Transferrin-Conjugated Pluronic Niosomes as a New Drug Delivery

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Transferrin-conjugated Pluronic niosomes as a new drug delivery system for anticancer therapy Lorena Tavano, Rita Muzzalupo, Loredana Mauro, Michele Pellegrino, Sebastiano Andò, and Nevio Picci Langmuir, Just Accepted Manuscript • DOI: 10.1021/la4021383 • Publication Date (Web): 16 Sep 2013 Downloaded from http://pubs.acs.org on September 21, 2013

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Graphical Abstract

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Transferrin-conjugated Pluronic niosomes as a new drug delivery system for anticancer therapy Lorena Tavanoa,b, Rita Muzzalupoa*, Loredana Mauroa, Michele Pellegrinoa, Sebastiano Andòa, Nevio Piccia a

Dipartimento di Farmacia e Scienze della Salute e della Nutrizione, Università della Calabria,

Edificio Polifunzionale, 87036 Arcavacata di Rende, Cosenza, Italia b

Dipartimento di Ingegneria Informatica, Modellistica, Elettronica e Sistemistica, Università della

Calabria, Via P. Bucci Cubo 39/C, 87036 Arcavacata di Rende, Cosenza, Italia

Corresponding author: [email protected]


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Abstract An efficient tumor targeted-niosomal delivery system for the vehiculation of doxorubicin hydrochloride as an anticancer agent was designed. Niosomes were prepared from a mixture of an opportunely modified Pluronic L64 surfactant and cholesterol as membrane additive and characterized in terms of sizes and related distribution functions and drug entrapment efficiency. After the preparation, Transferrin was conjugated to niosomes in order to produce Tf-niosomes and citotoxicity of the final formulation was studied. The specific uptake of Tf-niosomes into cells was evaluated incubating MCF-7 and MDA-MB-231 cells with fluorescently Rhodamine loaded Tfniosomes for various times and concentration intervals and further investigated by fluorescence microscopy. Results showed that doxorubicin can be easily encapsulated into niosomes, that are regular and spherical in shape. Moreover, Tf-conjugate niosomes demonstrated far greater extent of cellular uptake to MCF-7 and MDA-MB-231 cells, suggesting that they were mainly taken up by Tf-R mediated endocytosis. Doxorubicin-loaded niosomes anticancer activity was also achieved against MCF-7 and MDA-MB-231 tumoral cell lines and a significant reduction of viability in a dose- and time-related manner was observed. Finally our formulation could be potentially useful as target doxorubicin delivery system in anticancer therapy.

Keywords: Niosomes, Transferrin, Doxorubicin, Target, MCF-7 and MDA-MB-231 cell lines.


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1.Introduction Cancer therapy evolved from traditional routes in the last decades, because of the need to improve the therapeutic index of chemotherapy drugs [1]. Even if cancer cells are inherently more vulnerable than normal cells to the effect of chemotherapy agents, the drugs are nonselective and can cause injury to normal tissues [2]. Nowadays attempts are focused on efforts to kill cancer cells by more specific targeting while sparing normal cells. To achieve these goals, the idea is the development of novel carriers for both existing and new drugs and defining better therapeutic targets relative to the molecular changes in the cancer cells and their vasculature [3,4]. In general, solid tumors show hypervascular permeability and impaired lymphatic drainage [5]. Colloidal drug delivery systems can significantly accumulate in tumor by an active or passive process. Passive delivery refers to carriers transporting through leaky tumor capillary fenestrations into the tumor interstitium and cells by passive diffusion or convection [6], while active targeting involves drug delivery to a specific site based on molecular recognition [7]. One such approach is to couple a ligand (or antibody) to a delivery system so that the ligand can interact with its receptor (or antigen) at the target cell site. Then the therapeutic drug can be released from the carrier to initiate cytotoxic action on the target cell [8]. The vascular endothelium constitutes the inner layer of all blood vessels and has a large number of functions, among which to serve as a selective barrier for exchanging substances between blood and tissues [9]. On its surfaces, chemically differentiated microdomains that express receptors involved in specific transport of macromolecules occur [10] and among these, Transferrin receptors (Tf-R) localized in clathrin-coated pits and involved in receptor-mediated endocytosis and transcytosis of transferrin, were reported [11]. Human Transferrin (Tf) is a serum glycoprotein that transports ferric ions. Tf-R are over-expressed and feature a high turnover rate on tumor cells and showed a density of 10,000-100,000 molecules per cell commonly found on cancerian cells, while cell types with normal phenotype express Tf-R at low undetectable levels [12,13]. In addition, the high specificity of endocytic uptake of Tf by Tf-R has made it a subject of interest for targeted drug delivery via parenteral administration and for the delivery of Tf-R targeted drug conjugates across epithelial barriers [11]. Therefore, Tf-R is considered an effective successful target for specific drug delivery into tumor cells [14]. Many anti-cancer agents have been considered for conjugation to Tf by varying methods, including direct chemical linkage [15,16], encapsulation in vesicles [17,18], nanoparticles [19,20], dendrimers [21] and other kind of aggregates [22]. Conjugation of these drugs to Tf has the dual benefits of reducing drug toxicity in undesired tissues and increasing the targeting efficiency to the cancerous cells: a special note is that conjugation to Tf significantly enhances the effectiveness of these agents in many multi-drug resistant cell lines [23,24]. ACS Paragon Plus Environment

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Vesicular drug delivery systems coupled with Tf have been extensively studied and vesicles drug encapsulation has been demonstrated to allow specific tumors delivery of anticancer agents in vivo, reducing toxicity associated with these agents by altering the distribution of the drug [25]. Unfortunately, being colloidal systems, these vesicles could rapidly be cleared by the cells of the mononuclear phagocytes system (MPS) when given intravenously [26]. In this light, the combination of active targeting, based on the use of Tf as ligand, and passive targeting, based on the accumulation of delivery systems due to the enhanced retention, should provide a tumor-selective targeting strategy [27]. For example, the modification of the vesicles composition, incorporating polyethylene glycol, to make stealth vesicle drug carriers can prolong their residence time in the blood, increase their extravasation and accumulation in the pathological sites, like tumor, which are known to exhibit leaky vasculature [28, 29]. The dynamic PEO chains are claimed to prevent particles opsonization and render them ‘unrecognizable’ by reticulo-endothelial system (RES) and macrophages, and offer an interesting alternative to reduce significant hepatic uptake [30]. Recently novel target liposomes bearing polyethylene glycol (PEG), in which Tf is coupled to carrier have been described. The residence time of Tf-PEG-liposomes in the circulation is prolonged and reticulo-endothelial system (RES) uptake is low in tumor-bearing mice, resulting in enhanced extravasation of the carriers into solid tumors [14]. To our knowledge, there are no published data on the design of niosomes prepared from Pluronic surfactants, copolymers consisting of ethylene oxide (EO) and propylene oxide (PO) blocks arranged in a triblock structure, coupled with Tf and loading Doxorubicin, useful in anticancer theraphy. Our hypothesis that the coupling on the vesicles surface of Transferrin (which can bind specifically and with high affinity to its receptor on the cancerian cell membrane), is an efficient way for specific binding and internalization of drugs into the malignant cells. So, the design of this novel carrier could be one of the ideal solutions in the delivery of doxorubicin to tumors. To achieve this goal, we decided to change the Pluronic surfactant polar head by the oxidation of the terminal -CH2OH into -COOH, to link the niosomal carrier to the free amino groups of Tf by an amide bond. The advantage is that it is not necessary to modify the protein before coupling. Niosomes were prepared from oxidate Pluronic L64 (L64ox) surfactant and cholesterol as membrane additive. The obtained vesicles were characterized by their size, morphology and their Doxorubicin entrapment efficiency. Citotoxicity of both surfactant solution and empty niosomal system was also evaluated on MCF-7 and MDA-MB-231 human breast cancer cell lines. After this, Tf was coupled to carboxylic group of L64ox, in the presence of N-(3-dimethylaminopropyl)-Nethylcarbodiimide hydrochloride (EDC). The uptake levels of Transferrin-conjugated niosomes on


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MCF-7 and MDA-MB-231 cells were evaluated and in vitro anticancer activity was achieved to evaluate the potential of our vesicles as target drug delivery systems.

2.Materials and methods Chemicals Pluronic L64 was kindly donated by BASF (Mount Olive, NJ, USA). All reagents were purchased from Sigma-Aldrich (Milano, Italy). Chromium trioxide (CrO3), sulfuric acid (H2SO4) potassium hydroxide (KOH), and phenolphthalein indicator were used with no further purifications. Cholesterol,

Rhodamine,

Doxorubicin

hydrochloride,

N-(3-dimethylaminopropyl)-N-

ethylcarbodiimide hydrochloride (EDC), Transferrin and Sepharose CL-4B gel were purchased from Sigma-Aldrich (Sigma-Aldrich, Milan, Italy). Centrisart-10 concentrators (molecular weight cut off 10 kDa) were purchased from Vivascience, Hannover, Germany. The solvents are of high performance liquid chromatography grade. 2.1 Synthesis of L64ox Several studies reported that Pluronic surfactants themselves interact with multidrug-resistant cancer tumors, resulting in drastic sensitization of these tumors with respect to various anticancer agents [31-37]. The L64ox was synthesized according the procedure shown in Figure 1 and reported in our previous study [38]. Figure 1 should be inserted here

In brief, weighted amount of Pluronic surfactant were placed in acetone and the mixture was heated to obtain a clear and homogeneous solution. The solution was allowed to achieve room temperature and after 5.9 mL of Jones’s Reagent (containing 0.02 M CrO3) was added by stirring for 16 h. Finally, the reaction was quenched by adding 1.7 mL of isopropyl alcohol. To remove the chromium salts 1 g of activated charcoal was added to the suspension and stirred for 2 h. The suspension was filtered to obtain a colorless, clear acetone solution. At the end it was dried under reduced pressure for 24 h. The obtained L64ox dicarboxylic acid was quite viscous, ivory-like liquids and the yield were about 84%. Estimates of the number of acid groups were performed dissolving 1 g of L64ox in 10 mL of distilled water. The solution was titrated against 0.01 N KOH solution, in presence of


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phenolphthalein as indicator. The acid value was calculated in terms of mg of KOH required to neutralize 1 g of dicarboxylic acid. The percentage of carboxylic groups for L64ox was about 95%.

2.2 Preparation of niosomes Multilamellar niosomal vesicles (MLVs) were prepared by the hydration of lipidic film method [39]. Accurately weighed amounts of L64ox and cholesterol (0.04 mmol of L64ox, 0.01 mmol of cholesterol) were dissolved in chloroform in a round-bottom flask. After mixing, solvent was evaporated under reduced pressure and constant rotation to form a thin lipid film. The lipid film was then hydrated with 5 mL of distilled water (empty niosomes) or Rhodamine (1.28x10-3 M) or Doxorubicin (1.45x10-3 M) solution at 20°C for 30 minutes to form large multilamellar vesicles (MLV) at 10 mM total lipid concentration. After preparation, the dispersion was left to equilibrate at 25°C overnight, to allow complete annealing and partitioning of the drug between the lipid bilayer and the aqueous phase. Small unilamellar vesicles (SUV) were prepared starting from MLV by sonication in an ultrasonic bath for 30 min at 20°C. The purification of niosomes was carried out by exhaustive dialysis for 4 h, using Visking tubing (20/30), manipulated before use in according to Fenton’s method [40]. After purification, niosomes were stored at 4°C in the dark until used in subsequent experiments. 2.3. Characterization of niosomes 2.3.1. Morphology The morphology of hydrated niosome dispersions was examined by Transmission Electron Microscopy (TEM). A drop of dispersion was stratified onto a carbon-coated copper grid and left to adhere on the carbon substrate for about 1 min. The dispersion in excess was removed by a piece of filter paper. A drop of 2% phosphotungstic acid solution was stratified and, again, the solution in excess was removed by a tip of filter paper. The sample was air-dried and observed under a ZEISS EM 10 electron microscope at an accelerating voltage of 80 kV.

2.3.2. Size and distribution The niosomes size and distribution were determined by dynamic light scattering (DLS) analysis using 90 Plus Particle Size Analyzer, Brookhaven Instruments Corporation, New York, USA) at 25.0±0.1 °C by measuring the autocorrelation function at 90°. The laser was operating at 658 nm. The distribution size was directly obtained from the instrument fitting data by the inverse “Laplace transformation” method and by Contin [41]. The Polydispersity Index (P.I.) was used as a measure of the size distribution. P.I. less than 0.3 indicates a homogenous and monodisperse population in the case of colloidal systems [42]. All the samples were analyzed 24 h after their preparation and ACS Paragon Plus Environment

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before each following study. They were diluted with distilled water before the measurements. In particular 50 µL of each vesicle dispersion was diluted to 10 ml with distilled water. Each sample was measured three times and results are expressed as mean ±standard deviation.

2.3.3. Entrapment efficiency Drug encapsulation efficiency was determined using the dialysis technique for separating the nonentrapped drug from niosomes [43]. According to this thecnical, 3 mL of Doxorubicin-loaded niosomal dispersion were dropped into a dialysis bag (Spectra/Por MW cut-off 12,000 Canada) immersed in 100 mL of distilled water and magnetically stirred. Samples were dialyzed for 30 min each time and the dialysis was complete when no drug was detectable in the recipient solution. The percent of encapsulation efficiency (E%) was expressed as the percentage of the drug entrapped into niosomes referred to the initial amount of drug that is present in the non-dialyzed sample. It was determined by diluting 1 mL of dialyzed and 1 mL of non-dialyzed niosomes in 25 mL of methanol. This procedure is necessary to break the niosomal membrane. After, the measurement of absorbance of these solutions was performed at 480 nm. Absorption spectra were recorded with a UV±VIS JASCO V-530 spectrometer using 1 cm quartz cells. Each experiment was carried out in triplicate and results are expressed as mean ±standard deviation. 2.4 Preparation of Transferrin-conjugated niosomes Conjugation of Transferrin to the niosomes surfaces was achieved by adding 2 mg N-(3dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) per 1µmol of total surfactant/chol mixture and incubating the mixture for 6 h at room temperature. Excess EDC was removed by ultrafiltration using Centrisart-10 concentrators. In the next step, 125 µg Tf/mol lipid were added and incubated overnight at room temperature. Free Tf, was removed by passing the niosomes suspension through a Sepharose CL-4B gel column. In this method, the free amino groups of proteins are linked to the carboxylic carrier in the presence of EDC. The advantage is that it is not necessary to modify the protein before coupling [44]. 2.5 Cell culture MCF-7 and MDA-MB-231 cells, obtained from the American Type Culture Collection (Manassas, VA), were maintained in Dulbecco’s modified Eagle’s medium/Ham’s F-12 containing 5% fetal bovine serum and supplemented with 1% L-glutamine and 1% penicillin/streptomycin (Sigma, Milan, Italy). The cells were cultured in serum-free medium (SFM) for at least 24 h before treatments.


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2.6 Cytotoxicity assay The cytotoxic effects of L64ox as monomer or in the form of niosomal formulation on MCF-7 and MDA-MB-231 cells were evaluated with the MTT dye test (cell viability). The cells were seeded at a density of 1x104 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 SFM and L64ox or L64ox/Chol were added at 0.1, 1 and 10 µM. The toxicity experiments were carried out at 24, 48 and 72 h of incubation. Untreated MCF-7 and MDA-MB-231 cells were used as control. At the end of the incubation time, 50 µl of MTT tetrazolium salt (5 mg/ml dissolved in SFM) were added to each well and MCF-7 cells were incubated at 37°C and 5% CO2 for additional 4 h, to allow the formation of violet formazan crystals. 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: % ⁄ 100

Eq.1

where AT is the absorbance of treated cells and AU is the absorbance of untreated cells. Values of cell viability are expressed as the mean of at least three different experiments ± S.D.

2.7. Cellular uptake of Tf-niosomes Studies of uptake of the niosomal formulations were achieved in MCF-7 and MDA-MB-231 cells plated on coverslips. Two different types of formulations were tested in this assay: plain niosomes without any functionalization (L64ox/Chol) and Tf-conjugated niosomes (L64ox/Chol-Tf). Both formulations contained Rhodamine phalloidin (Invitrogen, Milan, Italy). The cells were washed with PBS followed by incubation with the niosomal formulations at the concentration of 10 µM for 24 h at 37°C. After incubation, cells were washed three times with ice cold PBS and fixed with 2% formaldehyde for 30 minutes at room temperature. Acridine orange staining was used for nuclei detection. Fluorescence was photographed with a Leica TCS SP2 confocal laser scanning microscope at X400 magnification. The fluorophores were imaged separately to ensure there was no excitation/emission wavelength overlap.

2.8. Evaluation of Doxorubicin-loaded Tf-niosomes antitumoral activity


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The effect of Doxorubicin-loaded Tf-niosomes (L64ox/Chol-D-Tf) was evaluated in MCF-7 and MDA-MB-231cells seeded at a density of 1x104 cells in 96-well tissue culture plates by MTT assay (above described). The cells were treated with L64ox/Chol-Tf, doxorubicin or doxorubicin-loaded Tf-niosomes at the concentration of 0.1, 1 and 10 µM for 24, 48, 72 and 96 h. Values of cell mortality and cell viability are expressed as the mean of at least three different experiments ± S.D.

2.9. Statistical analysis Data are expressed as mean ± S.D. of at least three independent experiments. Statistical analysis was performed using Student’s t-test. P-values ≤ 0.05 were considered statistically significant. 3.Results Doxorubicin is one of the most widely used broad spectrum anticancer agents and it has been in clinical use against a wide range of human cancers for decades [45]. Nevertheless, a number of issues critical to the therapeutic success and safety of the drug, such as cardiotoxicity, drug resistance, and specificity remain to be improved [46]. One of the most attractive strategy is to reduce the toxic effects of the drug releasing these agents specifically to the defined target cells by vesicular delivery systems [47]. This was obtained by using a ligand coupled to the surface of vesicle, which could be actively taken up via a receptor-mediated endocytic pathway [48]. Under these lights we decided to use L64, one of Pluronic surfactants, to obtain Tf-conjugated vesicular systems called niosomes, for the vehiculation of Doxorubicin as an anticancer drug. Rhodamine was also encapsulate to verify and evaluate the vesicles uptake into the malignant cells and their effective potential as target drug delivery systems. Vesicles were prepared by the thin layer evaporation technique, by weighing 117±5 mg of L64ox in the ratio 4:1 with cholesterol as additive. The Doxorubicin solution used in the preparation of loaded vesicles was 1.45x10-3 M. The particle size is one of the most important properties of vesicular drug delivery system because particle size shows a great effect on drug release characteristics and drug distribution in different organs of the body, particularly, tumor tissues. It has been reported that solid tumors show hypervascular permeability and impaired lymphatic drainage and thanks to this, niosomes can significantly accumulate in tumors by “filtration” mechanism [49]. The mean diameters of empty or Doxorubicin-loaded niosomes before coupling of Tf, also with the P.I. and Doxorubicin E%, are given in Tables 1.

Table 1 should be inserted here


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As reported in our previous study, L64ox surfactant needs membrane additive to give niosomes [30]. The non-modified L64 surfactant was reported to be able to produces vesicles without the addition of membrane additives [38], but the increase of hydrophilicity achieved by the oxidation of the L64 surfactant polar head assess changes in physico-chemical properties of molecules. Some surfactants, in fact, can not assemble to form vesicles do to their critical packing parameter, i.e. relative space requirements of the hydrophobic and the hydrophilic parts of the amphiphiles. The incorporation of cholesterol into the surfactant aggregates leads to appropriate molecular geometry and hydrophobicity also changing the fluidity of the hydrophobic chains in the bilayer, thus promoting the formation of surfactant vesicles [50]. In our case this bilayer composition gave rise to vesicles stable at room temperature over 6 months. No sedimentation, creaming or flocculation can be inferred. As reported in literature, the encapsulation of drug into the niosomal vesicles affects the particle size of the vesicles [51]. As shown in Table 1 the diameter of Doxorubicin loaded niosomes decreased respect to the empty one (from 450 nm to 350 nm). This is due to the nature of the encapsulated molecule and to the electrostatical attractions between the drug and the vesicular bilayer, that leads to an increase of vesicles cohesion [52,53]. The size of the niosomes after conjugation of Tf did not show significant alteration as reported in Table 1, indicating that the coupling process did not negatively affected the characteristic of the vesicles as reported in several works [54]. P.I. ranged from 0.275 to 0.243 and the size of the vesicular formulation as measured by DLS, shows one narrow distribution, indicating that the vesicles population is relatively homogenous in size. Doxorubicin E% was found to be around 37%. This value was related to the chemical nature of the drug and its interactions with niosomal matrix at the pH value of the experimental conditions. Doxorubicin is an amphiphilic molecule possessing an amino sugar moiety bound at C7 site, that bears the positive electrostatic charge localized at protonated amino nitrogen [47]. As the pKa of Doxorubicin is 8.2, most of the drug molecules are protonated at pH 7.4 and might be localized into the hydrophilic reservoir of the vesicles and partially adsorbed in the L64ox shell through electrostatic interactions. Several workers also reported that a conformational modification was achieved by the Doxorubicibn through interactions with vesicles: the dihydroxyanthraquinone moiety undergoes inside the bilayer and a prevalence of the hydrophobic interactions drug-membrane as compared to the electrostatic ones was obtained [55]. These hypothesis could explain the results achieved in our case. In fact, L64ox contains -COOH groups and some of these, at pH 7.4, could be dissociated into carboxyl anion, resulting in ionic repulsions among them and, consequently, in the conformational stretching. Conversely Doxorubicin containes protonated amino group, that allow the drug to participate in such charge-charge interactions, and results in a drug entrapment efficiency value of about 37%. No longer could Doxorubicin be


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associate with the bilayer due to saturation. Several studies reported that exceeding the limiting ratio of Doxorubicin into the vesicles did not result in a higher loading and was therefore useless [56]. As shown in the TEM, (Fig.2) niosomes were spherical and homogeneous in shape and the size correlated well with the results of the laser diffraction particle size. No drug crystal was observed.

Figure 2 should be inserted here

In vitro evaluation of L64ox toxicity The cytotoxic effects of the niosomes based on commercial L64 were previously evaluated with trypan blue dye exclusion assay (cell mortality) and MTT dye test (cell viability) by some of us (43). For this reason we decided to evaluate if the chemical modification of the surfactant (L64ox) affected the cellular viability. In this light we studied the effect of L64ox in the form of simple surfactant solution and niosomal formulation (L64ox and L64ox/Chol, respectively) on the survival of MCF-7 and MDA-MB-231 human breast cancer cells only by MTT assay. As shown, the addition of L64ox surfactant or L64ox/Chol niosomal formulation at the concentration range from 0.1 µM to 10 µM for 24, 48 and 72 h to MCF-7 (Fig.3A) or MDA-MB-231 (Fig. 3B) cells had no effect on cell viability. These results demonstrated that L64ox, as monomer or in the form of niosomal formulation, did not induce cytotoxic effects on the human breast cancer cell lines tested.


Uptake of Tf-niosomes into human breast cancer cells The previously prepared formulations were made targeted coupling Transferrin to the surface of the niosomal vesicles. We have chosen Tf-R as target molecule because of its well-defined fate in the endocytic pathway. As reported, iron-saturated Tf binds to EC surface receptors (Tf-R) and the complex is internalized via clathrin-coated pits that carry the complex to the endosomes. [57] Within this compartment, the iron is dissociated from the complex and the apoTf-Tf-R is recycled back to the plasmalemma where apoTf is removed due to the instability of apoTf-Tf-R at neutral pH. The Tf-R may be recycled to undergo further cycles of endocytosis. One can take advantage of the continuous recycling of the Tf-R from the surface to the endosomal compartment of the cell, to


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make it an efficient tool for internalization of drug-containing liposomes bearing Tf on the surface [57]. Moreover, it has been reported that human Transferrin receptor 1 (Tf-R1) is expressed on malignant cells at levels several fold higher than those on normal cells and its expression can be correlated with tumor stage or cancer progression. For instance, Tf-R is a potentially rational target for drug delivery. Evidence for cellular internalization and visual indications of the distribution patterns of the various formulations was obtained by confocal laser scanning microscopy analysis (Fig. 4) performed in breast cancer cells treated with 10 µM for 24 h of L64ox/Chol (L64ox/Chol-R) or Tf-conjugated niosomes (L64ox/Chol-R-Tf). Our experiments demonstrated that L64ox/Chol-R-Tf entered MCF-7 and MDA-MB-231 cells and its cellular uptake was higher than the unmodified counterparts (L64ox/Chol-R) (Fig.4). L64ox/Chol was used as negative control. Moreover, in the confocal microscopy studies were observed any visual signs of cell damage. These results suggested that the Transferrin receptor-mediated uptake of niosomes may be considered a potentially promising drug delivery system for tumor treatments.


Evaluation of Doxorubicin-loaded Tf-niosomes antitumoral activity In order to evaluate the antitumoral effect of L64ox/Chol-Tf and L64ox/Chol-D-Tf formulations, MTT assay was carried out in MCF-7 and MDA-MB-231 cells exposed to niosomes at the concentration of 0.1, 1 and 10 µM for 24, 48, 72 and 96 h. No significant change in viability was observed in the breast cancer cell lines treated with increased concentrations of empty niosomes conjugated with transferrin alone (L64ox/Chol-Tf) (Fig.5). On the contrary, a significant reduction (*p

Transferrin-Conjugated Pluronic Niosomes as a New Drug Delivery

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