Effects of gelucire content on stability, macrophage interaction and blood circulation of nanoparticles engineered from nanoemulsions

Colloids and Surfaces B: Biointerfaces 94 (2012) 259–265

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Effects of gelucire content on stability, macrophage interaction and blood circulation of nanoparticles engineered from nanoemulsions Daniel Wehrung, Werner J. Geldenhuys, Moses O. Oyewumi ∗ Department of Pharmaceutical Sciences, College of Pharmacy, Northeast Ohio Medical University, Rootstown, OH 44272, USA

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Article history: Received 19 December 2011 Received in revised form 1 February 2012 Accepted 1 February 2012 Available online 14 February 2012 Keywords: P-gp efflux Nanoemulsions Macrophages Colloidal stability Phagocytosis Cetyl alcohol

a b s t r a c t The main objective of the study is to investigate the efficacy of Gelucire 44/14 (gelucire) in facilitating formation of cetyl alcohol (CA)-based nanoparticle (NP) and to assess the effects on key NP properties and functions. NPs from oil-in-water nanoemulsion precursors were prepared using binary mixtures of CA and gelucire (CA/gelucire) containing gelucire at 0, 25, 50 and 75% (w/w). The sizes of gelucire-based NPs (128–183 nm) were five times lower than control NPs (made without gelucire). All the NPs (with or without gelucire component) did not activate macrophages as monitored by reactive oxygen species production. Results from differential scanning calorimetry, FT-IR and multimodal light scattering measurements demonstrated the involvement of gelucire component in achieving homogeneous CA/gelucire particle populations that were stable on storage. The P-glycoprotein (P-gp) function assay in MES-Dx5 cells showed the potential of gelucire-based NPs in inhibiting rhodamine 123 efflux. Similarly, the extent of NP uptake by macrophage (RAW 264.7 cell) was dependent on the amount of gelucire component (inverse relationship; R2 = 0.996). NPs made with CA/gelucire mixture (at 50%, w/w gelucire) were the most effective in blood circulation studies in BALB/c mice. Additional studies with paclitaxel-loaded NPs demonstrated that the retention of gelucire-based NPs in blood circulation was comparable to NPs coated with DSPE-PEG2000 (p > 0.6). The over-all work indicated the potential efficacy of gelucire as a safe and biocompatible excipient that can serve multiple functions in enhancing the performance of lipid-based NP drug delivery systems. Published by Elsevier B.V.

1. Introduction Colloidal nanocarriers (NCs) such as nanocapsules, nanoemulsions and nanoparticles have gained much attention as drug delivery systems on my many fronts [1]: (a) improving efficacy of drugs; (b) improving absorption of poorly water-soluble pharmaceuticals; (c) protecting sensitive drug molecules from degradation; and (d) modifying drug release and biodistribution. For many therapeutic applications, extensive uptake of NCs by the cells of the mononuclear phagocyte system (MPS) is a significant disadvantage. This is because uptake of NCs by the reticuloendothelial system (RES) will impair the ability of the delivery systems to target locations in non-RES tissues [2]. It is established that certain physical and chemical properties of the delivery systems (size, shape, and surface properties) will influence the rate and extent of RES uptake [3]. Strategies that have been employed to ensure retention of NCs in blood circulation include achieving and maintaining

∗ Corresponding author at: Department of Pharmaceutical Sciences, Northeast Ohio Medical University, 4209 State Route 44, Rootstown, OH 44272, USA. Tel.: +1 330 325 6669; fax: +1 330 325 5936. E-mail address: [email protected] (M.O. Oyewumi). 0927-7765/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.colsurfb.2012.02.005

colloidal stability as well as surface modification with hydrophilic groups. In this regard, the most common approach has been surface coating of NCs with PEGs exploiting the hydrophilicity, flexibility, and neutrality of PEG chains in surface modification of NCs. Thus PEG coatings on NCs are applied to decrease non-specific interaction with serum components, reduce macrophage uptake and increase blood circulation time [4]. The desired nature of NC interaction with macrophages will be dictated by the therapeutic target. For instance, extensive uptake by macrophages may be warranted in treatment of macrophageassociated pathologies or vaccination where macrophages serve as the therapeutic target [5]. While, in many other applications (pertaining to targeted delivery to disease sites such as tumors, inflammation), extensive phagocytic activities by macrophage (diverting NCs away from target) will be a major deterrent to achieving efficient targeted delivery. This is because, macrophages are involved in the host defense mechanism against pathogenic microorganisms, parasites and particles recognized as “foreign” or “strange” to the body system. Macrophages are phagocytic cells that participate in antigen presentation to T cells, when activated (by foreign particles or pathogens), macrophages produce oxygen and nitrogen-reactive metabolites (such as reactive oxygen species, nitric oxide, cytokines) with actions ranging from

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induction of cell growth to cell death [6,7]. Irrespective of the therapeutic target, accumulation and retention of effective concentration of drug at the site of action is highly desirable in achieving the desired clinical outcome. Since efflux of the therapeutic agent and/or the delivery system from target cells/sites can lead to therapeutic failure and/or drug resistance [8]. The most notable transporter protein responsible for efflux of drugs from cells is P-glycoprotein (P-gp) encoded by the multidrug resistance associated protein. A large number of P-gp inhibitors have been developed to improve therapeutic efficacy of drugs but their application have been curtailed due to associated toxicity and potential interference with drug biodistribution/pharmacokinetic properties [9,10]. In this regard, lipid nanoparticles (NPs) containing various concentrations of Gelucire 44/14 (gelucire) were developed with the goal of assessing if gelucire content will modify some key NP functions that are important to the performance as drug delivery systems. Gelucires are waxy amphiphilic excipients that are generally regarded as safe (GRAS) and are generally identified by their melting points and HLB (hydrophilic–lipophilic balance) values. They are saturated polyglycolized glycerides consisting of mono- and di-fatty acid esters of polyethylene glycol [11,12]. In addition to safety and biocompatible considerations, the rationale for selecting gelucire in NP preparation was based on many other attractive qualities that can facilitate lipid NP preparation with high drug loading while improving biocompatibility, and colloidal stability. This is substantiated from the fact that gelucires have been applied in many formulations and delivery systems as emulsifying, wetting, and stabilizing agent [11]. We are of the opinion that the amphiphilic property of gelucire is well suited for NP preparation that applied oil-in-water nanoemulsion as precursors. In this approach, binary mixtures of gelucire and cetyl alcohol (CA) were used as matrix materials for NP preparation from oil-in-water nanoemulsions [13]. Based on the aforementioned qualities of gelucire, it is envisaged that NPs prepared from gelucire-based nanoemulsions will possess desirable surface characteristics that will enhance colloidal stability, improve biocompatibility, overcome P-gp function and modify NP interaction with macrophages while sustaining NPs in blood circulation. To achieve these objectives, lipid-based NPs were developed from CA/gelucire-based (oil-in-water) nanoemulsions. NP characterization was carried out with the focus of investigating the effect of gelucire content on resultant NP size, size distribution, and colloidal stability. Also, P-gp efflux function assay was conducted based on rhodamine 123 accumulations in human sarcoma cell line (MES-Dx5). Nature of macrophage interaction was monitored based on the extent of NP uptake and macrophage activation while the blood profile studies were conducted in BALB/c mice.

2. Materials and methods 2.1. Materials Polyoxyethylene 20 sorbitan monooleate, sephadex G75, sephadex G25, cetyl alcohol, Dulbecco’s Modified Eagle medium (DMEM), Triton X-100, glycine, 3-(4,5-dimethyl-thiazol-2-yl), 2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma–Aldrich (St. Louis, MO). Fetal bovine serum was from USA Scientific (Orlando, FL). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-benzoxadiol-4-amine) (NBD) was from Avanti Polar Lipids (Alabaster, AL). Gelucire® 44/14 was obtained from Gattefosse˙ (Paramus, NJ). Paclitaxel (PTX) from LC Laboratories (Woburn, MA).

2.2. Preparation of gelucire-based nanoparticles (Gel-NPs) Gel-NPs were prepared from nanoemulsions according to earlier reported procedure of NP preparation from oil-in-water emulsified systems [13–15]. Briefly, nanoemulsion was prepared using binary mixtures of CA and gelucire (gelucire content ranged from 0, 25, 50 to 75%, w/w) as the oil phase (matrix materials) with or without Tween 80 as surfactant. To obtain nanoemulsions, 2–4 mg of the matrix materials was melted at 70 ◦ C together on a hotplate. The final preparation was obtained after addition of water (0.22 ␮m filtered) to make the final volume of 1 ml. Solid NPs were obtained by cooling the warm nanoemulsion to room temperature. To obtain fluorescent-labeled NPs, 5 ␮l of coumarin-6 (using a stock solution of 50 ␮g/ml) was added to the NP matrix before nanoemulsions were formed as earlier reported [14,15]. A similar procedure was employed to entrap PTX in NPs. Briefly, 500 ␮g of PTX as a solution in chloroform was added to the glass vial containing the NP matrix materials and the chloroform was evaporated prior to carrying out the remaining steps of NP preparation. To incorporate DSPE-PEG2000 into the NPs, DSPE-PEG2000 was added (5%, w/w) to the NP matrix prior to nanoemulsion preparation based on an earlier reported method [2]. 2.3. Characterization of Gel-NPs 2.3.1. Photon correlation spectroscopy (PCS) The particle sizes of the NPs were measured using a PSSNICOMP® particle sizer at 25 ◦ C. Prior to particle measurements, NP suspensions were diluted (1:5, v/v) with filtered water (0.22 ␮m filter, Nalgene International) to ensure that light scattering signals are within the sensitivity of the instrument. 2.3.2. Entrapment efficiency of PTX or fluorescence dyes in Gel-NPs Microcon Ultracel YM-100 centrifugal devices (MW cut off 100 kDa) (Millipore, Billerica, MA, USA) were used to determine entrapment efficiencies. Briefly, 400 ␮l of NP suspensions was added to the Microcon tube and spun for 30 min at 14,000 × g. The collected filtrate was labeled as free drug (not entrapped in NPs) while drug that was retained with NPs was labeled as entrapped. The percentage of drug entrapped was obtained from the ratio of amount of drug retained and total amount added to NP preparation. 2.3.3. Differential scanning calorimetry (DSC) DSC measurements were carried out using a diamond differential scanning calorimeter (Perkin Elmer Chicago, IL). The equipment was operated under nitrogen purge gas rate of 20 ml/min. Samples (1–8 mg) were weighed in aluminum pans and heated at a scanning rate of 10 ◦ C/min over the temperature range of 0–80 ◦ C followed by a cooling session. Thermal transitions during both heating and cooling cycles were obtained. 2.3.4. FT-IR measurements Fourier transform-infrared (FT-IR) spectra were obtained on a Bruker FT-IR spectrometer (Bruker Optics Inc., Manning Park, USA) using KBr disk method prepared with freeze-dried Gel-NP suspensions. The scanning range was 450–4000 cm−1 . 2.3.5. Transmission electron microscopy (TEM) The size and morphology of NPs was observed using Jeol electron microscope at the Northeast Ohio Medical University imaging facility according to the method earlier reported [2]. 2.3.6. Rhodamine (Rh) 123 assay for P-gp function A rhodamine assay was performed using a modified method [8]. Briefly, P-gp expressing MES-Dx5 (human sarcoma cell line) cells

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were seeded in black well plates at a density of 1 × 105 cells per well and co-treated for 30 min with 25 ␮l of NP preparation and rhodamine 123 (5 mM). The uptake was stopped and the cells were washed with ice-cold PBS. The intracellular fluorescence due to cell accumulation of Rh123 was measured.

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Table 1 DSC transition temperatures (temp) on heating and cooling cycles for CA alone and binary mixtures of CA/gelucire (gelucire content ranging from 10%, w/w to 75%, w/w). Samples

Transition temp Heating cycle

2.4. Macrophage activation studies Effects of incubating NPs with macrophages were investigated using RAW 264.7 cells (ATCC, Manassas, VA). The cells were plated at a density of 1 × 105 cells per well and incubated for 24 h with Gel-NPs. Hydrogen peroxide (100 ␮M) was used as a positive control. Reactive oxygen species (ROS) production was monitored by fluorescence intensity of dichlorofluorescein (DCF) based on intracellular oxidation of non-fluorescent 2,7-dichlorofluorescindiacetate (DCFH-DA) [16]. Initial experiments were carried out to confirm the effectiveness of DCF as a ROS probe by incubating cells with 100 ␮M of H2 O2 . After the incubation time, cells monolayer were rinsed with PBS and incubated with 5 ␮M of DCFH-DA for 60 min. Samples were removed from cells monolayer and replaced with pre-warmed growth medium. The fluorescence intensity due to internalized DCF was measured and compared to untreated control cells. Each data point is presented as percentage of ratio of fluorescence intensity between NP treated cells and cells treated with H2 O2 . 2.5. Microphage cell uptake RAW 264.7 cells were seeded (105 cell/well) in the complete growth medium and allowed to grow to reach 80% confluence. Afterwards, the growth medium was removed from each well and replaced with suspension of fluorescent-labeled Gel-NPs in the growth medium. After incubation of NPs with macrophages at 37 ◦ C, the medium was aspirated from each well and washed three times with PBS. Subsequently, the extent of NP uptake by macrophage was monitored by intracellular fluorescence. 2.6. Blood retention studies in BALB/c mice In vivo studies were carried out in BALB/c mice (20–23 g). The protocol was approved by the NEOMED Animal Care and Use Committee in accordance with NIH guidelines. The studies were conducted in two parts using either fluorescent-labeled Gel-NPs or PTX-loaded Gel-NPs. For the study with coumarin-6-labeled NPs, each mouse was injected (tail-vein administration) with 100 ␮l of NP suspensions (1 mg/kg animal body weight). At pre-defined time periods after NP administration, blood was collected from each mouse through tail vein. The fluorescence intensity (Fl) of coumarin-6 extracted by acetonitrile from blood was assayed at 485 nm/538 nm (Ex/Em) using a modified method earlier reported [17,18]. As a control, a separate study was conducted with coumarin-6 solution administered by tail vein injection. In the control studies, the coumarin-6 solution was prepared in DMSO and later diluted with phosphate buffered saline (PBS) before administration. Each data point was obtained from fluorescence intensity per gram of tissue (blood) obtained from mice administered with fluorescent-labeled NPs (Fl/g tissue-NPs ) after deducting the fluorescence intensity per gram of tissue (blood) obtained from injecting coumarin-6 solution (Fl/g tissue-control ). For the studies with PTX-loaded Gel-NPs, two types of NPs were used: (i) PTX-loaded Gel-NPs without DSPE-PEG2000 coating; and (ii) PTX-loaded Gel-NPs with DSPE-PEG2000 coating. The process of coating NPs surfaces with DSPE-PEG2000 was based on earlier reported method [2]. Mice were injected by tail vein administration with 100 ␮l (100 ␮g PTX/mouse) of PTX-loaded Gel-NPs. At predefined time post-injection, blood samples were collected through

CA CA/gelucire-10% (w/w) CA/gelucire-25% (w/w) CA/gelucire-50% (w/w) CA/gelucire-75% (w/w)

◦

39–44 C 37–44 ◦ C 18 ◦ C 35–44 ◦ C 16 ◦ C 35–41 ◦ C 19 ◦ C 36–38 ◦ C

Cooling cycle 56 ◦ C 56 ◦ C 56 ◦ C 54 ◦ C 50 ◦ C

the tail vein. PTX content in blood samples was extracted with acetonitrile and quantified by HPLC method [19] with the following conditions: Brown Lee SPERI-5-RP-18, 100 mm × 4.6 mm, and pore size of 5 ␮m. The mobile phase was water/acetonitrile (30/70, v/v) flow rate of 1 ml/min, and measured at a wavelength of 227 nm. Each data point was expressed as the concentration of PTX per gram of tissue (blood) normalized by the injected dose (PTX ␮g/g tissue/g injected dose). 2.7. Data analysis Statistical analyses of data were carried out using Student’s t-test and a single factor ANOVA followed by Student–Newman–Keuls post hoc test for multiple comparisons. Data were concluded to be statistically different at p-value of <0.05. 3. Results and discussion 3.1. Preparation and stability of Gel-NPs The key steps in NP preparation method are: (i) melting of the matrix materials; (ii) emulsification of dispersed oil droplets in the continuous phase; and (iii) solidification of the droplets (upon cooling) at room temperature to obtain solid NPs. Earlier reports demonstrated the feasibility of obtaining solid NPs using CA as the matrix material with surfactants such as Brij 78 and Tween 80 [2,14,20]. The current study is based on NP formation using binary mixtures of CA and gelucire (CA/gelucire). Initial experiments were focused on ascertaining the potential effect of gelucire addition on NP preparation. Potential interference of CA’s melting and cooling properties by gelucire (especially at high gelucire concentrations) may affect the physical construct of the resultant NPs to the extent of changing the form to nanocapsules or nanoemulsions. In this regard, the effect of gelucire content on thermal transition of CA was studied. DSC measurements were conducted using heating and cooling cycles so as to mirror the NP preparation process. On the heating cycle, the endothermic transition (melting) of CA alone occurred at a range of 39–44 ◦ C (Table 1). CA/gelucire mixtures showed endothermic transition at temperatures lower than with CA alone. Depending on the gelucire content in CA/gelucire mixtures, a second endothermic transition was observed at temperatures lower than the melting points of either CA or gelucire (Table 1). These secondary transitions at lower temperatures could be due to possible eutectic mixture formation which was pronounced at gelucire content of 25, 50 and 75% (w/w) (Table 1; Fig. 1). On the cooling cycle, CA alone and all the binary mixtures (CA/gelucire) showed exothermic transitions that possibly correspond to solidification of the melted matrix materials upon cooling (Table 1). Although the exothermic transition (on cooling cycle) of binary mixtures (CA and gelucire) occurred at lower temperatures

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Fig. 1. DSC thermographs of NP matrix material using physical mixture of CA/gelucire at: (A) gelucire content of 5% (w/w); and (B) gelucire content of 50% (w/w).

compared with CA alone, the general trend supported the feasibility of NP formation via gelucire containing oil-in-water nanoemulsion templates. Furthermore, CA/gelucire mixtures were applied in NP preparation (Table 2). It was observed that CA alone did not form nanoemulsions without the use of surfactant while the CA/gelucire mixtures (at 50%, w/w and 75%, w/w gelucire) resulted in stable NPs without the aid of surfactant. This may be ascribed to the amphiphilic properties of gelucire that most likely facilitated formation of stable nanoemulsion templates. Sizes and polydispersity indexes (PDI) of NPs prepared with various amounts of gelucire (0–75%, w/w) are shown in Fig. 2A indicating that the addition of gelucire facilitated NP formation as reflected from the low particle sizes and PDI. A representative TEM micrograph of Gel-NPs (25%, w/w gelucire content) showed that the NPs appeared spherical in shape with size of about 150 nm (Fig. 2B). There were no Table 2 Components of NPs prepared with CA alone and CA/gelucire mixtures (gelucire content in NP matrix ranged from 0%, w/w to 75%, w/w in a total volume of 1 ml). Components

CA (mg) Gelucire (mg) Polysorbate 80 (mM)

Gelucire content (w/w) in NP matrix 0%

25%

50%

50%

75%

75%

2 0 1.5

1.5 0.5 1.5

1 1 0

1 1 1.5

0.5 1.5 0

0.5 1.5 1.5

Fig. 2. (A) Sizes and polydispersity indexes of Gel-NPs and control NPs (mean ± SD; n = 3). (B) TEM micrographs showing the size and morphology of Gel-NPs (gelucire content of 50%, w/w). (C) Stability of control NPs and Gel-NPs on storage at 25 ◦ C.

apparent changes in morphology of NPs made with varying amounts of gelucire (data not shown). The data suggested that a potential benefit of gelucire content is in serving as a colloidal stabilizer or a protective barrier against flocculation or coalescence phenomena. The observation was supported by NP stability studies (Fig. 2C). The sizes of control NPs (made without gelucire) increased about 5 fold after storage at 25 ◦ C for seven days (Fig. 2C). However, Gel-NPs demonstrated stability of sizes upon storage and the trend was pronounced as gelucire content in NPs increased. For instance, the average size Gel-NPs (gelucire content 50%, w/w) of 142 nm on day 1 was maintained at 189 nm on day 7 (p = 0.051). A similar trend was observed at gelucire content of 75% (w/w) where sizes of the resultant NPs on days 1 and 7 (p = 0.14) were 119 nm and 146 nm, respectively. It is considered that a potential challenge to the application of gelucire as a colloidal stabilizer will be the possible formation of multimodal particle population that will not fit into the Gaussiantype distribution. This is because in aqueous solvents, depending on the concentration, gelucires (when applied alone) could be solubilized or dispersed forming micelles, microscopic globules or vesicles that could lead to multiple particle populations. In this

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Fig. 3. FT-IR spectra of (A) pure gelucire alone and (B) freeze-dried Gel-NPs (gelucire content of 50%, w/w).

regard, the data obtained from the NP stability studies (Fig. 2C) were analyzed using a multimodal (NICOMP) distribution model on the particle sizer in order to detect the presence of heterogeneous (multimodal) size distributions. The multimodal particle distribution analysis indicated (data not shown) that the gelucire component most likely contributed to formation of a single particle population and not as separate aggregates/micelles that would have created heterogeneous size distributions. 3.2. FT-IR measurements FT-IR measurements were carried out to ascertain if gelucire component contributed to the resultant Gel-NP construct/formation which will be evident from FT-IR spectra. In this study, Gel-NPs were prepared; purified and freeze-dried. Subsequently, the freeze-dried Gel-NP powder was used for FT-IR measurements while pure gelucire and control NPs (without gelucire) were used as controls. The key features from the gelucire spectrum (Fig. 3A) are: (a) a band at 1737 cm−1 for carbon–oxygen double bonds of an ester; and (b) a band at 2882.59 cm−1 corresponding to stretch band of carbon–hydrogen single bond. While the major features in FT-IR spectrum of CA alone (data not shown) are: a major band at 3200 cm−1 corresponding to O H stretch in addition to C H stretch at 2954, 2916 and 2848 cm−1 and C O

stretch band at 1063 cm−1 . A representative FT-IR spectrum for Gel-NPs (gelucire content of 50%, w/w) is shown in Fig. 3B. The key features in the FT-IR spectrum (Fig. 3B) that strongly indicated that gelucire was most likely involved in NP formation/construct are: (i) the presence of O H stretch band at 3479 cm−1 as contributed from CA; and (ii) a band at 1736 cm−1 of saturated ester group arising from gelucire component. The trend of the data suggested the involvement and retention of gelucire content in the cured NP construct. In this regard, it is envisaged that the lipophilic groups on gelucire most probably served as the link to the NP matrix while the hydrophilic groups will be displayed on NP surface. 3.3. P-gp efflux assay and macrophage interaction The P-pg function assay is an important assessment that has a great clinical relevance. P-gp is a member of the ATP-binding cassette transporter family that is able to efflux drugs and therapeutic agents against the concentration gradient thereby resulting in cellular drug accumulation deficit with possible associated therapeutic failure or drug resistance. The study was conducted in P-gp expressing MES-Dx5 cell line [10] using Rh123 as a substrate for P-gp. For positive control, Rh123 cell accumulation with verapamil treatment (P-gp efflux inhibitor) or CA control NPs prepared with Tween 80 (Tween 80 is a P-gp efflux inhibitor) were applied (Fig. 4).

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Fig. 4. P-gp assay using Rh123 intracellular accumulation (mean ± SD; n = 6–8) in MES-Dx5 cells upon treatment with Gel-NP samples at 50% (w/w) gelucire (CA/Gel50%) and at 75% (w/w) gelucire (CA/Gel-75%). Untreated cells were used as controls. Verapamil and CA-NPs made with Tween 80 (CA/T80) served as positive controls.

The results showed that Rh123 cell accumulation in untreated cells (negative control) was significantly lower than cells that were treated with Gel-NP samples (p < 0.001). The efficacy of gelucire in inhibiting P-gp efflux was expressed in that the intracellular levels of Rh123 with verapamil treatment was more than 2.4 fold lower than that observed with Gel-NPs (Fig. 4). Also, control NPs made with Tween 80 showed Rh123 accumulation that was comparable to that obtained with Gel-NPs (gelucire content 75%, w/w) (p = 0.14). Thus, a potential benefit of gelucire content in Gel-NPs will be as an inhibitor of P-gp efflux function as demonstrated from the intracellular retention of Rh123. Current follow-up studies are investigating the application of Gel-NPs as delivery systems for anticancer drugs with the potential of overcoming multi-drug resistance in some tumors. Another important aspect of NP properties that could be influenced by gelucire content is the nature of macrophage interaction as expressed by in vitro uptake and activation. For the uptake studies, NPs labeled with either coumarin-6 or NBD were used. To ensure that all the NPs were comparable based on the entrapped fluorescent label, some key initial studies were conducted to verify the entrapment efficiency and stability of the fluorescent-labeled NPs in cell growth medium after incubation at 37 ◦ C for 24 h (data not shown). The data from in vitro uptake studies show that control NPs (made without gelucire) had the highest levels of intracellular fluorescence intensity indicating the extent of NP uptake by macrophages (Fig. 5A). Compared with control NPs, all Gel-NPs resulted in significant reduction in macrophage uptake (p < 0.001). For instance, at gelucire content of 25% (w/w), the intracellular fluorescence after NP exposure to macrophages was 3 fold lower than in control NPs (made without gelucire). Analysis of macrophage uptake based on gelucire content (Fig. 5A) reflected an inverse relationship between the gelucire content in NPs and macrophage uptake (Fig. 5B). This is a strong indication that inclusion of gelucire in NPs could remarkably reduce the extent of NP uptake by macrophages. It was earlier reported that the extent of macrophage uptake is dependent on NP surface properties as well as sizes [21,22]. Based on the report, one can expect that small NP sizes will translate into less favorable macrophage uptake and vice versa. Further, the importance of NP surface properties is demonstrated in that phagocytic uptake of stealth NPs was very low compared to non-stealth [23]. Thus, one could ascribe the observed effects of gelucire in reducing macrophage uptake to either modification of NP surface properties or size reduction. As a follow-up, additional experiments were carried out to investigate if NP size or NP surface modification played influential roles in the observed gelucire effects on macrophage uptake. It was observed that application of control NPs and Gel-NPs that have comparable sizes did not negate

Fig. 5. (A) The extent of Gel-NP uptake by RAW 264.7 macrophage cells (mean ± SD; n = 5) as monitored by intracellular fluorescence (Fl) intensity. (B) Inverse relationship between macrophage uptake and gelucire component in NPs. (C) Effects of control NPs and Gel-NPs on macrophage activation (mean ± SD; n = 4) in comparison to hydrogen peroxide as a positive control.

the observed reduction of macrophage uptake in Gel-NPs. Thus, the effect of gelucire content in modifying NP surface most probably played a major role in macrophage uptake. Current studies are focused on investigating the rate; extent and mechanism of uptake by macrophages and the correlation to NP biocompatibility. Macrophage activation studies were conducted to assess the biocompatibility of NPs made with or without gelucire. Macrophage activation was monitored by ROS production using hydrogen peroxide as a positive control. This is based on earlier reports on production of ROS by macrophages during phagocytosis or when activated as part of the host-defense mechanism [10]. The level of ROS after each NP treatment is presented in comparison to hydrogen peroxide as the positive control (Fig. 5C). It was observed that all the NP samples (with or without gelucire) had negligible levels of ROS that were comparable to PBS as the negative control (p > 0.069). The data suggested that the NP samples were biocompatible and did not activate macrophages. Since all the NP samples have CA as the main component, the trend is supported by previous studies that demonstrated the safety and biocompatibility of CA as a NP matrix material [2,20]. However, additional follow-up

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the hydrophobic portion on the molecule while the hydrophilic groups (PEG) will be displayed on NP surface. We are currently investigating the performance of PTX-loaded Gel-NPs in delivery and antitumor efficacy studies. 4. Conclusion

Fig. 6. Blood levels in BALB/c mice (mean ± SD; n = 5–6 mice) of paclitaxel-loaded Gel-NPs (50%, w/w gelucire) and Gel-NPs coated with 5% (w/w) DSPE-PEG (Gel-NPsDSPE-PEG).

studies are warranted to monitor ROS production after NP incubation with macrophages at various time points as well as assess other mediators of macrophage activation such as nitrite and cytokine production. 3.4. Retention of gelucire-based NPs in blood circulation A unique benefit of gelucire-mediated NP surface modification could be in prolonging NP blood circulation time. This is because the stability of any delivery system in blood circulation will ensure that the nanocarriers can effectively deliver therapeutically effective concentration of the drug at the desired site of action. In this regard, blood circulation of any delivery system will be influenced by: (i) size stability in blood circulation; (ii) biocompatibility; (iii) NP surface properties; and (iv) RES uptake. The initial aspect of the study was carried out using coumarin-6 loaded Gel-NPs injected intravenously in BALB/c mice. The procedure was developed based on other studies that successfully applied fluorescent-labeled nanocarriers in blood profile studies [22,25]. Data obtained showed that Gel-NPs (gelucire-50%, w/w) had the highest level of blood fluorescence at 3 h post-injection. For all NP samples, the ranking of blood fluorescence intensity (3 h-post injection) from lowest to highest is as follows: control NPs (at 0%, w/w gelucire) < Gel-NPs (at 25%, w/w) < Gel-NPs (at 75%, w/w) < Gel-NPs (at 50%, w/w). Compared with control NPs, all the Gel-NPs showed higher levels in blood circulation. The highest blood retention of Gel-NPs (at gelucire content of 50%, w/w) is possibly an indication that apart from surface modification, the in vivo performance of Gel-NPs may be dependent on the ability of the resultant NPs to withstand the hydrodynamic pressure in blood circulation without premature drug release and disruption NP integrity. A follow up study was conducted using PTX-loaded Gel-NPs (50%, w/w gelucire) with or without DSPE-PEG2000 coating. The NPs were administered by tail vein injections in BALB/c mice. The retention of NPs in blood circulation was measured by blood levels of PTX (Fig. 6). It was observed that PTX blood levels from GelNPs without DSPE-PEG2000 coating were comparable to Gel-NPs that were coated with DSPE-PEG2000 (p > 0.6). The data strongly indicated the possible effectiveness of gelucire in achieving the desired NP surface modification that can sustain NPs in blood circulation. Thus, a potential benefit of the gelucire content as reported will be in providing stealth properties to NPs. This is supported by the amphiphatic nature of gelucires as polyglycolized glycerides consisting of mono- and di-fatty esters of PEG [24,25]. Thus, it is conceivable that gelucire can be anchored to NP matrix through

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