liposome composition, the liposome membrane resists penetration of DOX through the membrane which will decrease the release rate of DOX.²⁷ Hence, the cholesterol addition to lipid membrane decreases water permeability.²⁸ The same effect was also created by the incorporation of calcitriol into liposomal membrane which finally delayed the dissolution and release of aggregated DOX. This was a preferable outcome considering the in vivo conditions. When such a formulation is given into blood circulation it is desired that it will not release most of the anti-cancer drug before reaching the target cells or tissues. The additional PEG molecules incorporated into liposome membrane which were used to attach anti-CD20 to liposomes slightly decreased burst release and 1^st day release of DOX (Fig. 1).

Enhancement of doxorubicin cellular toxicity by calcitriol pretreatment

Prior to combinational calcitriol and DOX incubation, the effect of only calcitriol treatment on Namalwa cells was investigated after 24, 48 and 72 hours of incubations (Fig. 2). Calcitriol group with 10 μg per well concentration, created higher cytotoxicity (11.10 ± 3.67% and 24.34 ± 2.69%) after 24 and 48 hours. Following 72 hours of incubation, all calcitriol dose groups (2.5 μg per well, 5 μg per well and 10 μg per well) have created similar cytotoxic effects (20.12 ± 3.66%, 22.90 ± 2.48% and 18.84 ± 3.47%). Prior to liposome experiments, the possible enhancement effect of free calcitriol pretreatment on DOX cytotoxicity was investigated using free form of these agents. DOX cytotoxicity enhancement effect of calcitriol pretreatment was investigated on Namalwa cells at different calcitriol concentrations for three pretreatment periods; 24, 48 and 72 hours which were then exposed to 24 hours of free DOX at IC₅₀ dose (11.4 ± 0.46 μM). “Control group 1” had free DOX IC₅₀ dose without pretreatment of calcitriol. “Control group 2” had untreated Namalwa cells with which relative percent viabilities were calculated. As shown in Fig. 3a, cellular viability was between 55% and 65% for all groups when calcitriol pretreatment was applied for 24 hours prior to DOX treatment. When compared to free DOX group, no additional cytotoxicity was created by 24 hours of calcitriol pretreatment. Percent cellular toxicities decreased in all groups when 48 hours of calcitriol pretreatment was applied. The cytotoxicity created after 48 hours of calcitriol pretreatment was lower than 24 hours pretreatment. The main reason was the proliferation of Namalwa cells through pretreatment time (Fig. 3b) as doubling time of the Namalwa cells is around 35 h.²⁹ The highest cytotoxicity was detected when calcitriol concentration was 10 μg mL⁻¹ after 24 and 48 hours of pretreatments. However, until this point cytotoxicity was strongly related to DOX end exposure and no necessary enhancement was created by calcitriol pretreatment. However, 5 μg per well and 10 μg per well concentrations of calcitriol pretreatment for 72 hours enhanced DOX cytotoxicity up to 91.43 ± 0.55% and 93.21 ± 1.44%. Hence, the enhancement of DOX cytotoxicity on Namalwa cells upon 72 hours calcitriol pretreatment with concentrations of 5 and 10 μg per well was shown for the first time by our group. In the literature, anti-proliferative activity of calcitriol on tumor cells was studied as calcitriol alone and in combination with cancer drugs.^1,4

Fig. 2 Cytotoxicity of different calcitriol doses on Namalwa cells. * Significant difference between 2.5 and 10 μg per well dose groups (P < 0.05).

Fig. 3 Doxorubicin cytotoxicity enhancement by calcitriol pretreatments. Cytotoxicity results of (a) 24 hours of calcitriol pretreatment (b) 48 hours of calcitriol pretreatment (c) 72 hours of calcitriol pretreatment followed by DOX (IC₅₀) incubation for 24 hours. * Significantly different than dose groups having less than 5 μg per well and from free DOX group (P < 0.05).

Enhanced DOX cytotoxicity over breast cancer cell line by calcitriol treatment was reported by Ravid et al.⁴ The study also concluded that 72 hours of calcitriol (10 nM) pretreatment blocks the production of antioxidant enzymes which improves oxidative damage caused by DOX cell interaction. Therefore, the dose study for calcitriol pretreatment between 2 nM and 120 μM (0.00025 and 10 μg per well) concentrations was performed, to determine the effective concentration for Namalwa cells. Likewise, in the study of Siwińska et al., 72 hours of pretreatment with calcitriol was able to create synergistic cytotoxicity with DOX on human leukemia HL-60 cells and a 7 fold decrease in ID_{50,48 h} was reported.³⁰

Cellular toxicity of liposome formulations

For in vitro cytotoxicity experiments, liposome dose groups were formed with respect to phospholipid concentration (Table 1). Due to higher cytotoxicity created by CD20 targeted liposomes at 400 nmol mL⁻¹ concentration, it was not suitable to make comparison within groups at this dose. For this reason, maximum dose used for targeted liposomes was taken as 100 nmol mL⁻¹.

Table 1 Calculated liposomal drug contents of liposome groups

	DOX loaded	DOX/calcitriol co-loaded
Untargeted liposome groups (100 nmol mL^−1)
DOX	≅0.5 μg per well	≅0.5 μg per well
Calcitriol	—	≅ 0.09 μg per well
CD20 targeted liposome groups (100 nmol mL^−1)
DOX	≅0.35 μg per well	≅0.35 μg per well
Calcitriol	—	≅ 0.09 μg per well

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The ratio of DOX and calcitriol in co-loaded liposomes was 6 : 1 wt/wt. This ratio corresponds to 0.25 μg per well calcitriol pretreatment group in the free drug experiment. The DOX cytotoxicity enhancement created by 0.25 μg per well pretreatment with calcitriol was lost through 72 hours (Fig. 3). On the other hand, when delivered in liposome, less amount of calcitriol was enough to enhance DOX cytotoxicity through 72 hours (Fig. 4). When incubated in vitro, a portion of liposome was able to deliver drugs into the cells by unspecific fusion as concluded by confocal studies. That is why untargeted liposomal groups were able to create early and high drug cytotoxicity.

Fig. 4 Cytotoxicity comparison of DOX and DOX/calcitriol co-loaded liposome groups. Cytotoxicity created after (a) 24 hours (b) 48 hours (c) 72 hours treatment with DOX loaded and DOX/calcitriol co-loaded liposomes. * Significant difference between DOX loaded and DOX/calcitriol co-loaded liposomes at 24 h (P < 0.05). ** Significant difference between DOX loaded and DOX/calcitriol co-loaded liposomes at 72 h (P < 0.05).

IC₅₀ values of only DOX loaded and DOX/calcitriol co-loaded liposomes were presented in terms of liposome concentration and DOX content of the liposomes (Table 2). When co-loaded with calcitriol, DOX IC₅₀ decreased more than tenfold compared to only DOX loaded liposome.

Table 2 IC₅₀ values of untargeted liposome formulations

	DOX loaded liposome	DOX/calcitriol co-loaded liposome
Liposome (nmol mL^−1)	134.5 ± 6.2	11.25 ± 3.6
DOX content (μM)	5.8 ± 0.25	0.483 ± 0.16

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In order to observe the effect of calcitriol co-loading into liposomal formulation, Namalwa cells were incubated with DOX loaded or DOX/calcitriol co-loaded liposomes for 24, 48 and 72 hours. Cellular toxicities of DOX loaded and DOX/calcitriol co-loaded liposomes were presented in Fig. 4. Liposome dose treatments, as lipid dose concentrations ranging between 25 and 400 nmol lipid per mL were applied. Although calcitriol co-loaded groups showed enhanced cytotoxicity for all concentration groups, the effect was more leveled up when concentrations of 100 nmol mL⁻¹ and higher were used. After 24 hours (Fig. 4a) of incubation co-loaded groups (100, 200 and 400 nmol lipid per mL), showed high cellular toxicities (82.89 ± 5.32%, 83.22 ± 3.78% and 83.74 ± 3.44%) which were superior to only DOX loaded liposome groups. The 25, 50 and 100 nmol mL⁻¹ groups of only DOX loaded liposomes did not show dose-dependency. One of the reasons to that could be the DOX amount that was present in the groups. The 25, 50 and 100 nmol mL⁻¹ liposome groups respectively had 0.125, 0.25 and 0.5 μg per well total DOX contents which were less than IC₅₀ value of DOX. According to confocal study, liposomes were able to fuse to cell after 6 hours. Hence, it can be deduced that after 24 hours of treatment some portion of the delivered liposomes were able to fuse to cells. However, the liposomes with DOX amounts being lower than IC₅₀ was able to create only a restricted cytotoxicity at the first day of treatment and in the following days, groups increased their cytotoxicity further by fusion and DOX release. After 48 and 72 hours of treatment, (Fig. 4b and c) cytotoxicity difference between dose groups of only DOX loaded liposome groups were very small. This might be due to their similarity in cell fusion rates. After 48 hours of incubation, only DOX loaded liposome was able to reach to cytotoxicity level of co-loaded liposomes due to faster DOX release compared to co-loaded ones. It could also be seen that after 72 hours of incubation the cytotoxicity decreased as in the case of free DOX treatment (Fig. 3c). This outcome might be related with some drug resistance mechanisms after certain period of drug exposure at high concentration.

The additive effect of calcitriol co-loading was more obvious after 72 hours incubation. Co-loaded groups with 200 and 400 nmol mL⁻¹ preserved their high cellular toxicity (85.22 ± 4.38% and 83.55 ± 4.16%). In the free drug studies, upon calcitriol exposure, DOX was introduced after 24, 48 and 72 hours of free calcitriol pretreatment and enhancement effect was observed only after 72 hours but when cells were treated with co-loaded liposomal formulation cytotoxicity improvement was observed even after 24 hours. The reason for this change in effect was thought to be the higher cellular internalization of calcitriol and DOX in liposomal formulation than the free forms in pretreatment study. After 72 hours incubation with co-loaded liposomes, difference in cellular toxicities was more recognizable for 100, 200 and 400 nmol mL⁻¹ groups which were parallel to pretreatment experiment results. It was noticed that lower liposome concentration groups also showed higher toxicity at 72 h when co-loaded with calcitriol.

In the study of Frankenberger et al., cytotoxicity of calcitriol alone was investigated by liposome entrapment and similar concentration of calcitriol (24 nM liposomal or 50 nM free calcitriol) was related with the cytotoxicity over myelomonocytic leukemia cells through 4 day incubations.²² They reported 60% reduction in proliferation when HL60 cells were incubated with liposomal 1.25 Vit D₃ for 4 days. However, only about 20% cytotoxicity was observed when we treated Namalwa cells with free calcitriol doses (2.5 μg per well, 5 μg per well and 10 μg per well) for 3 days. This outcome demonstrated that calcitriol has minimal cytotoxic effect on Namalwa cells. Also, free calcitriol treatment in clinics is dose limited due to possible creation of hypercalcemia and relatively short circulation half-life of calcitriol (12–36 h), reported by pharmacokinetic studies.³¹ By this aspect, co-loaded liposomal DOX/calcitriol becomes a promising drug delivery system.

For comparison, cytotoxicity test was performed with free drug groups having the same amounts of encapsulated drug contents of liposome groups. This time there was no pretreatment group and cell treatment started with both calcitriol and DOX at the same time as mimicking the liposome treatment experiments (Fig. 5). In contrast with the co-loaded liposomes, no enhancement was observed when cells were treated with free calcitriol together with DOX. On the other hand, liposomal delivery of DOX and calcitriol has successfully promoted cytotoxicity starting at the 1^st day of treatment.

Fig. 5 Cytotoxicity created with free DOX and calcitriol treatments of 0.125 : 0.0225, 0.25 : 0.045, 0.5 : 0.09, 1 : 0.18 and 2 : 0.36 μg per well mimicking the drug content of the liposome groups 25, 50, 100, 200 and 400 nmol mL⁻¹. (a) 24 hours, (b) 48 hours and (c) 72 hours of treatment.

Finally, in vitro cytotoxicity test was carried out for anti-CD20 modified DOX and calcitriol liposome groups (Fig. 6). After 24 hours of incubation, empty targeted liposomes showed cytotoxicity around 20% which was caused by the anti-CD20 molecules on their surface. Anti-CD20 itself is an anti-cancer agent (Rituximab) used to treat lymphoma and has a therapeutic activity. When targeted to CD20, DOX loaded and DOX/calcitriol co-loaded liposome groups showed higher cytotoxicity on Namalwa cells after 72 hours of incubation than untargeted liposomes at 50 and 100 nmol mL⁻¹ concentrations. After 72 hours of incubation, while DOX loaded groups were able to create over 65% cytotoxicity, empty or only calcitriol loaded groups showed little or no cytotoxicity (Fig. 6). Calcitriol, when co-loaded with DOX into untargeted liposome, was able to enhance cytotoxicity of DOX and create higher cytotoxicity over only DOX loaded liposome groups. However, when targeted with non-internalizing antibody, DOX/calcitriol co-loading was not able to create any cytotoxicity enhancement over only DOX loaded CD20 targeted group. The reason for this was the specific interaction that causes CD20 targeted liposomes to be held on the surface which created a condition less likely for them to fuse with cell membrane unlike the untargeted liposomes. This is because anti-CD20 was reported as an antibody that was poorly internalized and able to sustain prolonged retention of bonded molecule on the cell surface with minimal intracellular accumulation.¹³ Non-specific internalization of untargeted liposomes was the reason of observed cytotoxicity enhancement effect of calcitriol even after 24 hours. All drug loaded groups showed that CD20 targeting created a superior cytotoxicity over Namalwa cells. However, due to non-internalization no additive effect of calcitriol on in vitro cytotoxicity of DOX was seen in the targeted liposomes.

Fig. 6 Effect of CD20 targeting of liposome groups on cytotoxicity against Namalwa cells (a) 24 hours treatment (b) 48 hours treatment (c) 72 hours treatment. * Significantly different from other dose groups except 50 nmol mL⁻¹ only DOX loaded group (P < 0.05). ** Significantly different from other dose groups (P < 0.05).

Laser scanning confocal microscopy

In order to evaluate early and high cytotoxicity achieved by untargeted liposomal formulation, liposome-cell association was investigated with laser scanning confocal microscopy, using Rh-PE labelled liposomes and DOX fluorescence. Both Rh-PE and DOX were excited at the same wavelength and same parameters were used to collect their emission. Rh-PE label was used to observe DOX free liposome groups and DOX fluorescence was used to detect fluorescence level of DOX loaded liposome groups and DOX distribution through cells. DOX/calcitriol co-loaded, only DOX loaded, only calcitriol loaded and empty liposomes were incubated with Namalwa cells for 6 hours (Fig. 7). Both DOX/calcitriol co-loaded and only DOX loaded liposomes showed high association with Namalwa cells (Fig. 7c and d). High fluorescence in the cells incubated with DOX loaded liposomes showed that DOX was able to get in the cells after 6 hours of incubation. Depth scan analysis also confirmed that both calcitriol free and calcitriol loaded liposomes were able to enter the cells after 6 hours of incubation (Fig. 8).

Fig. 7 Confocal images of Namalwa cells treated with Rh-PE-labeled liposome groups for 6 hours. (a) Empty liposomes free of DOX and calcitriol. (b) Only calcitriol loaded liposomes. (c) DOX loaded liposomes. (d) DOX/calcitriol co-loaded liposomes.

Fig. 8 Confocal depth scan analysis of Namalwa cells after 6 hours of incubation with liposomes labelled with Rh-PE. (a) DOX loaded liposomes. (b) DOX/calcitriol co-loaded liposomes.

Additional movie file shows confocal images of cell sections after 6 hours of incubation with CD20 targeted DOX loaded liposomes [see ESI† file 1]. The DOX accumulation inside the cells indicates that liposomes were able to interact and fuse to the cell membrane while delivering encapsulated DOX into the cell as also observed from cytotoxicity results. There was no detectable enhancement effect of calcitriol loading on liposome-cell association. Both DOX/calcitriol co-loaded and only DOX loaded liposomes were able to deliver DOX into Namalwa cells in the incubation time. By this aspect, the only reason of higher cytotoxicity achieved by DOX/calcitriol co-loaded liposomes was shown to be the result of combined anti-proliferative effect of DOX and calcitriol accumulation in cells. When anti-CD20 targeted liposome's interaction with Namalwa cells was observed under laser scanning confocal microscopy it was seen that most of the Namalwa cells had dark interiors which indicated DOX free areas. In contrast to untargeted liposomes, DOX delivered by targeted liposomes did not distribute through cells after 6 hours of incubation which explains the delayed cytotoxicity of targeted liposomes (Fig. 9). When delivered in targeted liposomes, calcitriol was unable to enhance DOX cytotoxicity. This result proved that untargeted DOX/calcitriol liposomes were able to deliver the drugs into cells by unspecific fusion and an enhanced cytotoxicity was created by combined anti-proliferative effect of DOX and calcitriol even after 24 hours of exposure.

Fig. 9 Confocal images of Namalwa cells treated with Rh-PE-labeled, anti-CD20 targeted liposome groups for 6 hours. (a) Empty liposomes free of DOX and calcitriol. (b) Only calcitriol loaded liposomes. (c) DOX loaded liposomes. (d) DOX/calcitriol co-loaded liposomes.

Conclusions

The new, dual loaded liposome formulation will enable high calcitriol and DOX administration to cancer cells in a single treatment without creating hypercalcemia risk while decreasing the released amount of the anti-cancer drug, DOX, during circulation in blood. Therefore, with this approach, the novel calcitriol and DOX co-loaded liposome formulation will provide superior cytotoxicity effect on Namalwa cells than the free form and only DOX loaded liposome treatments. In vitro studies demonstrated that the DOX IC₅₀ value for liposomal drug formulation decreased more than 10 fold when liposomes co-loaded with calcitriol. In order to explain whether the early cytotoxicity created by co-loaded liposomal group was the reason of cell fusion, effect of non-internalizing targeting was also investigated by modifying liposomal system with anti-CD20 antibody. Targeted liposome groups of DOX and DOX/calcitriol co-loaded group showed superior cytotoxicity over untargeted liposomes after 72 hours of in vitro treatment, while 50 and 100 nmol mL⁻¹ untargeted liposome groups cytotoxicity over Namalwa cells were decreased. However, there was no significant enhancement of DOX cytotoxicity by calcitriol co-loading detected when liposomes were targeted to CD20 antigen. It was concluded that targeting liposomes to non-internalizing cell surface antigen CD20, prevented the unspecific fusion of liposomes due to stable antibody–antigen attachment. On the other hand, in vitro conditions induced the nonspecific binding and fusion of untargeted liposomal groups thereby increasing the drug delivery into the cells.

Acknowledgements

This work was supported by the Middle East Technical University project BAP-08-11-2012-006 and Gulhane Medical Faculty, Scientific Research Center through the project 2012-37 by the financial means and laboratory facilities.

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Footnotes

† Electronic supplementary information (ESI) available: Additional movie file shows confocal images of cell sections after 6 hours of incubation with CD20 targeted DOX loaded liposomes. See DOI: 10.1039/c6ra19187h

‡ Current address: Hematology Unit, Memorial Hospital, Ankara, 06520 Turkey.

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