Biotinylated Cubosomes: A Versatile Tool for Active Targeting and

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Biotinylated Cubosomes: A Versatile Tool for Active Targeting and Codelivery of Paclitaxel and a Fluorescein-Based Lipid Dye Simone Aleandri,† Davide Bandera,† Raffaele Mezzenga,‡ and Ehud M. Landau*,† †

Department of Chemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland Department of Health Science & Technology, ETH Zurich, Schmelzbergstrasse 9, LFO, E23, 8092 Zürich, Switzerland

‡

S Supporting Information *

ABSTRACT: The functionalization of cubosomes with biotin is reported here as an alternative method for the preparation of drug delivery systems capable of active targeting specific receptors that are (over)expressed by cancer cells. We describe the design, synthesis, assembly, and characterization of these novel cubosome nanoparticles by small-angle X-ray scattering (SAXS) and dynamic laser light scattering (DLS) and show their application to human adenocarcinoma cell line HeLa. These cubosomes are stabilized and functionalized with a novel, designed biotin-based block copolymer and are able to simultaneously transport paclitaxel, a potent anticancer drug, and a hydrophobic fluorescent dye in the active targeting of cancer cells. Such biotinylated cubosomes are potentially applicable in diagnosis, drug delivery, and monitoring of the therapeutic response for active targeting versus cancer cells.

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have much lower viscosity in comparison to the bulk LCP.9 The latter are composed of bilayers that are curved in 3D space such that every point on their surface is a saddle point with a zero mean curvature. These structured yet flexible spongelike bilayers encompass a system of aqueous channels, forming nonbirefringent and optically transparent lipidic phases.10 Most LCPs and their dispersions used to date are based on monoacyl glycerols such as monoolein (MO), which is an uncharged lipid (Figure 1) whose headgroup structure does not impart steric stabilization to the cubosomes once formed in solution.11 Therefore, cubosomes need to be kinetically stabilized by steric means to prevent flocculation of the dispersion and thus improve the shelf life.12 Steric stabilization of such colloidal particles is typically achieved by the addition of a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymer.13 Pluronics are the most widespread class of steric stabilizers available in the market for lyotropic liquid-crystalline particles.14 Chong et al.12 investigated the ability of the pluronic series to stabilize MO-based cubosomes, demonstrating that Pluronic F108 (PF108) is the most effective stabilizer of such

INTRODUCTION One of the major challenges in drug delivery is the lack of control over the selectivity of drug and vehicle biodistribution toward target cells.1 Active targeting is a site-specific drug delivery strategy whereby the surface of the nanocarrier is decorated with ligands that bind specific receptors which are (over)expressed by cancer cells, thereby affecting drug release at a preselected biosite in a controlled manner.2−4 Targeted nanocarriers for cancer treatment thus offer a great potential advantage in tumor treatment due to the greater specificity of delivery. In comparison, in conventional drug delivery the active compound is distributed indiscriminately throughout the body, with arbitrary concentrations reaching both the target disease site and healthy tissue.5 An ideal drug delivery system should be biocompatible, biodegradable, incorporate the active agent without loss or alteration of its activity, and provide an efficient and controlled delivery mechanism6 to the specific location in vivo.7 Cubosomes constitute an alternative delivery system that offers the possibility to develop targeted therapeutic agents with improved bioavailability, biodistribution, pharmacokinetics, and safety profiles.8 Principally, cubosomes possess the same microstructure as the parent lipidic cubic phase (LCP), albeit their dispersions © 2015 American Chemical Society

Received: September 15, 2015 Revised: October 24, 2015 Published: October 29, 2015 12770

DOI: 10.1021/acs.langmuir.5b03469 Langmuir 2015, 31, 12770−12776

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Langmuir

Figure 1. Structures of PF108-B, MO-Fluo, and MO. Preparation of Cubosomes. Cubosomes (empty, loaded with PX, or doped with MO-Fluo) were prepared as described by Murgia et al.15 Briefly, a solution of the appropriate amount of MO in CHCl3 was evaporated to provide a film of MO on the wall of a round-bottomed flask. A solution of stabilizer (PF108, PF108-B, or a mixture of both at 1.65 mg/mL in PBS at pH 7.4) was added to the preformed films after overnight storage in a desiccator under reduced pressure (0.4 mbar) to yield the cubosome solution with the desired lipid concentration. The solution was vortex mixed and then dispersed using a Brenson digital sonifier 250 ultrasonic processor (cycle 0.9 s on/0.9 s off, amplitude 50%, 10 min). Subsequently, the cubosome dispersion was filtered through a 450 nm Acrodisc. The concentrations of MO and stabilizer in the resulting dispersions were typically 33 and 1.65 mg/mL, respectively. For the PX-loaded or MO-Fluo-doped cubosomes, a mixed film of MO and PX or MO-Fluo was prepared on the inside wall of a roundbottomed flask by the evaporation of organic solutions containing a defined amount of MO (dissolved in CHCl3) and PX (dissolved in DMSO) or MO-Fluo (dissolved in CHCl3) (PX/MO and MO-Fluo/ MO weight ratio kept at 1:150). Following hydration, the cubosomes were processed as described previously. Size Determination. Cubosome particle size determination was performed immediately after preparation and 1 month thereafter using a Zeta Sizer Nano ZS (Malvern Instruments, Malvern, U.K.) at 37 ± 0.1 °C. The scattered intensity was collected at an angle of 90°. The samples were housed in disposable polystyrene cuvettes of 1 cm optical path length with water as the solvent. For every point at least three independent samples were prepared, and each one was measured at least three times. The intensity size distribution of the cubosomes was typically unimodal; therefore, the autocorrelation function was analyzed according to the cumulant method. Small-Angle X-ray Scattering (SAXS). SAXS measurements were carried out to determine the phase identity and symmetry of the cubosomes. A microfocused Rigaku X-ray source of wavelength λ = 1.54 Å was used, operating at 45 kV and 88 mA. Scattered X-ray signals were collected on a gas-filled two-dimensional (2D) detector. Scattering vector q = (4π/λ)sin θ (with 2θ being the scattering angle) was calibrated using silver behenate. The sample-to-detector distance was 1 m, which provided a q range from 0.01 to 0.5 Å−1. Data were collected and azimuthally averaged using the Saxsgui software to yield the 1D intensity versus scattering vector q. Samples were equilibrated at 37 °C for 10 min prior to measurement, and the scattering intensity was collected for 5 h at 37 °C. Filtration. Excess nonincorporated MO-Fluo or PX was separated from the cubosomes by filtration using Amicon Ultra 0.5 mL centrifugal filters with a 30 kDa cutoff.8 About 0.2 mL of sample was placed in the Amicon cell and centrifuged at 10 000 rpm for 10 min. The encapsulation efficiency (E(%)) was calculated using the following equation

cubosomes, which retain their native diamond cubic phase (Pn3m) structure.15 Cubosomes fulfill a number of requirements including the controlled delivery of poorly water-soluble drugs and selective targeting of malignant cells to minimize side effects.16 While all living cells require vitamins and other nutrients for survival, cancer cells have a voracious appetite for these substances; consequently, the receptors involved in their uptake are overexpressed.17 Biotin (vitamin H or B-7) is a growth promoter at the cellular level, which serves as a cofactor for carboxylases during fatty acid biosynthesis, gluconeogenesis, and catabolism of several branched-chain amino acids. Its content in tumor cells is substantially higher than that in normal tissues.17 Biotin uptake into mammalian cells is mediated by a sodium-dependent multivitamin transporter (SMVT) and other unidentified transporters.18 Biotin receptors are overexpressed more than the folate and/or vitamin B-12 receptors in many cancer cells, e.g., ovarian cell lines.19,20 Fraser et al. have reported the use of biotin-functionalized amphiphiles in cubosome preparation and subsequent binding to avidin surfaces for sensing applications.21 To date, paclitaxel (PX) was found to be effective in treating a broad spectrum of advanced human cancer types including ovarian cancer.22 The commercial PX preparation (Taxol) is formulated in a vehicle composed of Cremophor EL (polyethoxylated castor oil used as a solubilizing surfactant) and dehydrated ethanol, which provides a homogeneous preparation. However, the diluted Cremophor EL/ethanol vehicle is toxic.23 To overcome this drawback, we present here an alternative biocompatible system based on cubosome nanoparticles composed of MO and loaded with PX. These cubosome dispersions are stabilized using a novel, designed biotin-conjugated stabilizer (PF108-B, Figure 1) that imparts to them better targeting capabilities toward cancer cells as compared to conventional cubosomes. Moreover, a fluorescent lipid, MO-Fluo (Figure 1), was synthesized and embedded in the cubosomes to follow the cellular internalization of the nanoparticles. All of the cubosome dispersions were characterized by SAXS and DLS techniques, the biotin functionalization was evaluated in vitro, and the targeting ability was tested in human adenocarcinoma cell line (HeLa).

2. EXPERIMENTAL SECTION Chemicals and Reagents. 1-Monooleoyl-sn-glycerol C18:1 (monoolein, MO) was purchased from Nu-Chek Prep, Inc. (MN, USA), and phosphate buffer solution (PBS (1X) pH 7.4) was purchased from Invitrogen. Paclitaxel (PX) and 4-hydroxyazobenzene2-carboxylic acid (HABA) were purchased from Sigma-Aldrich. Avidin was purchased from Thermo Fischer Scientific. All solutions were prepared using Mili-Q water (18.2 MΩ cm−1; Millipore, Bedford, MA). PF108-B and MO-Fluo were synthesized as part of this work and are described in the SI.

E(%) =

WT − WF × 100% WT

(1)

where WT is the weight of total MO-Fluo or PX in the cubosomes and WF the free MO-Fluo or PX in the ultrafiltrate detected after centrifugation. 12771

DOI: 10.1021/acs.langmuir.5b03469 Langmuir 2015, 31, 12770−12776

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Langmuir The MO-Fluo content was quantified by fluorescence spectroscopy at λexcitation = 470 nm and λemission = 520 nm using an Infinite auto 200 microplate reader (Tecan, Austria). The PX content was quantified using an HPLC method (C18 column 4.1 mm × 300 mm, 10 μm particle size; methanol/water 70:30 (v/v); UV detector at 227 nm; flow rate 1 mL/min).24 Specifically, the PX concentration was determined after Amicon filtration, lyophilization of the recovered aqueous solution, and resuspension in methanol before injection. Determination of the Biotin to PF108 Ratio. HABA (4′hydroxyazobenzene-2-carboxylic acid) was used to estimate the molar ratio of biotin to PF108. Briefly, a solution of PF108-B (0.08 mg/mL) was added to a mixture of HABA (H) and avidin (A). The avidin− HABA solution (H/A) was prepared by mixing 9.9 mL of 2.42 mg/mL (H) solution with 0.1 mL of 0.5 mg/mL (A) solution. Because of its higher affinity for avidin, biotin displaces HABA from its interaction with avidin, and the absorption at 500 nm decreases proportionately. Thus, an unknown amount of biotin present in solution can be detected by measuring the absorbance of the (H/A) solution before and after the addition of the PF108-B solution (H/A/B).25 The steps followed in the calculation to evaluate the molar ratio of biotin to PF108 are reported in the SI. Colorimetric Determination of the Interaction of PF108-BBased Cubosomes with Avidin. A cubosome solution (stabilized with PF108 or with 50% PF108 and 50% PF108-B, CB) at various concentrations was added to an (H/A) solution prepared as reported above. Biotin displaces HABA from its interaction with avidin, and the absorption at 500 nm decreases proportionately. The change in absorbance at 500 nm was monitored after 15 min of incubation at 37 °C and evaluated using eq 2 ΔA500 = AH/A − AH/A/C

was then added. Following a 4 min incubation, the cells were harvested by adding 1 mL of PBS and treated with probe-type ultrasonication (five times) to obtain the cell lysate. The cell lysate was centrifuged at 10 000 rpm for 15 min, and the supernatant was submitted to a fluorescence assay by using an Infinite auto 200 microplate reader (Tecan, Austria) at λ excitation = 470 nm and λ emission = 520 nm. The cellular uptake percentage was calculated from eq 3 cellular uptake (%) =

I × 100 I0

(3)

where I is the fluorescence intensity at different times and I0 is the initial fluorescence intensity of the fluorescent cubosomes. For the cell viability and cellular uptake experiments, statistical analysis was carried out as follows: Data are expressed as the mean ± SD derived from five independent experiments (formulations). Statistically significant differences are indicated by *** (p < 0.001) and * (p < 0.05) via the t test vs untreated control cells and among differently treated cells.

3. RESULTS AND DISCUSSION Preparation of Biotinylated PF108 and MO-Fluo. We present here the use of designed MO-based cubosomes as nanocarriers for active paclitaxel (PX) targeting to HeLa cancer cells. To achieve this goal, the surfactant commonly used to stabilize cubosomes, PF108, was conjugated with biotin (Scheme S2, SI), a targeting ligand which shows affinity for the sodium-dependent multivitamin transporter (SMVT)18 that is overexpressed in the membrane of tumor cells. The synthesis of PF108-biotin (PF108-B) was carried out as shown in Scheme S2 in the SI. Briefly, intermediate PF108-NH2 was prepared according to Caltagirone et al.,16 following by its reaction with NHS, DCC, Et3N, and biotin to obtain final product PF108-B after dialysis purification. To quantify the biotin label incorporation, a solution containing the biotinylated PF108 molecule was added to a mixture of HABA and avidin. Because of its higher affinity for avidin, biotin displaces HABA from its interaction with avidin, and the absorption at 500 nm decreases proportionately. By measuring the absorbance of the HABA−avidin solution before and after the addition of the biotin-containing sample, an unknown amount of biotin present in solution can be evaluated. Following dialysis purification of PF108-B, the molar ratio of biotin per PF108 was found to be 2.145 (calculation steps in the SI). In order to ascertain the absence of unreacted biotin and thereby the quality of the dialysis purification, the following negative proof was performed: A solution of free biotin at the same concentration as that used in the preparation of PF108-B was loaded into the porous membrane tubing and dialyzed for 3 days. Following dialysis, no biotin was detected by colorimetric assay or by 1H NMR. Moreover, in order to fluorescently follow the cellular uptake of cubosomes, fluorescent lipid MO-Fluo was synthesized (Scheme S1, SI) and embedded in the lipid bilayers. MO-Fluo is an oleic acid derivative in which the fluorescein dicarboxylic acid was conjugated to the amine headgroup of the lipid by an amide bond. Following the preparation, the fluorescent lipid was characterized in solution (results not shown) and finally embedded in the cubosomes at 1 wt %. Its entrapment efficacy (E(%)), evaluated using a filtration method described above, is 90 ± 5%. Cubosome Characterization. To evaluate the effect of the novel stabilizer on the phase identity and unit cell size of the system, cubosomes were prepared and analyzed with smallangle X-ray scattering (SAXS), see SI, Figures S1−S6. In the

(2)

where AH/A and AH/A/C are the absorbance values of the (H/A) solutions at 500 nm before and after the addition of cubosomes, respectively. Agglutination Assay. The agglutination of cubosomes (stabilized with PF108 or with 50% PF108 and 50% PF108-B, CB) at 0.15 mg/ mL in the presence of a 0.5 mg/mL avidin solution in PBS was established from the time-dependent change of the particle size in suspension, as evaluated by DLS measurements at 37 °C. Scans were carried out immediately after mixing and at selected times within the first 2 h. Cell Culture. HeLa cells were grown in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, USA) with high glucose, supplemented with 10% (v/v) fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) (Invitrogen) in a 5% CO2 incubator at 37 °C. Cells were seeded in 35 mm dishes, and experiments were carried out 48 h after seeding, when cells had reached 90% confluence. For competitive experiments, cells were grown in biotin-enriched media (1 mM). Viability Assay. HeLa cells were seeded in 96-well plates (5000 cells per well), cultured overnight, and subsequently treated with toxicant solutions in the cell medium for 24 h. Specifically, in the case of PX, the drug was solubilized in DMSO, and aliquots of the DMSO solution were diluted using the cell medium in order to obtain solutions at different concentrations. Following the removal of the extracellular particle suspension with fresh serum-free medium, cells were incubated with 10 μL CCK-8 (Invitrogen) for 4 h at 37 °C. Absorbance was measured at 450 nm using an Infinite 200 microplate reader (Infinite 200, Tecan, Austria). Results are shown as the percent of cell viability in comparison to nontreated control cells. Data are expressed as the mean ± SD from at least three independent experiments (involving triplicate analyses for each sample). Cellular Uptake. HeLa cells were seeded in a 24-well plate at a density of 10 000 cells per well in 1 mL of growth medium and incubated for 24 h. Subsequently, cells were incubated with an MOFluo-loaded cubosome suspension (0.15 mg/mL cubosome concentration and 1:150 MO-Fluo/lipid weight ratio) in a growth medium for various times. To assay the cellular uptake, cells were washed twice with PBS (pH 7.4), and 40 μL of trypsin PBS solution (2.5 mg/mL) 12772

DOI: 10.1021/acs.langmuir.5b03469 Langmuir 2015, 31, 12770−12776

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Langmuir SAXS regime, sharp Bragg reflections characteristic of the longrange positional order are detected. SAXS experiments were performed on either empty cubosomes or on the formulations that contain PX (PX/lipid 1:150 weight ratio). The cubosomes were stabilized with either PF108 (C), PF108-B, or a 1/1 mixture of PF108 and PF108-B (CB). Additionally, as discussed above, MO-Fluo was embedded at 1% w/w in the cubosomes for fluorescent detection. Table 1 summarizes the Table 1. Samples, Phase Identity, Crystallographic Unit Cell Parameters, Size, and Polydispersity Index (PDI) Observed for the Cubosome Dispersionsa sample PF108 (C) PF108-B PF108 + PF108-B (50:50 wt %) (CB) C + MO-Fluo CB + MO-Fluo C-PX CB-PX CB-PX + MO-Fluo a

phase identity

unit cell (Å)

size (nm)

PDI

Pn3m Pn3m Pn3m

100.9 98.7 99.8

131.5 ± 10 210 ± 8 132 ± 7

0.04 0.33 0.013

Pn3m Pn3m Pn3m Pn3m Pn3m

100.3 99.7 99.2 99.4 100.1

130.5 135.5 137.5 140.4 138.7

± ± ± ± ±

3 5 4 10 6

Figure 2. ΔA at 500 nm for HABA/avidin solution in the presence of various concentrations of biotinylated cubosomes (CB) and nonbiotinylated cubosomes (C) (black and white circles, respectively). Data are expressed as the mean ± SD from three independent experiments.

0,08 0.02 0.03 0.05 0.04

ΔA) as a function of increasing the concentration of biotinylated cubosomes. In contrast, cubosomes stabilized with PF108 alone do not show a similar decrease in absorbance. Because of its higher affinity for avidin, the biotin moiety exposed on the cubosome surface displaces HABA from its interaction with avidin, and the absorption at 500 nm decreases proportionately. The decrease in absorption is directly correlated with the increase in biotinylated cubosome concentration and therefore with the amount of biotin in solution. The results obtained from colorimetric experiments were supported and confirmed by agglutination experiments carried out by size measurements on CB (150 μg/mL) in the presence of avidin. This method of analysis of the cubosome suspensions in the presence of avidin is based on measuring the time-dependent sample size. Size changes, observed over 90 min after the addition of avidin (Figure 3 and Figure S11) and complete particle precipitation observed after 24 h indicated cubosome agglutination. In the case of PF108 cubosomes, no size changes or precipitation was observed. Thus, the agglutination method confirmed that the biotin ligand, exposed

Abbreviations are in parentheses.

SAXS results. All investigated samples exhibited a cubic Pn3m phase, with a unit cell parameter of approximately 100 Å.12 The concentrations of MO and stabilizer in the resulting dispersions were typically 33 and 1.65 mg/mL, respectively. Increasing the stabilizer concentration from 1.65 to 2 and 3 mg/mL resulted in the formation of the bicontinuous cubic double diamond and primitive phases, with space groups Pn3m and Im3m, respectively, as shown by SAXS26,27 (SI, Figures S7−S9). Interestingly, the type of stabilizer used to stabilize the nanoparticles strongly affected the mean particle size (Table 1), which increased from 131.5 nm (100% PF108) to 210 nm (100% PF108-B). Size analysis suggests that the conjugation of biotin to the terminal end of PF108 changes the stabilizing effect of the Pluronic.28 In contrast, a 1/1 mixture of PF108 and PF108-B did not affect the mean particle size of the dispersions, which was 132 nm. This formulation was found to be stable for 1 month (SI, Figure S10). On the basis of these results this formulation (CB) was selected for further investigations. In summary, SAXS and DLS measurements demonstrate that the presence of MO-Fluo and PX coloaded into the cubosomes (CB) does not change the phase identity, the unit cell size, or the size of the formulation. Cubosomes can generally entrap hydrophobic molecules and release them after a certain time.29 The encapsulation efficiency depends on the method of preparation, on the lipid type, and on the lipid/drug ratio used.30 The entrapment efficacy of PX in the cubosomes (PX/MO 1:150 weight ratio) is 94 ± 4%. The lipid bilayers of cubosomes (C and CB) provide a suitable environment, which enhances the solubility of the hydrophobic molecule of PX by the association of the drug within membrane bilayers. Decoration of the cubosome surface with PF108B does not change the capability of the cubosome’s lipid bilayer to host hydrophobic molecules. Biotin-Affinity Assay. The presence of biotin on the surface of the cubosomes was evaluated by both colorimetric and agglutination assays. As shown in Figure 2 (black circles), the absorption of HABA decreases (depicted as an increase in

Figure 3. Time-dependent change in the size of biotinylated cubosomes (CB) and nonbiotinylated cubosomes (C) (black circles and white squares, respectively) in the presence of avidin. Data are expressed as the mean ± SD from three independent experiments. 12773

DOI: 10.1021/acs.langmuir.5b03469 Langmuir 2015, 31, 12770−12776

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fluorescence intensity was measured following incubation. Figure 5 shows the cellular uptake percentage of fluorescent

on the surface of cubosomes, is well accessible to the target receptor.24,31,32 Cell Viability. The HeLa cell line was used to evaluate the activity of paclitaxel-loaded cubosomes. The effects of paclitaxel (PX), paclitaxel-loaded cubosomes (C-PX), and paclitaxelloaded biotinylated cubosomes (CB-PX) on HeLa cell viability were investigated. Initially, the effect of various concentrations of empty cubosomes (C and CB) on HeLa cell viability was studied (SI, Figure S12). Cells incubated for 24 h with empty cubosomes did not evoke any cytotoxicity, as shown by cell viability values that remained >95% (up to 150 μg/mL). The IC50 of PX (dissolved in the cell medium) after 24 h of incubation on HeLa was found to be 6 μg/mL, which is in accordance with the value reported in the literature33 (SI, Figure S13). Therefore, for further experiments, concentrations of 150 and 1 μg/mL cubosomes and PX, respectively, were used. Because of the relatively low solubility of PX in water, PXloaded cubosomes were more effective against tumor cells at the tested concentrations (Figure 4). Moreover, CB-PX causes

Figure 5. Time-dependent cellular uptake of biotinylated cubosomes (CB), nonbiotinylated cubosomes (C), and biotinylated cubosomes (CB) in the presence of free biotin (white, black, and gray bars, respectively). Data are expressed as the mean ± SD from five independent experiments. Statistically significant differences are indicated by *** (p < 0.001) via the t test vs nontargeted cubosome (C) treated cells.

cubosomes as a function of incubation time. No difference in uptake between CB and C was observed up to 2 h. At 4 and 24 h, the order of cellular uptake capability for cubosomes was CB (62% after 24 h) > C (44% after 24 h). This result indicates that the cubosome targeting strategy increases the antitumoral activity of PX due to a remarkable increase in the cellular uptake.17 To provide further confirmation, the biotin receptor was presaturated with excess biotin (1 mM) as described above. Even in this case (Figure 5, gray bars), in accordance with the cytotoxicity experiment, once the biotin receptors are saturated, the cellular uptake decreases and becomes similar to that observed with C. This observation leads us to conclude that the biotin functionality present in CB plays an important role in terms of mediating the cellular uptake of cubosomes, and depends on the level of overexpressed biotin receptors present in the cancer cells.

Figure 4. Cell viability of HeLa cells after 24 h of incubation with different toxicants: Nonbiotinylated cubosomes (C), biotinylated cubosomes (CB), paclitaxel (PX), nonbiotinylated cubosomes loaded with paclitaxel (C-PX), and biotinylated cubosomes loaded with paclitaxel (CB-PX). Data are expressed as the mean ± SD from five independent experiments. Statistically significant differences are indicated by *** (p < 0.001) and * (p < 0.05) via the t test vs untreated-control cells and among differently treated cells.

4. CONCLUSIONS An alternative method for the efficient active targeting of specific receptors that are (over)expressed by cancer cells is presented herein. Biotinylated stabilizer PF108 (PF108-B) and a fluorescent lipid (MO-Fluo) were designed and synthesized as additives in the fabrication of fluorescent targeted cubosomes for the active delivery of paclitaxel versus human adenocarcinoma cell line HeLa. PX-loaded biotinylated cubosomes (CBPX) were characterized by chemical and physical techniques, notably small-angle X-ray scattering (SAXS) and dynamic laser light scattering (DLS), and their delivery efficacy on HeLa cells was established. Compared to PX or to the nontargeted cubosomes, biotinylated cubosomes displaying a high degree of active functional biotin on their surface markedly increased the antitumor activity of PX at a concentration of 1 μg/mL. Moreover, the biotin ligand promotes cancer cell uptake via receptor-mediated endocytosis.34 These results suggest that the efficacy of PX against tumor cells may be increased, and the

a greater decrease in cell viability (29%) than does C-PX (45%), demonstrating that biotinylated cubosomes are efficiently taken up into cells by receptor-mediated endocytosis. To provide further confirmation, the biotin receptors were pretreated with excess biotin (1 mM) prior to incubating with cubosomes. This treatment was expected to block the receptors and reduce the binding by CB.19 The results are in accordance with expectations and reveal that once the biotin receptors are saturated, the cell viability increases and reaches levels that are similar to those observed with C-PX. Cellular Uptakes. To elucidate the relationship between the cytotoxicity and the capability of cubosomes to penetrate the cell, the cellular uptake of CB doped with MO-Fluo was measured and compared to that of nontargeted cubosomes, C. It was found that the presence of MO-Fluo (1 wt %) in the formulation did not cause any cytotoxicity on HeLa cell after 24 h of incubation (SI, Figure S14). Cells were incubated with 150 μg/mL fluorescent cubosomes for various times, and the 12774

DOI: 10.1021/acs.langmuir.5b03469 Langmuir 2015, 31, 12770−12776

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toxicity of PX can be decreased by using the biotinylated cubosome targeting method. Such biotinylated cubosomes are potentially applicable in diagnosis, drug delivery, and monitoring of therapeutic responses.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b03469. Description of the MO-Fluo and PF108-B syntheses, evaluation of the molar ratio of biotin per mole of PF108, SAXS patterns, stability studies, cell viability of empty cubosomes and MO-Fluo cubosomes, and IC50 of PX (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Polish-Swiss Joint Research Programme grant PSPB-079/2010 to E.M.L. and by SNF Sinergia grant CRSII2_154451 to E.M.L. and R.M.

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REFERENCES

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DOI: 10.1021/acs.langmuir.5b03469 Langmuir 2015, 31, 12770−12776

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DOI: 10.1021/acs.langmuir.5b03469 Langmuir 2015, 31, 12770−12776