Article pubs.acs.org/Langmuir
Further Evolution of Multifunctional Niosomes Based on Pluronic Surfactant: Dual Active Targeting and Drug Combination Properties Lorena Tavano,† Loredana Mauro,† Giuseppina Daniela Naimo,† Leonardo Bruno,‡ Nevio Picci,† Sebastiano Andò,† and Rita Muzzalupo*,† †
Department of Pharmacy, Health and Nutritional Sciences and ‡Department of Biology, Ecology and Earth Sciences, University of Calabria, Via Pietro Bucci, Ed. Polifunzionale, 87036 Arcavacata di Rende, Italy ABSTRACT: The loading of chemotherapics into smart nanocarriers that simultaneously possess more than one useful property for specifically targeting a tumor site improves their therapeutic effectiveness, reducing their side effects. Hence, we proposed a combined approach for the treatment of human breast cancer (BC) consisting of the co-encapsulation of doxorubicin and curcumin or doxorubicin and quercetin into multifunctional niosomes, which results in prolonged blood circulation and an ability to spontaneously accumulate at the tumor site (passive target) and to recognize and bind the tumor cells through dual ligand−receptor interactions (active target). The drug-loaded vesicles showed high stability and good capability of loading doxorubicin and antioxidants alone or in combination. Their diameter was around 400 nm. The drugs released from the vesicles were found to be controlled and sustained for over 24 h, with a strong dependence on the co-presence of the loaded molecules. Transferrin and/or folic acid were conjugated on the external surface of the niosomes as ligands, considerably improving the cellular uptake into MCF-7 and MDAMB-231 malignant cells when compared with the uptake of nonconjugated samples. In vitro evaluation of anticancer activity demonstrated the strong potential of niosomes loaded with a doxorubicin/curcumin combination as useful devices in breast tumor treatment. These features hold great promise for the development of multifunctional devices that combine several advantages such as biocompatibility, stealth properties, loading capability, and active targeting, moving toward the development of more specific and efficient carriers for personalized tumoral therapy. flavonoids could be of clinical benefit, promoting cytotoxicity in tumor cells while protecting normal cells against doxorubicininduced damage.6−8 On the other hand, the vehiculation of chemotherapics into multifunctional nanocarriers that simultaneously possess more than one useful property of specifically targeting the tumor site in the same system improves therapeutic effectiveness and reduces the side effects of the drug.9,10 Among the macromolecular systems useful for targeted drug delivery, niosomes are the most extensively studied and possess characteristics that are most suitable for the development of multifunctional devices.11 Niosomes are very versatile because of their structure and can be differently designed and modified in such a way that they exhibit combinations of some of the following properties: longevity in blood, specific target to the tumor, response to internal/external stimuli, and promotion of intracellular cargo delivery.11 Thus, we proposed a combined approach for the treatment of human BC consisting of the co-encapsulation of both a
1. INTRODUCTION Breast cancer (BC) is the most common cancer and the second major cause of death in women.1 Despite advances in early detection and therapeutic approaches in the past few years, BC continues to have a poor prognosis.2 The high mortality related to BC is mostly due to the low therapeutic selectivity of antitumor agents and the high rate of metastasis and resurgence.3 In most advanced breast tumors, the leading anthracycline doxorubicin is the main option, but its therapeutic effectiveness is greatly limited by conventional toxicities, dose-dependent cardiotoxicity, and development of multidrug resistance.4 Thus, to improve the therapy regimen for this anticancer drug, it is necessary to design new and more tumor-specific doxorubicin-based combination therapies, targeting several cellular pathways and reducing the dose necessary for efficacy and adverse side effects. A combination therapy with two or more drugs that promotes synergism and vehiculation into multifunctional nanocarriers has shown significant promise in cancer treatment. More recently, it has been demonstrated that the efficacy of doxorubicin increased if administered in combination with antioxidant molecules because this drug is claimed to induce oxidative stress.5 Several studies, in fact, reported that combining doxorubicin and © XXXX American Chemical Society
Received: June 1, 2016 Revised: July 14, 2016
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DOI: 10.1021/acs.langmuir.6b02063 Langmuir XXXX, XXX, XXX−XXX
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temperature. The film was then hydrated with 5 mL of distilled water at 20 °C for 30 min to form large empty MLVs at 10 mM total lipid concentration. Doxorubicin- or rhodamine-loaded vesicles were obtained by hydrating the thin lipid film with 5 mL of doxorubicin hydrochloride or rhodamine (3.45 × 10−4 and 4.16 × 10−4 M, respectively) aqueous solution at 20 °C for 30 min. To obtain singleantioxidant-loaded niosomes, 2 mL of curcumin or quercetin ethanolic solution (2.18 × 10−3 and 2.71 × 10−3 M, respectively) was added to the initial chloroform mixture. After mixing, the solvent was evaporated under reduced pressure, and the obtained film was then hydrated with 5 mL of distilled water, whereas niosomes loaded with doxorubicin + curcumin and doxorubicin + quercetin were obtained by hydrating the same film with 5 mL of doxorubicin aqueous solution at 20 °C for 30 min. After preparation, all dispersions were left to equilibrate at 25 °C overnight to allow complete annealing and partitioning of the drug between the bilayer and aqueous phase. Small unilamellar vesicles (SUVs) were prepared starting from MLVs by sonication in an ultrasonic bath for 30 min at 20 °C. The purification of niosomes was carried out by a flow of niosome suspensions across a Sepharose CL-4B gel column. After purification, the niosomes were stored in the dark at 4 °C until their use in subsequent experiments. 2.5. Characterization of Niosomes. Formation and morphology of vesicles were checked by transmission electron microscopy (TEM). A thin aqueous film was stratified onto a carbon-coated copper grid and left to adhere to the carbon substrate for about 1 min. The excess dispersion was removed by filter paper. The resulting thin film was stained with a drop of 2% phosphotungstic acid solution, and the sample was air-dried and observed under a ZEISS EM 900 electron microscope at an accelerating voltage of 80 kV. The average diameter and width of size distribution of the niosomes (polydispersity index, PDI) were determined by dynamic light scattering (DLS), using a 90 Plus Particle Size Analyzer (Brookhaven Instruments Corporation, New York, USA) at 25.0 ± 0.1 °C. We measured the autocorrelation function at 90°. The laser beam was operating at 658 nm. PDI was used as a measure of the size distribution. It was directly obtained from the instrumental data-fitting procedures by the inverse Laplace transformation and by Contin methods.15 The samples were analyzed 24 h after preparation and before each of the following characterization steps. They were diluted with distilled water before the measurements were run. Each vesicle dispersion (50 μL) was diluted to 10 mL with distilled water. Each sample was measured six times, and the results were expressed as mean ± standard deviation (SD). Stability of the niosomes was evaluated by measuring the average size of the vesicles and PDI over a period of several months at room temperature (about 25 °C). 2.5.1. Preparation of Tf-, FA-, and Tf-FA-Conjugated Niosomes. As reported previously,12 conjugation of Tf and/or FA to the surface of the niosomes was achieved by adding 1.29 × 10−5 mol N-(3dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) per 1 μmol of total lipid and incubating the mixture for 6 h at room temperature. Excess EDC was removed by ultrafiltration using Centrisart-10 concentrators (molecular weight cut off 10 kDa, Vivascience, Hannover, Germany). In the next step, 125 μg of Tf/ mol lipid and/or 125 μg of FA/mol lipid were added and incubated overnight at room temperature. The niosomes were then purified by free ligands using size exclusion chromatography on a Sepharose CL4B glass column. 2.5.2. Entrapment Efficiency. The encapsulation efficiency of a drug was expressed as the percentage of the drug entrapped into purified niosomes referred to the total amount of drug present in a nonpurified sample.16 It was determined by diluting 1 mL each of purified and nonpurified niosomes in 25 mL of methanol, followed by the measurement of absorbance of these solutions at the corresponding drug wavelengths (480, 426, and 380 nm for doxorubicin, curcumin, and quercetin, respectively). Methanol allows the breaking of niosomal membranes and the release of the encapsulated drug. Each experiment was carried out in triplicate, and the results were expressed as mean ± SD. 2.6. In Vitro Release Studies. The release of drugs from the niosomes was examined under sink conditions. Aliquots of niosomal
chemotherapic and an antioxidant into multifunctional niosomes, which results in prolonged circulation in the blood, the ability to spontaneously accumulate at the tumor site (passive target), and the ability to recognize and bind tumor cells through ligand−receptor interactions (active target). In particular, to further enhance carrier accumulation in pathological sites and promote drug-specific delivery, the effect of the simultaneous conjugation of two ligands on the surface of the niosomes was also investigated and compared with that obtained with single-ligand-endowed vesicles. Considering the results reported in our previous work,12 niosomes were prepared from the opportunely modified Pluronic L64 surfactant, which is claimed to prevent vesicle opsonization and therefore promote long circulation times and accumulation at the tumor site. The chemical modification of the surfactant makes it possible to link the free amino groups of transferrin (Tf) and/or folic acid (FA), whose receptors are overexpressed and feature a high turnover rate on tumor cells, on the external surface of the niosomes, improving cellular uptake into MCF-7 and MDA-MB-231 malignant cells and making the vesicles tumor-specific. Further, we decided to improve our carriers coencapsulating doxorubicin and curcumin or quercetin and evaluate the effect of their co-presence on the physicochemical and biological properties of the formulations. In vitro anticancer activity was achieved to evaluate the potential synergic anticancer activity of the optimized multifunctional niosomes.
2. MATERIALS AND METHODS 2.1. Chemicals. Pluronic L64 was kindly donated by BASF (Mount Olive, NJ, USA). All reagents were purchased from SigmaAldrich (St. Louis, MO, USA). Chromium trioxide (CrO3), sulfuric acid (H2SO4), potassium hydroxide (KOH), and phenolphthalein indicator were used with no further purification. Cholesterol, doxorubicin hydrochloride, curcumin, quercetin, rhodamine, N-(3dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), Tf, FA, and Sepharose CL-4B gel were purchased from Sigma-Aldrich (Milan, Italy). All solvents were of high-performance liquid chromatography grade. 2.2. Cell Culture. MCF-7, MDA-MB-231, and MCF-10A cell lines were acquired from American Type Culture Collection (Manassas, VA, USA), where they were authenticated, stored according to supplier’s instructions, and used within 4−6 months after frozen aliquots were resuscitated. MCF-7 and MDA-MB-231 human BC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/ Nutrient Mixture F-12 Ham (DMEM/F-12) supplemented with 5% fetal bovine serum (FBS) containing L-glutamine (1%) and penicillin/ streptomycin (1%). The MCF-10A cell line is a nontumorigenic human epithelial breast cell line. MCF-10A cells were cultured in DMEM/F-12 supplemented with 5% horse serum (HS), L-glutamine (1%), penicillin/streptomycin (1%), 100 ng/mL cholera toxin, hydrocortisone (0.5 mg/mL), insulin (10 mg/mL), and epidermal growth factor (EGF) (20 ng/mL). All cells were grown at 37 °C in a 5% CO2 humidified incubator. Before each experiment, cells were grown in a phenol red-free medium, containing 5% charcoal-stripped FBS (cs-FBS), for at least 24 h. 2.3. Synthesis of L64ox. The oxidation of the Pluronic terminal −CH2OH group into a −COOH group was obtained by the reaction with Jones reagent, according to the procedure reported in our previous work.13 2.4. Preparation of Niosomes. Multilamellar vesicles (MLVs) were prepared by a modification of the hydration of lipidic film method.14 The surfactant and cholesterol were dissolved in 10 mL of chloroform in a round-bottom flask (8 × 10−3 and 2 × 10−3 M, respectively). The organic solvent was vacuum-evaporated, and the resulting lipidic film was dried under vacuum for 1 day at room B
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Langmuir suspension (1 mL) were placed in dialysis bags (Visking dialysis tubes, 20/30) and suspended in 50 mL of water−ethanol solution (4:1, pH 6) at 37 °C under gentle magnetic stirring. At predetermined time intervals, 2 mL of the medium was withdrawn while maintaining the volume of the receptor compartment with an equal volume of fresh water−ethanol solution. The total number of active molecules in the withdrawn samples was determined using ultraviolet−visible (UV−vis) spectrometry, at the corresponding drug wavelengths. All experimental procedures were repeated three times, and the results were in agreement within ±4% standard error. Release of the active molecules was also investigated in the same way. 2.7. Cellular Uptake of Ligand-Conjugated Niosomes. Studies on the uptake of the niosomal formulations were conducted in MCF-7, MDA-MB-231, and MCF-10 cells plated on coverslips. Different types of formulations were tested in this assay, as reported in Table 1.
quercetin as single agents or co-encapsulated. The niosomes were stable over the long term; no sedimentation, creaming, or flocculation was observed. The size and PDI did not change up to 6 months. The size and particle distribution of the formulations were measured using DLS, and the hydrodynamic diameters obtained and PDI are shown in Table 2. The stability of the Table 2. Hydrodynamic Diameters, PDI, Entrapment Efficiency, and Drug Moles/mL of Loaded Vesicular Systems, at 25 °Ca label
Table 1. Details of Tested Ligand-Conjugated Niosomes for Cellular Uptake label L64ox Tf-L64ox FA-L64ox Tf-FA-L64ox
formulation L64ox-Chol L64ox-Chol L64ox-Chol L64ox-Chol
8:2 8:2 8:2 8:2
rhodamine rhodamine rhodamine rhodamine
± ± ± ±
450 371 429 346
13 10 12 15
0.247 0.253 0.234 0.249
Q-L64ox D-Q-L64ox
437 ± 12 380 ± 13
0.281 0.298
E% 31.8 ± 3.1 12.5 ± 1.8 D 9.4 ± 0.8 C 13.5 ± 1.2 33.3 ± 3.0 D 13.2 ± 1.2 Q 33.6 ± 2.8
drug moles (mL) 1.06 1.08 3.28 1.17 3.57 4.50 3.62
× × × × × × ×
10−7 10−7 10−8 10−7 10−7 10−8 10−7
Values represent mean ± SD (n = 3). D = doxorubicin, C = curcumin, Q = quercetin. a
All formulations contained rhodamine. The cells were washed with phosphate-buffered saline (PBS) followed by incubation with the niosomal formulations at a concentration of 10 μM for 24 h at 37 °C. After incubation, the cells were washed three times with ice-cold PBS and fixed with 2% formaldehyde for 30 min at room temperature. Acridine orange staining was used for nuclei detection. Fluorescence was photographed with a Leica TCS SP2 confocal laser scanning microscope at ×400 magnification. The fluorophores were imaged separately to ensure that there was no excitation/emission wavelength overlap. 2.8. Antitumoral Activity of Drug-Loaded Multifunctional Niosomes. The effect of all drug-loaded niosomes was evaluated in MCF-7, MDA-MB-231, and MCF-10 cells using an MTT dye test (cell viability). The cells were seeded at a density of 1 × 104 cells in 96-well tissue-culture plates for 24 h at 37 °C and 5% CO2 to allow the adhesion of the cells. After 24 h incubation, the culture medium was replaced with cs-FBS, and the niosomal formulations were added at 10 μM. The toxicity experiments were carried out at 96 h of incubation. At the end of the incubation time, 50 μL of MTT tetrazolium salt (5 mg/mL dissolved in serum-free medium, SFM) was added to each well, and the cells were incubated at 37 °C and 5% CO2 for additional 4 h to allow the formation of violet formazan crystals. Dimethyl sulfoxide (DMSO) (150 μL) was added to solubilize the formazan crystals in the cells. The absorbance was measured with the Multiskan EX (Thermo Scientific) at a test wavelength of 570 nm, with a reference wavelength of 690 nm. The percentage of viable cells, directly proportional to the amount of formazan crystals formed, was calculated using the following equation % cell viability = AT/AU × 100
PDI
L64ox D-L64ox C-L64ox D-C-L64ox
ligand transferrin folic acid transferrin and folic acid
diameter (nm)
niosomes was not altered after drug encapsulation (alone or in combination); this was due to the ionic association between the hydroxylic groups of surfactants and the drugs, ensuring a strong interaction with the neighboring molecules (essentially by hydrogen bonds). As shown, L64ox gives larger vesicles when no drug is encapsulated in the preparation (450 nm). The encapsulation of hydrophobic or hydrophilic compounds into vesicular systems was claimed to strongly affect the size of the niosomes because of their affinity for the niosomal bilayers that influence the drugs partition therein. In our case, diameters decreased up to 371 and 429 nm in the case of D-L64ox and C-L64ox samples, respectively. When doxorubicin and curcumin were co-encapsulated, vesicles with a smaller hydrodynamic diameter of 346 nm were obtained. The same behavior was achieved with quercetin: in this case, the reduction in vesicle size was smaller with respect to that obtained in the presence of curcumin. These results may be due to the different interactions occurring between hydrophobic and hydrophilic molecules with colloidal systems. Several works reported that the diameter of the vesicles decreases when a hydrophilic drug is encapsulated; this is ascribed to the electrostatic attractions between the drug and the vesicular bilayer, leading to an increase in vesicular cohesion.17 Doxorubicin is an amphiphilic molecule possessing an amino sugar moiety bound at the C7 site, resulting in an optimal incorporation in the aqueous core of the niosomes because of the attractive interactions between its hydroxylic groups and the niosomal matrix that is rich in PEO−PPO groups.18 In the case of lipophilic compounds, they were completely entrapped in the bilayer, contributing and competing with the surfactant molecules in the formation of the vesicles bilayer, and they may reduce their vesicles size.19 In fact, hydrophobic compounds were claimed to lead to H bonding between their hydroxyl groups and niosomal matrices, resulting in an increase in the niosomal cohesion and then a decrease in the diameter. The size of the niosomes after the conjugation of Tf and/or FA did not show significant alteration,
(1)
where AT is the absorbance of the treated cells and AU is the absorbance of the untreated cells. Values of cell viability are expressed as the mean of at least six different experiments ± SD. 2.9. Statistical Analysis of Data. Each datum point represents the mean ± SD of three different experiments. Data were analyzed by Student’s t test using the GraphPad Prism 4 software program. *p < 0.05 was considered as statistically significant.
3. RESULTS AND DISCUSSION As reported previously, L64ox and cholesterol in a molar ratio of 4:1 were able to produce stable niosomes. All niosomal vesicles were found to be opalescent and homogeneous both in the absence and presence of doxorubicin and curcumin and C
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Figure 1. Cumulative percentage of released drugs versus time at 37 °C: (■) curcumin in C-L64ox; (◆) doxorubicin in D-L64ox; (▲) curcumin in D-C-L64ox; (●) doxorubicin in D-C-L64ox (mean ± SD; n = 3).
been shown to maintain desirable, synergistic drug ratios as well as to coordinate the release of the encapsulated drug combinations to achieve increased antitumor activity.20 Unfortunately, the stability of long-circulating carriers may not always be favorable for drug delivery, whereby these devices entrap high amounts of chemotherapics and accumulate inside the tumor, but they may not be able to release the drug easily to kill malignant cells.21 For this reason, we tested the ability of our multifunctional niosomes to release the entrapped drugs, simulating parenteral administration. The drug release kinetics from all carriers were investigated over a time period of 24 h in a water−ethanol solution at pH 6 and 37 °C, under experimental conditions similar to those of a tumor. Doxorubicin and curcumin release profiles from single-drugloaded and dual-drug-loaded formulations showed a biphasic release pattern (Figure 1): an initial burst release lasting few hours followed by a phase of slow release. More specifically, the release of doxorubicin from D-L64ox showed a release percentage of 29% after 1 h, whereas about 66 and 81% were released after 4 and 24 h, respectively. The cumulative drug percentages released from D-C-L64ox were found to be 10, 80, and 94% at the corresponding times. Curcumin releases from single-drug-loaded and dual-drug-loaded samples differed greatly: approximately 8, 22, and 50% of the antioxidant was released after 1, 4, and 24 h from C-L64ox, whereas the percentages increase up to 17, 62, and 90% in the case of D-CL64ox. Apparently, this means that the co-encapsulation increased the amount of drug released from the niosomal formulations. Anyway, we must consider the entrapment efficiency values of all samples. In the case of curcumin, because E% was very similar (12 and 13% for C-L64ox and D-C-L64ox, respectively), the previous hypothesis was valid. For doxorubicin-based samples, because the encapsulated quantities were significantly different (31 and 9%, for D-L64ox and D-C-L64ox, respectively), higher cumulative amounts of released drug were obtained by single-drug-loaded vesicles with respect to the D-C-L64ox sample (8.58 × 10−8 and 3.08 × 10−8 moles, respectively). The total amount of drugs that was released from the D-C-L64ox formulation was higher (1.35 × 10−7 moles). The release of drugs from niosomes is mostly driven by diffusion mechanisms across the bilayer, whereby the movement of such molecules is dependent upon the composition of the niosomal membrane, its fluidity, and the interaction
indicating that the coupling process did not negatively affect the characteristic of the vesicles as reported previously. Another important parameter for a possible application of the colloidal drug delivery system is the loading capacity of the drug within the carriers: the surfactant vesicle encapsulation efficiency is a product of the stability of the dispersion, the ionization state of the drugs, the method and factors governing vesicle loading, and the intrinsic properties of the vesicle.17 A major advantage of niosomes is their ability to simultaneously contain both water- and lipid-soluble drugs. As reported in Table 2, our carriers showed a similar affinity for doxorubicin and curcumin as loaded molecules, corresponding to 1.06 × 10−7 and 1.08 × 10−7 moles, respectively, entrapped in the vesicles, whereas a higher E% value was obtained in the case of quercetin (3.57 × 10−7 moles). This is an unexpected result because curcumin and quercetin are similar in terms of their molecular weight, solubility, pKa, and all physicochemical properties. The co-encapsulation of doxorubicin and the antioxidant led to a slight increase in the total amount of drug present in the vesicles, and more interestingly, the variation in E% of each molecule follows a particular behavior. In the case of D-CL64ox, doxorubicin’s E% decreased from 31 to 9%, whereas curcumin remained constant. The same trend was observed for D-Q-L64ox: entrapped moles of doxorubicin decreased from 1.06 × 10−7 to 4.50 × 10−8 moles, whereas quercetin E% kept the same value. Such peculiar behavior has been reported in the literature by our research group for Tween 60-based niosomes co-encapsulating antioxidants.21 As Tween 60 is a nonionic surfactant rich in PEO portions like Pluronic L64, we can postulate again that the competition between surfactants and lipophilic antioxidants for the niosomal bilayer constitution determines a maximum and optimal amount of drug that can be accommodated into the bilayer, without affecting niosome formation and regardless of the addition of hydrophilic molecules. In addition, it seemed that doxorubicin did not have much affinity for the niosomal matrix constituted by the surfactant and curcumin or quercetin. As reported in several studies,18 exceeding the limiting ratio of doxorubicin in the vesicles did not result in a higher loading and was therefore useless. 3.1. In Vitro Release Study. Extended drug retention and retarded release are important items to achieve in order to develop an optimal drug delivery system.17 Niosomes have D
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Langmuir between the drug and L64. From these results, we can assume that the observed initial burst release of doxorubicin might be due to drug molecules that were adsorbed onto the vesicles surface or loosely incorporated into the aqueous core, independent of the bilayer composition. Because it forms part of the bilayer, it is more difficult for curcumin to leave it, but its co-encapsulation with a hydrophilic molecule such as doxorubicin, which easily passes across the bilayer and with which curcumin possesses hydrophilic/hydrophobic interactions, could represent an assistance for its release, allowing an increase in curcumin efflux from the niosomes. Cumulative doxorubicin and quercetin release profiles from single-drug-loaded and dual-drug-loaded formulations are illustrated in Figure 2.
Figure 3. Effect of niosomal formulations on normal and BC cell viability. The cells were incubated with the niosomal formulations at a concentration of 10 μM for 96 h and then analyzed for cell survival, using the MTT assay. Each value represents the mean ± SD of three independent experiments, performed with quadruplicate cultures. The results are expressed as the percentage of the control (assumed to be 100%).
alized with Tf and/or FA. Tf is a plasma glycoprotein used by cells to acquire iron via a process involving receptor endocytosis. There is evidence for overexpression of the receptor for Tf on tumor cells, including prostate and BC cells.22 Folate, also known as pteroylglutamate, is a nonimmunogenic water-soluble B vitamin that is critical for DNA synthesis, methylation, and repair (folate is used to synthesize thymine). The folate receptors, glycosylphosphatidylinositolanchored cell surface receptors, are overexpressed in a vast majority of cancer tissues, whereas their expression is limited in healthy tissues and organs. Folate receptors are highly expressed in ovarian, cervical, brain, lung, kidney, colorectal, and breast tumors.23 After Tf or folate attaches to the specific receptors located within caveolae, it is internalized through the endocytotic pathway. The variation of the pH in the endosome induces the Tf or folate to dissociate from the receptors, which can be recycled back to the membrane.24 This mechanism is exploited to efficiently release drugs into cancer cells. To investigate whether the functionalized niosomes could be internalized by the cells, confocal laser scanning microscopy analysis was conducted in all cell lines used. Our results showed a consistent positive distribution of all formulations in the BC cells (Figure 4). On the contrary, in MCF-10A normal cells, the uptake of the niosomes was less efficient. 3.4. Antitumoral Effect of Functionalized Niosomes Loaded with Doxorubicin and Curcumin. After uptake of all of the formulations tested, we assessed whether they could represent a reliable tool for drug-targeting purposes. Indeed, we decided to take advantage of the increased expression of Tf and FA receptors in cancer cells for a targeted chemotherapeutic drug effect by niosomal formulations. Doxorubicin is a first-line chemotherapeutic drug for BC, although it has limits in clinical use because of the development of resistance by tumor cells and toxicity for healthy tissues.25 Thus, new therapeutic combinations have been developed to improve doxorubicin effects at lower concentrations of the drug to ensure protective effects for nontumoral cells.26 Curcumin may represent such an agent. The anticancer potential of curcumin stems from its ability to suppress proliferation; modulate transcription factors (e.g., NF-kappa B, AP-1, and Egr-1); downregulate the expression of COX2, LOX, iNOS, MMP-9, uPA, cytokines (TNF, IL-1, and IL-6), growth factor receptors (EGFR and
Figure 2. Cumulative percentage of released drugs versus time at 37 °C: (◆) quercetin in Q-L64ox; (■) doxorubicin in D-L64ox; (▲) quercetin D-Q-L64ox; (●) doxorubicin in D-Q-L64ox (mean ± SD; n = 3).
As reported, the results were not in line with those obtained with curcumin-based formulations. The amount of drug released from single-drug-loaded formulations was always higher than that obtained from D-Q-L64ox, in terms of both percentages and moles. In particular, 8.48 × 10−8 and 2.49 × 10−8 moles of doxorubicin were released from D-L64ox and DQ-L64ox samples, whereas the amount of quercetin released decreased from single-drug-loaded formulations to dual-drugloaded formulations. Probably, the physicochemical interactions between quercetin and L64 resulted in a bilayer that was more cohesive and less permeable to doxorubicin. Furthermore, no interactions favoring the chemotherapic efflux from the aqueous core occurred between doxorubicin and quercetin. Considering the physicochemical characterization and the in vitro release tests, we decided to carry out biological studies only on the formulations containing doxorubicin and curcumin. 3.2. In Vitro Evaluation of L64ox Toxicity. For the experiments, we selected two human breast cancer cell lines, MCF-7 cells that are less aggressive and MDA-MB-231 cells that are highly aggressive, and a breast-derived cell line, MCF10A cells that are nontumoral. All niosomal formulations were preliminarily tested for toxicity against BC and normal cells. We studied the survival by MTT assay in our cellular models. As similarly reported,12 L64 did not affect cell survival and all L64derived formulations tested were clearly nontoxic at the concentration of 10 μM at 96 h (Figure 3). For this reason, in all experimental procedures, we used this nontoxic amount of niosomes. 3.3. Evaluation of Niosome Uptake Using Fluorescence Microscopy Analysis. The niosomes were functionE
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Figure 4. Confocal microscopic analysis of uptake of the niosomal formulations in BC and normal cells. Intracellular localization of the niosomal formulations in MCF-7, MDA-MB-231, and MCF-10A cells. The cells were treated with 10 μM niosomes for 24 h. The cell nuclei were counterstained with acridine orange for 10 min. The fluorescence of rhodamine was excited at 555 nm and detected at a wavelength of 580 nm; for acridine orange, the fluorescence was excited at 460 nm and detected at a wavelength of 650 nm. The images are representative of three different experiments.
HER2), cell cycle proteins p21 and cyclin D, and cell surface adhesion molecules; and inhibit the activity of c-Jun N-terminal kinase, protein tyrosine kinases, and protein serine/threonine kinases of a wide variety of tumor cells and cancer stem cells.27 Preliminary experiments were performed on MCF-7 and MDA-MB-231 cells treated with D-C-L64ox and D-L64ox, respectively, to evaluate cytotoxicity. Results showed enhanced cytotoxicity (4 times higher) in cells treated with doxorubicin/ curcumin niosomes, suggesting synergistic effects of this combination in the niosomal carriers. Our results were consistent with the previous reports of enhanced efficacy of doxorubicin in nanodevices, which might be related to its passive or active tumor-targeting abilities resulting in reduced systemic toxicity and overcoming drug resistance.28,29 On this basis, only multitarget niosomes containing a combination of doxorubicin and curcumin were tested for effects on cell viability. Thus, the MTT assay was performed in BC and normal cells after 96 h of exposure to the niosomal formulations at a concentration of 10 μM. Our results showed that the niosomes significantly decreased cell survival in both BC cell lines more efficiently with respect to that caused by comparable amounts of free doxorubicin and curcumin (Figure 5). On the contrary, normal breast cells showed no difference between the growth inhibition observed in niosomes loaded with doxorubicin and curcumin and free-drug-treated samples with respect to control.
Figure 5. Effect of doxorubicin/curcumin-loaded niosomal formulations on breast cell viability. Cells were treated with 10 μM of the different niosomal formulations, free doxorubicin and curcumin, or left untreated (control, C) for 96 h. Cell viability was assayed by MTT assay. The results are expressed as mean ± SD of three experiments, each performed in quadruplicate, and is expressed as the percentage of the control (assumed to be 100%). *p < 0.05 vs control; *p < 0.05 vs free doxorubicin/curcumin-treated cells.
the surface of the niosomes was generated by amide bonds between the −COOH groups of L64ox and the −NH2 group of the target moieties, as shown schematically in Figure 6. In the case of FA, the condensation reaction may proceed in two different ways. The involvement of the primary amine makes the pteridine group no longer recognized by the receptor,30 whereas if the reaction involves the secondary amine,31 such a group is free to interact with FA-R, and the carrier can be effectively internalized by the cells. This might explain the lower effectiveness of FA as a target molecule. This disadvantage could be avoided by choosing a different conjugation strategy (i.e., exploiting the −COOH group of FA and introducing amino functionalities on the surface of the niosomes), but the main objective of our work was to optimize
4. GENERAL REMARKS It has to be noted that among the formulations tested, the most efficient reduction in cell viability was observed in BC cells exposed to the Tf-L64ox formulation, whereas the worst performance was achieved by the FA-L64ox sample. The combined formulation gave intermediate results. These results may be explained as follows: The conjugation of Tf and FA to F
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231 malignant cells with respect to that of the nonconjugated samples. Promising results were provided by in vitro evaluations of the anticancer activity. The collected data demonstrated the strong potential of niosomes loaded with doxorubicin and curcumin combination as devices useful in breast tumor treatments with respect to the formulations containing only doxorubicin, thereby confirming the synergistic effects of this combination. Unexpectedly, the most efficient reduction in cell viability was observed in BC cells exposed to Tf-L64ox, followed by Tf-FA-L64ox and FA-L64ox samples. These results may be due to the condensation reaction between the −COOH groups of L64ox and the two different amino groups of FA, one of which may reduce the ligand−receptor recognition required for cellular uptake. These features hold great promise for the development of multifunctional devices that combine several advantages, such as biocompatibility, stealth properties, loading capability, and active targeting, in moving toward the development of more specific and efficient carriers for personalized tumoral therapy.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: 0039 0984 493173. Fax: 0039 0984 493298. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank MIUR, the Italian Ministry for University for financial support (Grant # EX-60%). Moreover, the project has been co-funded with support from the Commission European Social Fund and Region of Calabria (Italy).
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Figure 6. Schematic representation of the condensation reaction between target moieties and niosomes.
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5. CONCLUSION The niosomes obtained from Pluronic L64ox, coupled with Tf and FA and their combination, have been used as multifunctional devices for controlled release of doxorubicin. These systems possess coordinated action of stealth, targeting, and internalizing in tumor cells to achieve intracellular drug delivery. Also co-loading of doxorubicin with curcumin or quercetin was used as a further valid strategy to overcome the problems related to traditional chemotherapy. The drug-loaded vesicles were around 400 nm in diameter, and they were small enough to extravasate from the blood into the tumor interstitial space. High vesicle stability was observed when dispersions were stored at 4 °C, with negligible degradation after 6 months. The niosomes demonstrated good capability in loading doxorubicin and curcumin/quercetin alone or in combination, depending on the drug’s affinity for the niosomal matrix. Drugs released from the vesicles were found to be controlled and sustained over 24 h, with a strong dependence on the copresence of loaded molecules. The methodology of vesicle conjugation with Tf was improved and also coupled to that of FA to give potential dual ligand-endowed niosomes, without additional toxicity. Indeed, the dual ligand-endowed niosomes showed improved cellular uptake into MCF-7 and MDA-MBG
DOI: 10.1021/acs.langmuir.6b02063 Langmuir XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.langmuir.6b02063 Langmuir XXXX, XXX, XXX−XXX
