Ionic Surfactants for the Formulation of

May 15, 2014 - Nanostructured lipid carriers are colloidal particles of a binary mixture of .... in NLCs and detected weight of CMT-8 in supernatant, ...
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Mixture of Nonionic/Ionic Surfactants for the Formulation of Nanostructured Lipid Carriers: Effects on Physical Properties Shuangni Zhao,† Xiaomin Yang,† Vasil M. Garamus,‡ Ulrich A. Handge,∥ Luthringer Bérengère,‡ Lin Zhao,§ Gabriele Salamon,‡ Regine Willumeit,‡ Aihua Zou,*,† and Saijun Fan§ †

Shanghai Key Laboratory of Functional Materials Chemistry, State Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East China University of Science and Technology, Shanghai 200237, P. R. China ‡ Helmholtz-Zentrum Geesthacht: Centre for Materials and Coastal Research, Institute of Materials Research, Max-Planck-Strasse 1, 21502 Geesthacht, Germany § School of Radiation Medicine and Protection, Soochow University Medical College, Suzhou 215123, P. R. China ∥ Helmholtz-Zentrum Geesthacht: Centre for Materials and Coastal Research, Institute of Polymer Research, Max-Planck-Strasse 1, 21502 Geesthacht, Germany ABSTRACT: The objective of the present work was to investigate the effects of the mixture of nonionic/ionic surfactants on nanostructured lipid carriers (NLCs). Nonionic surfactant (polyethylene−poly(propylene glycol), Pluronic F68) and ionic surfactant (octenylsuccinic acid modified gum arabic, GA-OSA) were chosen as emulsifier for NLCs. The NLCs systems, which were composed of lipid matrix, modified 4-dedimethylaminosancycline (CMT-8), and various emulsifier agents, were characterized with dynamic light scattering (DLS), high performance liquid chromatography (HPLC), transmission electron microscopy (TEM), small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), in vitro release, and phagocytosis assay. This mixture of nonionic/ionic surfactants showed significant effects on physical properties including particle size, polydispersity index (PDI), entrapment efficiency, and particle morphology. Compared with single stabilizer, this mixed nonionic/ionic surfactant system provided NLCs with better drug carrier properties including prolonged release profile and low phagocytosis by phagocyte. We expect that these explorations can provide a new strategy for the development of lipid nanoparticles as drug delivery.

1. INTRODUCTION Nanostructured lipid carriers are colloidal particles of a binary mixture of solid lipids and spatially different liquid lipids.1 As a biocompatible colloidal carrier, the most outstanding advantage of NLCs is the high drug loading and drug entrapment efficiency for lipophilic drug.2,3 However, nanostructured lipid carriers are heterogeneous systems and thermodynamically unstable, and therefore, they have a tendency to lose physical stability during storage.4 Consequently, stability studies are necessary before large-scale applications of NLCs. Recently, adding surfactants, screening raw materials and modifying preparation methods have been used for long-term stable NLCs. The shell of NLCs is composed of significant amount of surfactants, so the surface of NLCs can be modified by choosing emulsifier agents. Nonionic stabilizers are very popular for environmental awareness and their low sensitive interaction with physiological hurdles during drug delivery.5 Ethylene oxide-based surfactant is one of the nonionic surfactants; its poly(ethylene glycol) (PEG) avoids protein adsorption on NLCs, hence increasing circulation time of intravenously injected NLCs.6 However, use © 2014 American Chemical Society

of nonionic surfactants as only stabilizers usually requires relatively high amount for effective stabilization. Electrostatic stabilizers are known to impart favorable electrical potential to nanoparticles, leading to higher zeta potential (indicator of long stability). But higher concentrations of electrostatic stabilizers were reported to decrease the zeta potential of nanoparticles due to a reduction in the thickness of the diffuse layer.7 Consequently, electrostatic stabilizers are suggested to be used in combination with nonionic surfactants. The emulsification process can be separated into two stages: the initial pre-emulsion stage and the crystallization stage. According to the literature, ionic emulsifiers are more efficient during the initial pre-emulsion stage.8 Compared with nonionic stabilizers, they can more spontaneously and faster act as emulsifiers. In general, some shear forces and enough amounts of stabilizers may increase the process efficiency. During the second stage the coverage of the hydrophobic crystalline Received: May 16, 2013 Revised: May 15, 2014 Published: May 15, 2014 6920

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Figure 1. Chemical structure of Pluronic F68 and GA-OSA. (a) Pluronic F68. (b) GA-OSA, A = arabinosyl; ● = 3-linked Galp (Galp attached); ○ = 6-linked Galp (Galp or Glcp attached) or end-group; R1 = Rha → 4GlcA (Rha occasionally absent, or replaced by Me, or by Araf); R2 = Gal → 3 Ara; R3 = Ara → 3 Ara → 3 Ara...18,19 China); capric/caprylic triglycerides (MCT, Aladdin Chemical Reagent Co. Ltd., China); octenylsuccinic acid modified gum Arabic (GA-OSA, Guangzhou TIC GUMS, China); polyethylene−poly(propylene glycol) (Pluronic F68, Adamas Reagent Co. Ltd., China); trehalose (Aladdin Chemical Reagent Co. Ltd., China); 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, China); and freshly prepared distilled water were used. 2.2. Preparation of CMT-8 Loaded Nanostructured Lipid Carriers (CMT-8/NLCs). NLCs were prepared by high-pressure homogenization (HPH). The aqueous and lipid phase were separately prepared. In brief, 2500 mg of lipid mixture (lipid phase) was maintained at 70 °C to prevent the recrystallization of lipids during the process, and then CMT-8 was added to the lipid phase which was stirred until completely dissolved. At the same time, 5 mg/mL surfactant aqueous solution (100 mL) was maintained at the same temperature. Then the aqueous phase was added to the lipid phase with intense stirring (10 000 rpm for 1 min; Turrax T25, Fluko, Germany). These dispersions were processed through an HPH (ATS Engineering, Canada) with five homogenization cycles at 600 bar. The nanodispersions were cooled overnight to obtain the nanostructured lipid carriers. All samples were lyophilized for long stability. Appropriate amounts of trehalose (3% w/v in water) were used to dilute the NLC dispersions. The samples were frozen at −78 °C for 10 h before being lyophilized for 36 h. The mean particle size and polydispersity index of NLCs were measured by dynamic light scattering at 25 °C using a Nano-ZS90 system (Malvern Instruments Ltd., UK) with a measurement angle of 90° after the freeze-dried powders were rehydrated with phosphate buffer solution (PBS, pH 7.4). Zeta potential determination was carried out for freshly prepared samples by dynamic light scattering (Delsa Nano C, Malvern Instruments, Malvern, UK). An overview of the CMT-8/NLCs formulation details is given in Table 1. 2.3. Entrapment Efficiency. The entrapment efficiency of CMT-8 in the NLCs was determined by calculating the total amount of drug and subtracting that of the free drug in supernatant.20 The separation of the two phases was carried out by ultrafiltration, using Amicon

surface on the formed lipid core takes place, and it is logical to assume that the nonionic surfactants are more efficient.9 Combination of ionic and nonionic stabilizers is preferred for emulsification technology; therefore, the mixed nonionic/ionic surfactants may provide NLCs with some special properties due to their steric and electrostatic effects. Pluronic F68 (Figure 1a) is one of the most popular nonionic stabilizers which are used to prepare drug carriers. In the present study, it is chosen for its long PEG chains, which are known for providing a stealth character to nanocarriers and slowing their elimination from the bloodstream after intravenous injection. 10 GA-OSA (Figure 1b) is a natural biopolymer which is mainly composed of six carbohydrate moieties and protein, and it is a nondigestible polysaccharide which can reach the large intestine without digestion. In the small intestine, it can be classified as dietary fiber. Attributed to its good emulsifying and film forming properties, GA-OSA is being widely used for industrial purposes as a stabilizer, a thickener, an emulsifier in food, cosmetic, and pharmaceutical industry. Recently, GA-OSA has been used as drug carrier since it is considered as a physiologically harmless substance.11−13 In the present work, GA-OSA is intended to be used as the coemulsifier with Pluronic F68 for the NLCs preparation. CMT-8 is analogue of tetracycline, which is used clinically as antibiotic and antitumor drug.14−16 However, despite its beneficial pharmacological effects, CMT-8 suffers from poor water solubility and limited bioavailability, as it is difficult to transport into the target tissues and organs effectively. In addition, CMT-8 injection can cause serious adverse effects including nausea, vomiting, and liver function abnormalities.17 The first aim of the present work is to identify optimal formulation of CMT-8/NLCs with appropriate size, high drug loading, and sustained release profiles for prolonging the blood circulation, enhancing anticancer effects and reducing adverse effects of CMT-8. The second aim is to elucidate the influence of different emulsifiers on NLCs and to gain a first set of data on the physical stability, crystalline state, morphology, cell viability, and phagocytosis.

Table 1. Details of CMT-8/NLCs Formulations lipids (mg)

2. MATERIALS AND METHODS 2.1. Materials. Modified 4-dedimethylaminosancycline (CMT-8, CollaGenex Pharmaceuticals Inc., Newtown, PA); stearic acid (SA, LingFeng Chemical Reagent Co. Ltd., China); monoglyceride (MGE, composed of ∼50% of monoglycerides, together with variable quantities of di- and triacylglycerols, Aladdin Chemical Reagent Co. Ltd., China); oleic acid (OA, Aladdin Chemical Reagent Co. Ltd., 6921

emulsifier (mg)

batch

MGE

SA

MCT

OA

1 2 3 4 5 6

1074 1074 1074 1074 1074 1074

596 596 596 596 596 596

593 593 593 593 593 593

237 237 237 237 237 237

Pluronic F68

500 250 125

GAOSA

CMT-8 (mg)

100 250 500

30 30 30 30 30 30

250 125

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Ultra-4 centrifugal filter units (MWCO100 KD, Chaoyan, Shanghai, China). To determine the free CMT-8, 0.1 mL of CMT-8/NLCs was dispersed in 1.9 mL of 0.2 wt % Tween 80 distilled water and shaken (VORTEX-5, Instruments factory of Qilinbeier, China) for 5 min to dissolve the free drug. Then the diluted solution was centrifuged at 6000g for 30 min (KDC-140HR, Anhui USTC Zonkia Scientific Instruments, China). The supernatant after centrifugation was filtrated through 0.22 μm membrane filters, and then the drug content in the filtrate was detected by high-performance liquid chromatography (HPLC, Agilent, Austria) using the following experimental conditions: Diamond C18 column (150 nm × 4.6 nm i.d., pore size 5 μm), the mobile phase MeOH:H2O (0.5% trifluoroacetic acid, v/v) = 30:70 (v/ v), flow rate = 1.5 mL/min, and wavelength = 360 nm. The total drug content in CMT-8/NLCs was determined as follows: 0.1 mL of CMT8/NLC was diluted by 1.9 mL of methanol to dissolve the lipid ingredient, and then obtained suspension was measured by HPLC with the same procedure. The drug entrapment efficiency (EE) of CMT-8/NLCs was calculated as follows:

EE% =

with 100 μL of medium at a density of 8 × 103 cells per well. After 12 h, cells were exposed to various concentrations of blank NLCs for 24 h. MTT solution was directly added to the media in each well, with a final concentration of 0.5 mg/mL, and incubated for 4 h at 37 °C. The resulting formazan crystals were solubilized with 150 μL of DMSO. The absorbance was measured using an enzyme-linked immunosorbent assay reader at 570 nm, with the absorbance at 630 nm as the background correction. The effect on cell proliferation was expressed as the percent cell viability. Untreated cells were taken as 100% viable. 2.9. In Vitro Release Studies. The in vitro release of CMT-8 from CMT-8/NLCs was conducted by dialysis bag diffusion.21 The free CMT-8 was first removed from freshly prepared CMT-8/NLCs by ultrafiltration as described in section 2.3, and then 5 mL of CMT-8/ NLCs was put into a preswelled dialysis bag with 7 kDa MW cutoff. The dialysis bag was incubated in release medium (PBS, pH 7.4, 20 mL) with 0.5% (v/v) of Tween 80 to enhance the solubility of released free CMT-8 and to avoid its aggregation at 37 °C under horizontal shaking. At predetermined time points, the dialysis bag was taken out and placed into a new container containing fresh release medium (20 mL). The content of CMT-8 in release medium was determined by HPLC as described in section 2.3. The release rate of CMT-8 was expressed as the mass of CMT-8 in release medium divided by time. All measurements were carried out in triplicate. 2.10. Phagocytosis Assay. The promonocytic leukemia cell line U937 was purchased from the European Collection of Cell Cultures (ECACC, Salisbury, United Kingdom). U937 cells were cultured with DMEM and RPMI 1640 medium (Life Sciences, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS, PAA Laboratories GmbH, Linz, Austria), 1% penicillin, and 100 mg mL−1 streptomycin (Life Sciences, Karlsruhe, Germany) and under cell culture conditions (37 °C, 5% CO2 and 95% humidity-controlled atmosphere). In this study U937 cells were treated with CMT-8/NLCs (2.5 μM calculated by CMT-8). After 24 h incubation, U937 cells were washed with PBS to remove the excess of nonincorporated nanoparticles and lysed by the freeze/thaw procedure. Methanol was used to dissolve nanoparticles. After cell debris removal (centrifugation step), the amount of phagocytozed nanoparticles was measured by HPLC as described in section 2.3. All experiments were carried out in triplicate. CMT-8/NLCs phagocytosis was calculated as the amount of CMT8 in phagocytozed nanoparticles divided by the original amount of CMT-8.

Wtotal − Wfree × 100% Wtotal

where Wtotal and Wfree are the weight of drug added in NLCs and detected weight of CMT-8 in supernatant, respectively. 2.4. Transmission Electron Microscopy. The morphology of NLCs was determined by a transmission electron microscope (TEM) (JEOL-1400, Jeol, Tokyo, Japan). Prior to analysis, samples were diluted with distilled water and then loaded on Formvar-coated copper grids, dried at room temperature overnight, and negatively stained with the aqueous solution of sodium phosphotungestic acid 2% (w/v). 2.5. Rheological Analysis. The rheological experiments in the oscillatory mode (frequency sweeps) were performed on a MCR 501 rheometer by Anton Paar Physica (Graz, Austria), equipped with a parallel plates geometry (plate diameter 60 mm). For each sample, at first strain sweeps were performed at different frequency levels to determine the linear viscoelastic region. Then the storage modulus (G′), loss modulus (G″), and complex viscosity (η*) were measured as a function of frequency over a range from 0.1 to 15 Hz at a constant strain amplitude of 0.2% within the linear viscoelastic range. All measurements were carried out at a temperature of 25 °C. 2.6. Small-Angle X-ray Scattering Characterization. The SAXS measurements were performed at laboratory SAXS instrument (Nanostar, Bruker AXS GmbH, Karlsruhe, Germany). Instrument includes IμS microfocus X-ray source with power of 30 W (used wavelength Cu Kα) and VÅNTEC-2000 detector (14 × 14 cm2 and 2048 × 2048 pixels). The sample-to-detector distance is 108.3 cm and accessible q range from 0.1 to 2.3 nm−1. Dry CMT-8/NLCs were measured in vacuum at T = 25 °C. 2.7. Differential Scanning Calorimetry Analysis. DSC analysis was performed using a DSC 1 (Mettler-Toledo, Greifensee, Switzerland). Approximately 8 mg of powered sample was weighed into an aluminum pan. DSC experiments were performed under a nitrogen purge, using an empty pan as reference. The thermal analysis profiles consisted of a first heating interval from 25 to 120 °C and a subsequent cooling interval to −20 °C. The final heating interval ranged from −20 to 80 °C at a rate of 10 K/min. The final heating interval was analyzed. The DSC measurements were carried out with the following samples: (a) lyophilized nanostructured lipid carriers with different surfactants; (b) lipid mixture; (c) pure CMT-8. 2.8. In Vitro Cell Viability Test. The HeLa human cervical cancer cell line was purchased from American Type Culture Collection (Manassas, VA). The cells were seeded into cell culture dishes containing DMEM supplemented with 10% new calf serum, Lglutamine (5 mmol/L), nonessential amino acids (5 mmol/L), penicillin (100 U/mL), 205, and streptomycin (100 U/mL) (Invitrogen, Carlsbad, CA) at 37 °C in a humidified 5% CO2 atmosphere. Cell viability was measured by MTT assay to determine the cytotoxicity of blank NLCs. HeLa cells were plated in 96-well plates

3. RESULTS AND DISCUSSION 3.1. Preparation of CMT-8 Loaded Nanostructured Lipid Carriers. CMT-8 loaded NLCs were successfully prepared by HPH and then freeze-dried with trehalose as cryoprotectants. The lyophilized CMT-8/NLCs particles could be easily redispersed in PBS buffer. According to our preexperiments, four different lipids including stearic acid (SA), monoglyceride (MGE), oleic acid (OA), and capric/caprylic triglycerides (MCT) were chosen for their physical compatibility of CMT-8 (data not shown). Being liquid at room temperature, OA and MCT are believed to disrupt the crystalline domains with the solid lipid (SA/MGE) matrices.22 It should be noted that MGE is composed of ∼50% of monoglycerides and therefore depicts some surface tension activity. GA-OSA and ethylene oxide-based surfactants Pluronic F68 were added as electrostatic and steric emulsifiers, respectively. According to our former work, 5 mg/mL Pluronic F68 had better emulsification efficiency; consequently, sample 4 (Table 1) was prepared, and the GA-OSA was chosen as the cosurfactant to improve the emulsifying efficiency of Pluronic F68. Because of the less knowledge of GA-OSA in lipid nanoparticles, the effects of GA-OSA’s concentration on CMT8/NLCs were also studied (Table 1). Table 2 summarizes the average particle diameters and PDI of NLCs, where the diameters of all samples increased slightly 6922

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Table 2. Average Particle Diameters (Particle Size ± SD) and Polydispersity Index (PDI ± SD) of CMT-8/NLCs (1 and 30 days) particle size ± SD batch 1 2 3 4 5 6

1 day 205.0 152.9 170.6 181.6 133.9 130.5

± ± ± ± ± ±

PDI ± SD

30 days 5.5 2.0 2.2 3.5 2.4 6.3

260.2 190.0 172.5 200.5 169.0 163.9

± ± ± ± ± ±

3.2 1.4 3.2 5.5 2.0 4.1

1 day 0.12 0.18 0.11 0.11 0.32 0.32

± ± ± ± ± ±

0.10 0.03 0.03 0.01 0.04 0.07

30 days 0.32 0.25 0.27 0.15 0.42 0.32

± ± ± ± ± ±

0.05 0.06 0.15 0.02 0.10 0.12

during the storage of 30 days; indeed, all samples maintained a size of 60%; samples 1, 3, and 5 had significant reduction after 30 days of preparation, while it remained stable at almost 90% for samples 2 and 4. For single Pluronic F68 (sample 4), its PEG chain is expected to sustain the leakage of CMT-8 from NLCs during storage. The crystalline structure of lipid matrix is a key factor in determining

Figure 2. Entrapment efficiency (EE) of formulations in Table 1 measured by HPLC on the day of preparation and 30 days after preparation.

whether drug can be expelled or firmly incorporated for long time. The decreasing of entrapment efficiency could be explained by the creation of a perfect β-modification (most stable modification) of lipid matrix during storage and leads to drug leaking. Similar results were observed by Zhang.26 Except for the crystalline changes of lipid matrix, the solubility of drug in lipid matrix and the polarity of drug are significant for entrapment efficiency.27 3.3. TEM Examination. According to recent studies, it is clear that the morphology of nanoparticles plays an important role in drug delivery,28 so morphology observation is very necessary. TEM measurement was employed to study the morphology of NLCs with different surface chemistry. It was obvious in Figure 3 that CMT-8/NLCs were of spherical or

Figure 3. TEM graphs of CMT-8/NLCs with different surfactant formulations: (A) Pluronic F68 (sample 4); (B) GA-OSA (sample 3); (C) Pluronic F68/GA-OSA (sample 5). White numbers indicate the measured diameter of particles. 6923

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Figure 4. Results of frequency sweeps: storage modulus (G′), loss modulus (G″), and complex viscosity (η*) of the CMT-8/NLCs with different surfactant formulations as a function of frequency ( f, 0.1−15 Hz). The measurement temperature was 25 °C.

3.5. SAXS Characterization. Scattering methods were applied to characterize the 3-dimensional structure of NLCs. In SAXS experiments, fluctuations of the electron density give rise to characteristic patterns for the scattering of incident X-rays. The scattering intensity [I(q)] can be expressed against the scattering vector (q): q = (4π/λ) sin(θ/2), where θ and λ are the scattering angle and wavelength of the incident X-rays, respectively. At a q range, an evidence for fractals in the submicrometer and nanometer scale can be conveniently derived from smallangle scattering based on well-known dimensional analysis.36 The power law of the scattering intensity I(q) can be described as I(q) ∼ q−α. This exponent indicates that the microscopic structure as revealed by scattering experiments can be understood as mass fractals or surface fractals. Mass fractals mean open structures, whereas surface fractals describe dense materials with rough surfaces. When the angular coefficient of the log I(q) versus log q plot is determined, its relationship with the dimensions of mass and surface fractal, Dv and Ds, is α = 2 × Dv − Ds. If 1 < α < 3, the curve is associated with mass fractal. In this system, the surface and the bulk are not uniformly dense; thus, the surface and the inner part of structure have the same fractal dimension (Dv = Ds). When 3 < α < 4, the materials have surface-fractal behavior with dense core (Dv = 3) and Ds = 6 − α. For nonfractal materials (α = 4), they have uniformly dense and perfectly smooth surface (Dv = 3, Ds = 2). In this study, samples with different surface chemistry were investigated by SAXS. Figure 5 shows that for all samples there are two peaks (q = 1.86 nm−1, q = 2.26 nm−1), which means that all samples are cubic in 3-dimensional structures.37 The low q part of SAXS was also investigated. α for samples 1, 2, and 3 (Figure 5: (1), (2), and (3), that emulsified by GA-OSA) are 3.6, 3.7, and 3.5, respectively. It means that particles which were stabilized only by ionic stabilizer (GA-OSA) have dense

oval shape with wide size distribution. It was reported that elongated particles could avoid phagocytosis in a particular orientation,29,30 and these particles had higher contact surface area, so they show higher accumulation at the target, which means higher targeting ability compared to spheres.31−33 Studies showed that elongated particles were internalized more slowly than spherical ones.34 In Figure 3, it also can be seen that the particle size of CMT-8/NLCs stabilized with the combination of Pluronic F68 and GA-OSA (Figure 3C) is obviously smaller than those with pure Pluronic F68 or GAOSA, which further confirmed the results of DLS in section 3.1. 3.4. Rheological Analysis. Semisolid systems such as NLC aqueous dispersions combine solid and liquid properties within the same material. Before the frequency sweeps, the linear viscoelastic region was determined by an amplitude sweep at a frequency of 1 Hz. The viscoelastic character at frequencies from 0.1 to 15 Hz at constant strain amplitude of 0.2% is presented in Figure 4. Both G′ and G″, as well as the magnitude of η*, were affected by the type and concentration of the surfactant in the formulation.35 Figure 4 displays the results for three different NLC formulations (samples 3, 4, and 5). Both G′ and G″ increase with frequency. The storage modulus (G′) is smaller than the loss modulus (G″), indicating a predominantly viscous response or liquidlike behavior. The comparison of the storage modulus (G′) for all samples reveals that the storage modulus of sample 4 decreases with decreasing frequency to a larger extent than for the samples 3 and 5. This effect shows that the ionic surfactant GA-OSA at the interface of NLC particle leads to weaker flocculated networks or the disappearance of the network structure and enhanced dispersion stability. The addition of ionic emulsifier GA-OSA would result in the repulsive forces between the NLC particles, which will overcome the van der Waals attractive forces, causing stabilization of the particulate in suspension. 6924

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over from fractal to nonfractal only for samples 5 and 6, which means that there are in mixture the particles of nano size (few nanometers). Therefore, it can be concluded that the mixture of nonionic/ionic surfactants should increase PDI value and decrease size of the nanoparticles, which are agreement with the results in Table 2 and Figure 3. 3.6. Analysis of DSC Experiments. As mentioned before, the crystalline regions of the lipid matrix bring significant effects on the entrapment efficiency of CMT-8/NLCs. In the present work, DSC experiments were carried out 30 days after sample preparation to offer a close look at the crystallization and thermal behavior of the lipid nanoparticles. There are two main purposes of this test: (i) observation of the influence of stabilizer system on the melting behavior and changes in crystallinity; (ii) inspecting whether or not the crystallinity was different in the lipid nanoparticles compared with raw materials. It has been reported that lipid molecules recrystallize in different polymorphic forms, i.e., the unstable α, the metastable β′, and the most stable β modification.38 Figure 6A presents the DSC data for all six samples. Obviously, all samples depict two peaks at around 28 and 47 °C. According to the literature data, the first peak was attributed to the α-polymorphic form (thermodynamic instable modification) and the second peak belonged to the β-polymorphic form (stable modification).1,39 The concentration of GA-OSA had significant effects on the blending behavior of NLCs. As demonstrated in Figure 6 (A, samples 1−3), with increasing amount of GA-OSA, both peaks around 30 and 50 °C became sharp (decrease in difference between melting temperature and onset) and the peak height increased; thus the crystallinity was increased. That is to say, the crystallinity was mainly determined by the concentration of GA-OSA. Figure 6B summarizes the relationships between raw materials and CMT-8/NLCs (sample 5). The characteristic peaks of lipids (around 1, 15, 27, and 50 °C) were found in the DSC curves of lipid mixture. No peak of pure CMT-8 was detected in the DSC thermograms of CMT-8/NLCs compared with the thermograms of the raw materials, which can be ascribed to the amorphous or molecularly dispersed structure of CMT-8 in the lipid matrix. Compared to the lipid physical mixture, CMT-8/NLCs have a sharper heating peak, a lower melting point, and melting enthalpy. A possible reason for this effect is the large surface-to-volume ratio of these colloidal particles. 3.7. In Vitro Release Studies. The drug release behavior of CMT-8/NLCs was characterized using a dialysis bag in PBS

Figure 5. SAXS curves for folie freeze-dried CMT-8/NLCs at 25 °C.

core and rough surface for length scale from 1 to 70 nm; the core has a Euclidean dimension. The slope of sample 4 (Figure 5 (4)) remained at 4; thus, formulation that emulsified by nonionic stabilizer (Pluronic F68) demonstrated uniformly dense core and perfectly smooth surface. Compared with other samples with single emulsifier, slope changes from surface fractal (slightly higher than 3) to smooth surface (slope is equal to 4) at samples 5 and 6 (Figure 5: (5), (6)) were observed. The position of this change so-called cross over can be connected with size of primary aggregates which form surface fractals and are not fractal. The estimated radius of primary aggregates is ∼1.5 nm (sample 5) and ∼2 nm (sample 6) for higher length scale up to 70 nm; these samples are surface fractal. Small bump (sample 5) around q = 0.46 nm−1 can be attributed to interference maximum between subunits with average distance around 15 nm. Moreover, it can be seen cross

Figure 6. Heating curves of DSC experiments for samples shown in Table 1. (A) All formulations. (B) DSC curves for pure CMT-8, lipid mixture, and CMT-8/NLCs (sample 5). 6925

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Figure 7. In vitro drug release profiles of CMT-8/NLCs (n = 3). Sample details are shown in Table 1.

with 0.5% of Tween 80 at 37 °C. In Figure 7A, the association between emulsifier type and drug release was studied. In general, CMT-8/NLCs showed a constant rate of drug release. The release behavior of CMT-8 from the mixed lipid matrices mainly depended on diffusion or bulk erosion. Sample 3 was stabilized with pure GA-OSA, and the polysaccharide of GAOSA may facilitate sustained release of drugs from dosage forms. Thus, it showed comparatively slower release rate than sample 4. It was obvious that sample 5 owning best prolonged release properties, which meant that the mixture of nonionic/ ionic surfactants favored sustained release profile of CMT-8/ NLCs. The release profiles can be modified by controlling lipid matrix, emulsifier concentration, and production parameters (e.g., temperature). Hence, the relationship between concentration of both nonionic and ionic emulsifier agents and drug release was investigated. As shown in Figure 7B, a higher fraction of emulsifier leads to nonsustained release in agreement with previous studies.40,41 3.8. In Vitro Cell Viability Test and Phagocytosis Assay. MTT was used to investigate the effect of blank NLCs on HeLa human cervical cancer cell line metabolic activity. Three samples with the same carrier composition of samples 3, 4, and 5 without CMT-8 (samples 3′, 4′, and 5′) were determined by MTT assay. As demonstrated in Figure 8, no dramatic effect on cell viability was observed for sample 3′ (single GA-OSA), sample 4′ (single Pluronic F68), and sample 5′ (the mixture of GA-OSA and Pluronic F68) cell viability remains around 75−80% in all cases. Thus, GA-OSA would be

suitable to prepare drug carriers as the well-known Pluronic F68.42,43 The effects of shape and particle size on cellular incorporation are a pertinent theme in drug delivery as therapeutic-carrying nanoparticles.44 Champion and Mitragotri carried out a pioneering work in this field. They found that the shape was the major factor deciding on whether phagocysis will occur. In this study, U937 was employed to investigate the influence of surface properties of CMT-8/NLCs on cellular internalization.45−47 The results in Figure 9 show obvious difference among formulations. Formulation of the NLCs has a strong impact on

Figure 9. Phagocytosis of CMT-8/NLCs by U937 cell line after 24 h incubation (n = 3). Results are expressed as the uptake amount of CMT-8/NLCs normalized by the initial amount of CMT-8/NLCs.

their U937 incorporation (Figure 9). NLCs stabilized only with Pluronic F68 exhibit the highest incorporation rate. With increasing of GA-OSA content, the phagocytosis is decreasing (sample 1 > sample 2 > sample 3). However, addition of Pluronic F68 seems to counteract this effect (e.g., sample 5 (250 mg of Pluronic F68 and 250 mg of GA-OSA) is more incorporated than sample 2 (250 mg of GA-OSA)). The polysaccharide-like structure of GA-OSA may explain this effect, by disturbing the interaction of the NLCs with the cellular membrane. In turn, addition of Pluronic F68 decreased particle size (see Table 2) and may ease the interaction. Samples emulsified with single GA-OSA or the combination of Pluronic F68/GA-OSA have lower phagocytosis compared to sample 4 (emulsified with single Pluronic F68)that is to say, GA-OSA cannot trigger immunological responses and macrophages cannot recognize these particles; the likely explanation is the polysaccharide structure of GA-OSA. In

Figure 8. In vitro cytotoxicity tests of surfactants on HeLa cells for 24 h. Cell viability is expressed as the percentage of untreated controls. Data are given as mean ± SD (n = 6). 6926

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(2) Liu, D. H.; Liu, Z. H.; Wang, L. L.; Zhang, C.; Zhang, N. Nanostructured Lipid Carriers As Novel Carrier for Parenteral Delivery of Docetaxel. Colloids Surf., B 2011, 85, 262−269. (3) Tian, B. C.; Zhang, W. J.; Xu, H. M.; Hao, M. X.; Liu, Y. B.; Yang, X. G.; Pan, W. S.; Liu, X. H. Further Investigation of Nanostructured Lipid Carriers As an Ocular Delivery System: In Vivo Transcorneal Mechanism and in Vitro Release Study. Colloids Surf., B 2013, 102, 251−256. (4) Huang, Z. R.; Hua, S. C.; Yang, L. Y.; Fang, J. Y. Development and Evaluation of Lipid Nanoparticles for Camptothecin Delivery: A Comparison of Solid Lipid Nanoparticles, Nanostructured Lipid Carriers, and Lipid Emulsion. Acta Pharmacol. Sin. 2008, 29, 1094− 1102. (5) Tadros, T. E. Applied Surfactants-Principles and Applications; Wiley-VCH: Weinheim, 2005; p 634. (6) Moghimi, S.; Hunter, A.; Murray, J. Long-Circulating and TargetSpecific Nanoparticles: Theory to Practice. Pharmacol. Rev. 2001, 53, 283. (7) Tan, S. W.; Billa, N.; Roberts, C. R.; Burley, J. C. Surfactant Effects on the Physical Characteristics of Amphotericin B-containing Nanostructured Lipid Carriers. Colloids. Surf. Physicochem. Eng. Asp. 2010, 372, 73−79. (8) Helgason, T.; Awad, T. S.; Kristbergsson, K.; McClements, D. J.; Weiss, J. Effect of Surfactants Surface Coverage on Formation of Solid Lipid Nanoparticles (SLN). J. Colloid Interface Sci. 2009, 33, 75−81. (9) Goddard, E. D. Polymer/Surfactant Interaction: Interfacial Aspects. J. Colloid Interface Sci. 2002, 256, 228−285. (10) Delmas, T.; Couffin, A. C.; Bayle, P. A.; Crecy, F.; Neumann, E.; Vinet, F.; Bardet, M.; Bibette, J.; Texier, I. Preparation and Characterization of Highly Stable Lipid Nanoparticles with Amorphous Core of Tuneable Viscosity. J. Colloid Interface Sci. 2011, 360, 471−481. (11) Kshirsagar, A. C.; Singhal, R. S. Optimization of Starch Oleate Derivatives from Native Corn and Hydrolyzed Corn Starch by Response Surface Methodology. Carbohydr. Polym. 2007, 69, 455− 461. (12) Savitha Prashanth, M. R.; Parvathy, K. S.; Susheelamma, N. S.; Hari Prashanth, K. V.; Tharanathan, R. N.; Cha, A. Galactomannan EstersA Simple, Costeffective Method of Preparation and Characterization. Food Hydrocolloids 2006, 20, 1198−1205. (13) Montenegro, M. A.; Boiero, M.; Valle, L.; Borsarelli, L.; Gum, C. D. Arabic: More Than an Edible Emulsifier. Prod. Appl. Biopolym. 2012, 1−26. (14) Golub, L. M.; Ramamurthy, N. S.; McNamara, T. F.; Greenwald, R. A.; Rifkin, B. R. Tetracyclines Inhibit Connective Tissue Breakdown: New Therapeutic Implications for an Old Family of Drugs. Crit. Rev. Oral Biol. Med. 1991, 2, 297−321. (15) Liu, Y.; Ramamurthy, N.; Marecek, J.; Lee, H. M.; Chen, J. L.; Rayan, M. E.; Rifkin, B. R.; Golub, L. M. The Lipophilicity, Pharmacokinetics, and Cellular Uptake of Different ChemicallyModified Tetracycline (CMTs). Curr. Med. Chem. 2001, 8, 243−252. (16) Seftor, R. E.; Seftor, E. A.; De Larco, J. E.; Kleiner, D. E.; Leferson, J.; Stetler-Stevenson, W. G.; McNamara, T. F.; Golub, L. M.; Hendrix, M. J. Chemically Modified Tetracyclines Inhibit Human Melanoma Cell Invasion and Metastasis. Clin. Exp. Metastasis 1998, 16, 217−225. (17) Syed, S.; Takimoto, C.; Hidalgo, M.; Rizzo, J.; Kuhn, J. G.; Hammond, L. A.; Schwartz, G.; Tolcher, A.; Patnaik, A.; Eckhardt, S. G.; Rowinsky, E. K. A Phase I and Pharmacokinetic Study of Col-3 (Metastat), an Oral Tetracycline Derivative with Potentmatrix Metalloproteinase and Antitumor Properties. Clin. Cancer Res. 2004, 10, 6512−6521. (18) Sarkar, S.; Singhal, R. S. Esterification of Guar Gum Hydrolysate and Gum Arabic with N-octenyl Succinic Anhydride and Oleic Acid and its Evaluation as Wall Material in Microencapsulation. Carbohydr. Polym. 2011, 86, 1723−1731. (19) Stephen, A. M.; Churms, S. E. In Food Polysaccharides and Their Applications; Stephen, A. M., Ed.; VCH: New York, 1995; p 377.

addition, the phagocytosis of CMT-8/NLCs decreased with increasing of GA-OSA (sample 1 > sample 2 > sample 3) also proved the effect of GA-OSA for escaping from recognition by macrophages. GA-OSA is composed of polysaccharide, which increase the hydrophility of the NLC’s surface, protecting it from protein opsonisationa process that marks the NLC for removal from the circulation by specialized macrophages.48 In addition, GA-OSA is adhesive, especially for mucosal surfaces, which has been used for targeting specific organs or cells and prolonging the drug residence time.49

4. CONCLUSIONS In summary, stable CMT-8/NLCs with high entrapment efficiency and low phagocytosis by phagocyte can be prepared when proper concentrations of both nonionic and ionic surfactants were used. The analysis of DSC experiments revealed that the emulsifier agents changed the melting behavior and crystallinity of NLCs. At low concentration, GA-OSA was homogeneously dispersed in the lipid blends, but this phenomenon disappeared when the concentration of GAOSA exceeded a certain value. SAXS clearly demonstrated that the 3-dimensional structure of all CMT-8/NLCs was cubic. Furthermore, addition of nonionic surfactant Pluronic F68 resulted in CMT-8/NLC formulations of primary smooth aggregates of size around few nanometers and the uniformly dense and perfectly smooth surface at higher fraction of Pluronic F68, while formulations stabilized with pure GA-OSA had dense core and rough surface. In vitro release studies provided the information that the mixture of nonionic/ionic surfactants was in favor for sustained release. Phagocytosis assay showed that GA-OSA can decrease the phagocytosis of NLCs from macrophages; the likely reason was polysaccharide composition of GA-OSA. Generally, these studies revealed that once proper concentration was selected, the mixture of nonionic/ionic surfactants was more suitable than single stabilizer in terms of physical stability, entrapment efficiency, in vitro release, and phagocysis. In vitro and in vivo investigations will be further studied to collect sufficient data for the clinical application of CMT-8/NLC formulations with the mixture of nonionic/ionic surfactants.

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AUTHOR INFORMATION

Corresponding Author

*Tel +86-21-64252231; e-mail [email protected] (A.Z.). Author Contributions

S.Z. and X.Y. contributed equally to this article. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. Lihui Zhou is acknowledged for help during TEM measurements and Ivonne Ternes for DSC experiments. Aihua Zou gratefully acknowledges the support of this work by the Alexander von Humboldt Foundation. We gratefully acknowledge the support of this work by Chinese National Natural Science Foundation (201003047) and Fundamental Research Funds for the Central Universities.

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REFERENCES

(1) Rainer, M. H. Lipid Nanoparticles: Recent Advances. Adv. Drug Delivery Rev. 2007, 59, 375−376. 6927

dx.doi.org/10.1021/la501141m | Langmuir 2014, 30, 6920−6928

Langmuir

Article

(20) Liu, X.; Wang, Z. L.; Feng, R. H.; Hu, Y.; Huang, G. H. A Novel Approach for Systematic Delivery of a Hydrophobic Anti-Leukemia Agent Tamibarotene Mediated by Nanostructured Lipid Carrier. J. Biomed. Nanotechnol. 2013, 9, 1586−1593. (21) Xu, Z. H.; Chen, L. L.; Gu, W. W.; Gao, Y.; Lin, L. P.; Zhang, Z. W.; Xi, Y.; Li, Y. P. The Performance of Docrtaxel-loaded Solid Lipid Nanoparticles Targeted to Hepatocellular Carcinoma. Biomaterials 2009, 30, 226−232. (22) Jenning, V.; Thunemanm, A. F.; Gohla, S. H. Characterisation of a Novel Solid Lipid Nanoparticle Carrier System Based on Binary Mixtures of Liquid and Solid Lipids. Int. J. Pharm. 2000, 199, 167− 177. (23) Bunjes, H.; Westesen, K.; Koch, M. H. J. Crystallization Tendency and Polymorphic Transitions in Triglyceride Nanoparticles. Int. J. Pharm. 1996, 129, 159−173. (24) Han, F.; Li, S. M.; Yin, R.; Liu, H. Z.; Xu, L. Effect of Surfactants on the Formation and Characterization of a New Type of Colloidal Drug Delivery System: Nanostructured Lipid Carriers. Colloids Surf., A 2008, 315, 210−216. (25) Ma, C. M.; Xia, Y. Mixed Adsorption of Sodium Dodecyl Sulfate and Ethoxylated Nonylphenols on Carbon Black and the Stability of Carbon Black Dispersions in Mixed Solutions of Sodium Dodecyl Sulfate and Ethoxylated Nonylghenols. Colloids Surf. 1992, 66, 215− 221. (26) Zhang, X.; Pan, W.; Gan, L.; Zhu, C.; Gan, Y.; Nie, S. Preparation of a Dispersible PEGylate Nanostructured Lipid Carriers (NLCs) Loaded with 10-hydroxycamptothecin by Spray-drying. Chem. Pharm. Bull. (Tokyo) 2008, 56, 1645−1650. (27) Jenning, V.; Gohla, S. H. Encapsulation of Retinoids in Solid Lipid Nanoparticles (SLN). J. Microencapsul. 2001, 18, 149−158. (28) Doshi, N.; Mitragotri, S. Designer Biomaterials for Nanomedicine. Adv. Funct. Mater. 2009, 19, 3843−3854. (29) Champion, J.; Mitragotri, S. Role of Target Geometry in Phagocytosis. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 4930. (30) Doshi, N.; Mitragotri, S. Macrophages Recognize Size and Shape of Their Targets. PLoS One 2010, 5, e10051. (31) Muro, S.; Garnacho, C.; Champion, J. A.; Leferovinch, J.; Gajewski, C.; Schuchman, E. H. Control of Endothelial Targeting and Intracellular Delivery of Therapeutic Enzymes by Modulating the Size and Shape of ICAM-1-Targeted Carriers. Mol. Ther. 2008, 16, 1450− 1458. (32) Zhang, K.; Fang, H.; Chen, Z.; Taylor, J. S.; Wooley, K. L. Shape Effects of Nanoparticles Conjugated with Cell-penetrating Peptides (HIV Tat PTD) on CHO Cell Uptake. Bioconjugate Chem. 2008, 19, 1880−1887. (33) Yoo, J. W.; Doshi, N.; Mitragotri, S. Endocytosis and Intercellular Distribution of PLGA Particles in Endothelial Cells: Effect of Particle Geometry. Macromol. Rapid Commun. 2010, 31, 142−148. (34) Kodali, V.; Roos, W.; Spatz, J.; Curtis, J. Cell-Assisted Assembly of Colloidal Crystallities. Soft Matter 2007, 3, 337−358. (35) Zaman, A. A.; Singh, P.; Moudgil, B. M. Impact of Selfassembled Surfactant Structures on Rheology of Concentrated Nanoparticle Dispersions. J. Colloid Interface Sci. 2002, 251, 381−387. (36) Schmidt, P. W. Some Fundamental Concepts and Techniques Useful in Small-Angle Scattering Studies of Disordered Solids. In Modern Aspects of Small-Angle Scattering; Brumberger, H., Ed.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1995; pp 1−56. (37) Brandenburg, K.; Richter, W.; Koch, M. H. J.; Meyer, H. W.; Seydel, U. Characterization of the Nonlamellar Cubic and HII Structures of Lipid A From Salmonella Enterica Serovar Minnesota by X-ray Diffraction and Freeze-fracture Electron Microscopy. Chem. Phys. Lipids 1998, 91, 53−69. (38) Freitas, C.; Muller, R. H. Correlation between Long-Term Stability of Solid Lipid Nanoparticles (SLN) and Crystallinity of the Lipid Phase. Eur. J. Pharm. Biopharm. 1999, 47, 125−132. (39) Saupe, A.; Wissing, S. A.; Lenk, A.; Schmidt, C.; Muller, R. H. Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers

(NLC)-Structural Investigations on Two Different Carrier Systems. Biomed. Mater. Eng. 2005, 15, 393−402. (40) Shoji, F.; Kazuhiko, J.; Masahiro, N. Preparation of and Drug Release from W/O/W Type Double Emulsions Containing Anticancer Agents. Chem. Pharm. Bull. 1983, 31 (11), 4048−4056. (41) Puglia, C.; Blasi, P.; Rizza, L.; Schoubben, A.; Bonina, F.; Rossi, C.; Ricci, M. Lipid Nanoparticles for Prolonged Topical Delivery: An in Vitro and in Vivo Investigation. Int. J. Pharm. 2008, 357, 295−304. (42) Yang, X. M.; Zhao, L.; Laszlo, A.; Garamus, V. M.; Zou, A. H.; Willumeit, R.; Fan, S. J. Preparation and Characterization of 4Dedimethylamino Sancycline (CMT-3) Loaded Nanostructured Lipid Carrier (CMT-3/NLC) Formulations. Int. J. Pharm. 2013, 450, 225− 234. (43) Chen, Y. Y.; Yang, X. M.; Zhao, L.; Laszlo, A.; Garamus, V. M.; Willumeit, R.; Zou, A. H. Preparation and Characterization of Nanostructured Lipid Carrier for a Poorly Soluble Drug. Colloids Surf., A 2014, 455, 36−43. (44) Mitragotri, S. In Drug Delivery, Shape Does Matter. Pharm. Res. 2009, 26, 232−234. (45) Wen, L. Z.; Xiao, G.; Hui, B.; Rui, H. Y.; Chen, D. D.; Liu, J. P. Nanostructured Lipid Carriers Constituted from High-density Lipoprotein Components for Delivery of a Lipophilic Cardiovascular Drug. Int. J. Pharm. 2010, 391, 313−321. (46) Andreas, E.; Swapan, K. C.; Ali, A.; Dagmar, R.; Jürgen, L.; Peter, N. Influence of Spacer Length on Interaction of Mannosylated Liposomes with Human Phagocytic Cells. Pharm. Res. 2003, 20, 51− 57. (47) Malcolm, C. S.; George, S. B.; David, I. S.; Miles, D. H. Remodelling of the PDE4 cAMP Phosphodiesterase Isoform Profile upon Monocyte-Macrophage Differentiation of Human U937 Cells. Br. J. Pharmacol. 2004, 142, 339−351. (48) Pain, D.; Das, P. K.; Ghosh, P. C.; Bachhawat, B. K. Increased Circulatory Half-Life of Liposomes after Conjugation with Dextran. J. Biosci. 1984, 6, 811−816. (49) Shoshy, M.; Dan, P. Polysaccharides as Building Blocks for Nanotherapeutics. Chem. Soc. Rev. 2012, 41, 2623−2640.

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