http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, 2014; 31(6): 600–608 ! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2014.898709
Cellular uptake of Nigella sativa oil-PLGA microparticle by PC-12 cell line Abd Almonem Doolaanea1, Nur ‘Izzati Mansor1, Nurul Hafizah Mohd Nor1, and Farahidah Mohamed1,2,3 1
Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University Malaysia (IIUM), Kuantan, Malaysia, International Institute of Halal Research & Training (INHART), Kulliyyah of Engineering, Kuala Lumpur, Malaysia, and 3IKOP Sdn. Bhd., Pilot Plant Pharmaceutical Manufacturing, Faculty of Pharmacy, IIUM, Kuantan, Malaysia
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Abstract
Keywords
The aim of this study is to investigate the cell uptake of Nigella sativa oil (NSO)-PLGA microparticle by neuron-like PC-12 cells in comparison to surfactants; hydrophilic (Tween 80 & Triton X100) and hydrophobic (Span 80). Solvent evaporation was used to precisely control the size, zeta potential and morphology of the particle. The results revealed varying efficiencies of the cell uptake by PC-12 cells, which may be partially attributed to the surface hydrophobicity of the microparticles. Interestingly, the uptake efficiency of PC-12 cells was higher with the more hydrophilic microparticle. NSO microparticle showed evidence of being preferably internalised by mitotic cells. Tween 80 microparticle showed the highest cell uptake efficiency with a concentration-dependent pattern suggesting its use as uptake enhancer for nonscavenging cells. In conclusion, PC-12 cells can take up NSO-PLGA microparticle which may have potential in the treatment of neurodegenerative disease.
Hydrophobicity, microparticles, neurodegenerative, Nigella sativa oil, PLGA, surfactant, uptake
Introduction Neurodegenerative diseases include a variety of conditions characterised by the loss of neuronal activities among which the Alzheimer’s and Parkinson’s diseases are the most common neurodegenerative diseases worldwide (Sahni et al., 2011). It was reported that more than 24.3 million people were affected by Alzheimer’s disease with at least one new case reported every 7 s. In turn, it is one of the most severe progressive burden in the disease management globally (Ferri et al., 2006). In addition, dementia cases were predicted to increase up to 71% and 300% in the developed countries and developing countries, respectively, by 2040 (Ferri et al., 2006). The blood–brain barrier (BBB) is presented by the cerebrovascular endothelium sealed with tight junctions. Extra structures are present like supportive cells such as pericytes, astrocyte endfeet, and a discontinuous basal membrane (or basal lamina). The BBB is the primary impediment for the delivery of large or hydrophilic substances into the brain (Krol, 2012). It was reported that a sustained-release of drug was achievable by direct delivery to the brain interstitium using a biodegradable polymer as the micro-carrier while the fate of the drug is associated with the rate of local binding and internalisation (Garcia-Garcia et al., 2005). The superiority of the micro-carrier over other delivery strategies is notably due to its size, which easily allows direct implantation of the micro-carrier containing drugs by stereotaxy in the discrete functional areas of the brain without damaging the surrounding
Address for correspondence: Farahidah Mohamed, Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University Malaysia (IIUM), Kuantan, Malaysia, Malaysia. Tel: +60133299016. Fax: 609 571 6775. E-mail: [email protected]
History Received 4 September 2013 Revised 16 January 2014 Accepted 10 February 2014 Published online 2 April 2014
tissue. Besides, this implantation was more convenient than the insertion of catheters and pumps via open surgery with feasible multiple and repeat dosing (Patel et al., 2009). The GliadelÕ , a polymer-mediated implant drug delivery system, delivering antineoplastic agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU; also known as carmustine) to the brain tumours, represents the first successful drug developed from this microencapsulation technology. It was also the first new treatment procedure approved by the FDA for malignant gliomas (Wang et al., 2002). There are numerous compounds of plant origin that exhibit neuro-protective and neuro-regenerative effects. These include Nigella sativa, curcumin, catechins and resveratrol (Sahni et al., 2011). Curcumin is one of the most extensively studied phytochemicals for its benefits in neuro-degenerative diseases (Dikshit et al., 2006; Yang et al., 2008; Zhao et al., 2011). Whereas, N. sativa, an annual herbaceous plant belonging to the Ranuculacea family had received greater attention than curcumin since it is an ancient herb and an established ethno-medicine. Mature seeds of N. sativa have been consumed for edible and medicinal purposes. The seeds and their oil/extracts have been documented to have multiple medical benefits, amongst them neuroregeneration is of great concern. In a review, Paarakh (2010) reported that N. sativa was demonstrated to have antitumour, antidiabetic, antidepressant, anti-inflammatory, antioxidant, anticonvulsant, antinociceptive and anxiolytic properties. It was predicted that the list of its therapeutic effects is endless. It has been shown that N. sativa volatile oil caused morphological improvement on the degenerative neurons in the frontal cortex and brain stem following chronic toluene exposure in rats (Kanter, 2008). In a separate study, Azzubaidi et al. (2012) demonstrated that N. sativa fixed oil produced a noticeable spatial cognitive preservation in rats when challenged with chronic cerebral hypoperfusion.
Cell uptake of Nigella sativa oil-PLGA microparticle
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DOI: 10.3109/02652048.2014.898709
Microencapsulation of the active substances has been proven to enhance efficacy of therapeutic agent of interest by providing a sustained release coupled with potential of targeting to the desired site. While several researches have reported on curcumin microencapsulation, researches investigating microencapsulation of N. sativa oil (NSO) are profoundly lacking particularly those intended for brain delivery. Similarly, several studies have been conducted on the cell uptake by white blood cells or epithelial cells whereas studies using other cell types are still lacking. Therefore, this study aimed to investigate the cell uptake of poly (lactic-co-glycolic acid) (PLGA) microparticles modified with NSO and other surfactants (Span 80, Tween 80 and Triton X100) by neuron-like cell line, i.e. PC-12 cells. These microparticles are intended to be used for the treatment of neurodegenerative diseases. PLGA was used as the encapsulating polymer because of its well-known biocompatibility and biodegradability (Bala et al., 2004). Coumarin-6 was used as a fluorescent probe to quantify the cell uptake and to aid visualisation of the microparticles in the cell culture. PC-12 cell line was established in 1976 as a useful model system for neurobiological and neurochemical studies (Greene and Tischler, 1976). In several studies, PC-12 cell line has served as an in-vitro model for neurodegenerative diseases like Alzheimer’s disease (Shearman et al., 1994; Ge and Lahiri, 2002; Leutz et al., 2002), Parkinson’s disease (Elkon et al., 2001; Ryu et al., 2002) and Huntington’s disease (Peters et al., 2002). It was also used as a model for neurotoxicological studies (Shafer and Atchison, 1991), as an ischemic tolerance model (Hillion et al., 2005) and for cell signalling studies (Vaudry et al., 2002).
Materials PLGA5004 [lactic to glycolic acid ratio is 50:50 and the intrinsic viscosity (IV) is 0.4 dl/g] was obtained from PURAC (Gorinchem, Netherland). Polyvinyl alcohol (PVA) was obtained from BDH Chemicals (Poole, England). Dichloromethane (DCM) and acetone (AC) were purchased from Sigma-Aldrich (Steinheim, Germany). Span 80, Tween 80 and Triton X100 were supplied by Merck (Hohen-brunn, Germany). Nigella sativa oil, manufactured by cold-press was obtained from Hemani (Karachi, Pakistan). All other chemicals used in this study were of analytical grade unless otherwise stated. Fabrication of microparticle Microparticles were prepared by a modified double emulsionsolvent evaporation method (Ismail et al., 2012) according to the formulation as shown in Table 1. The oil phase was prepared by dissolving PLGA in a mixture of DCM and acetone followed by the addition of coumarin-6 and either NSO or the respective surfactants as surface modifiers to incur either hydrophobic or hydrophilic properties to the microparticles. The primary W/O Table 1. Formulation of the PLGA microparticles loaded with N. sativa oil and different surfactants.
Internal aqueous phase External aqueous phase and hardening
emulsion was obtained by homogenising the internal aqueous phase consisted of distilled water with the oil phase at 14 000 rpm for 1 min using IKAÕ T10 basic homogeniser (IKA Werke GmbH and Co., Staufen, KG, Germany). The primary emulsion was directly transferred to an aqueous PVA solution and homogenised at 14 000 rpm for 3 min to yield the secondary W/O/W emulsion. The latter was stirred for 2 h to evaporate the solvents while hardening the microparticles. Finally, the microparticles were collected by centrifugation at 5000 g for 3 min, washed three times with distilled water then lyophilised. Characterisation of the microparticles Particle size analysis Particle size distribution of the microparticles was measured by laser diffraction using Laser Particle Size Analyser BT-9300H (Dandong Bettersize Instruments, Dandong, China) and expressed as volume median diameter (D 50%). The span value was used to express particle size distribution (polydispersity) and calculated as the difference in particle diameters at 90% (D 90%) and 10% (D 10%) cumulative volume, divided by the D 50%. Surface morphology Microparticles were sprinkled onto aluminium stubs, pre-pasted with carbon adhesive tapes. Samples were sputter-coated with gold-palladium and viewed using Carl Zeiss EvoÕ 50 Scanning Electron Microscope (Oberkochen, Germany). Zeta potential analysis
Materials and methods
Oil phase
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PLGA5004 Coumarin-6 Dichloromethane Acetone Formulation-1: Span 80 Formulation-2: Tween 80 Formulation-3: Triton X100 Formulation-4: NSO H2O Polyvinyl alcohol 3%
100 mg 100 mg 1 ml 1 ml 22 ml
200 ml 22 ml
Zeta potentials for the microparticles in different pH values ranging from 3.0 to 9.0 were obtained by Zetasizer Nano Z (Malvern Instruments Ltd, Malvern, Worcestershire, UK) equipped with a Malvern autotitrator MPT-2. Briefly, about 1 mg of microparticles were suspended in 10 ml deionised water then the suspension was titrated with either 0.25 M HCl or 0.25 M NaOH to the desired pH starting from pH 3.0 to pH 9.0. Cell culture and cellular uptake measurement PC-12 cell line (American Type Tissue Culture, ATCC) was grown in 12-well plates supplied with Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO) and 10% FBS until 80% confluency. For each formulation, microparticles were suspended in serum-free DMEM and added to the wells at different concentrations (1, 2.5 and 5 mg microparticles per 1 ml medium). After incubation for 4 h at 37 C and 5% CO2, the cells were washed three times with icecold phosphate buffered saline (PBS) and the images were captured with a Nikon Eclipse Ti fluorescence microscope. After that, cellular uptake was quantified and normalised to total cell protein according to a modified method previously described by Davda and Labhasetwar (2002), as the following: Total cell protein First, a standard curve for a protein was constructed using a series of accurately prepared standard solutions of bovine serum albumin (BSA) (CalBiochem, San Diego, CA). The quantification was based on Bradford method (Bradford, 1976) using Bioquant reagent (Merck) according to the manufacturer’s instructions. From every standard solution, 10 ml were added to 100 ml of Bioquant reagent in 96-well plate and after 2 min the absorbance was measured at 595 nm using Tecan Infinite 200 microplate reader (Tecan Austria GmbH, Gro¨dig, Austria). The standard curve exhibited linearity in the tested range of BSA concentrations (0.01–0.1 mg/ml) with R2 ¼ 0.981. The cells in each well were lysed by adding 200 ml Promega cell culture lysis buffer
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(CCLR) (Promega, Madison, WI). Total cell protein in each well was quantified by adding 10 ml of the lysate to 100 ml of Bioquant reagent prior to measuring the absorbance at 595 nm as mentioned above.
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Quantification of the cellular uptake efficiency The fluorescent dye coumarin-6 was encapsulated in the microparticles and then extracted from each well following appropriate incubation time. To achieve that, the remaining lysate and the cell debris in each well were lyophilised, then 1 ml of methanol was added and incubated at 37 C for 24 h at 100 rpm using rotary incubator shaker (Innova 4000; New Brunswick Scientific Inc., Edison, NJ). A volume of 200 ml of the coumarin-6 that had dissolved into methanol was transferred to a black 96-well plate and the measurement of the fluorescence intensity was carried out using a fluorescence spectrophotometer (Perkin Elmer Corp., Norwalk, CT) at 450 nm excitation and 505 nm emission. To correlate the fluorescence intensity to the amount of microparticles, a standard curve for the fluorescence from the microparticles was obtained by adding known amounts of the microparticles containing coumarin-6 to 80% confluent PC-12 cells. After 4 h incubation time, the cells were lysed and lyophilised. Coumarin-6 was extracted with methanol and the fluorescence intensity from every known microparticle-cell mixture was quantified as mentioned above. Standard curves obtained from these known mixtures were shown in Figure 1. To overcome the possibility of encapsulating different amounts of coumarin-6 in the different formulations, a separate standard curve was obtained for each formulation. Finally, the cellular uptake efficiency was represented as the mass of microparticles (mg) relative to the mass of total cell protein (mg). Statistical analysis All data were reported as mean of triplicates ± standard deviation. One-way ANOVA with 95% confidence interval (p50.05) followed by Tukey post-test was applied to acquire
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significant differences between variables using SPSS (ver. 18) software (Chicago, IL).
Results and discussion Several studies reported the effect of the particle size on the cellular uptake by different cell lines (Tabata and Ikada, 1988a; Rudt and Mu¨ller, 1992, 1993b; Desai et al., 1997; Champion et al., 2008; Patel et al., 2012). The optimal particle size for cell uptake seems to be cell type-dependent but this has been paradoxically reported in the literature. Tabata and Ikada (1988a) found that the maximal phagocytosis of polystyrene and phenylated polyacrolein microspheres by mouse peritoneal macrophages occurred for the particle size of range 1.0–2.0 mm. Similar size range (2–3 mm) was found to be an optimal range for polystyrene microspheres to be phagocytosed by rat alveolar macrophages (Champion et al., 2008). In contrast, Foged et al. (2005) studied the uptake of 0.04–15 mm polystyrene particle by human dendritic cells and found that particles of diameters 0.5 mm and below were optimal for the cell uptake. Desai et al. (1997) used intestinal epithelial Caco-2 cells to study the uptake of PLGA microparticles of mean diameters 0.1 mm, 1 mm, and 10 mm containing bovine serum albumin as a model protein and 6-coumarin as a fluorescent marker. It was found that the small diameter microparticles (0.1 mm) had significantly greater uptake compared to larger diameter microparticles. These discrepancies may be due to the different cell lines employed in each study and also different types of polymers used in the microparticle fabrication. It also suggests that different uptake mechanisms were involved in different cell types. To date, most of the microparticle uptake studies used white blood cells (mostly macrophages and dendritic cell) and epithelial cells. The reasons behind this may include the trend in developing vaccine delivery using microparticle (Eldridge et al., 1990; O’Hagan, 1998) in addition to the role of macrophages (and other scavenging cells) in clearing the microparticles that need to be delineated to avoid rapid clearance (Juliano, 1988). In addition, the importance of rapid uptake by epithelial cells which were
Figure 1. Standard curves of the fluorescence intensity against microparticle concentrations. Known amounts of microparticles containing coumarin-6 were added to 80% confluent PC-12 cells in 12-well plates. After 4 h incubation, the cells were lysed, lyophilised then the coumarin-6 was extracted with methanol. The fluorescence intensity was measured at 450 nm excitation and 505 nm emissions. A: Span 80Span 80-loaded microparticles, B: Tween 80-loaded microparticles, C: Triton X100-loaded microparticles, D: NSO-loaded microparticles.
Cell uptake of Nigella sativa oil-PLGA microparticle
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DOI: 10.3109/02652048.2014.898709
desirable for therapeutic microparticles delivered through oral or pulmonary delivery (Desai et al., 1997; Edwards et al., 1998) were also prevalent. In this study, PC-12 cells were used as the model cell line in view of the microparticles’ usage as micro-carrier for therapeutics intended for the treatment or prevention of neurodegenerative diseases, and as such the testing on a neuron-like cell line is highly relevant. More importantly, we had successfully demonstrated that our modified method of microparticle preparation was able to precisely control the particle size to yield similar size range of particles regardless of type of surfactants. This was important to eliminate the influence of the particle size and hence investigation of other factors can be further evaluated. We fabricated all the four formulations to have similar particle size (D 50% was in the a narrow range from 3.82 ± 0.07 mm to 4.01 ± 0.10 mm) with no significant difference among all the formulations except for the difference between Span 80 microparticles and Triton X100 microparticles at D 10% and D 50% (p ¼ 0.020 and p ¼ 0.022, respectively). The polydispersity index was less than 0.5 with no significant difference (Table 2). Moreover, the four microparticle formulations exhibited smooth surface morphology (Figure 2) and comparable zeta-potential
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values in the range from pH 3.0 to 9.0 except for a slightly lower zeta-potential value for Span 80Span 80 (at pH &7.0, Span 80 microparticles displayed 13.2 mV compared to 5.5 mV for NSO microparticles) (Figure 3). Similar microparticles’ characteristics will eliminate the influence of zeta potential (Foged et al., 2005; He et al., 2010) on the cellular uptake efficiency. By controlling aforementioned characteristics, this study was able to focus on the influence of surface modification by means of surfactant types on the cell uptake of the microparticles. Coumarin-6 was employed here due to the previous studies (Desai et al., 1997; Qaddoumi et al., 2004; Tahara et al., 2010) that reported its reliable characteristic suitable for cellular and tissue uptake study of PLGA micro/nanoparticles. A recently published study (Panyam et al., 2003) on Coumarin-6 demonstrated that it did not cause acute toxicity and for that reason we did not perform the cell viability assay. The coumarin-6 was found stable inside the PLGA micro/nanoparticle post-encapsulation and upon contact with the cellular media will not instantly be released prior to being taken up by the micro/nanoparticles (Panyam et al., 2003). The study also reported that less than 0.5% of the dye was released when exposed to either physiological pH buffer or lipid environment mimicking cell membrane or even
Table 2. Particle size and polydispersity index (span value) of the microparticles fabricated with different surfactants and NSO. Formulation
D 10% (mm)
D 50% (mm)
D 90% (mm)
Span value
Span 80 microparticles Tween 80 microparticles Triton X100 microparticles NSO microparticles
3.02 ± 0.11 3.05 ± 0.10 3.16 ± 0.03* 3.12 ± 0.02
3.82 ± 0.07 3.84 ± 0.06 4.01 ± 0.10* 3.95 ± 0.16
4.72 ± 0.03 4.72 ± 0.03 5.02 ± 0.22 4.95 ± 0.50
0.44 ± 0.03 0.44 ± 0.03 0.46 ± 0.04 0.46 ± 0.11
Note: (*) indicates significant difference (p50.05) compared to Span 80 microparticles.
Figure 2. Scanning electron microscope images for PLGA microparticles fabricated with NSO and other surfactants. All scale bars are equivalent to 10 mm.
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Figure 3. Zeta-potential for PLGA microparticles fabricated with NSO and different ?surfactants. The measurements were carried out by suspending the microparticles in distilled water followed by titrating with either 0.25 M HCl or 0.25 M NaOH for corresponding pH 3.0 to 9.0.
acidic pH. The latter pH is useful in indicating that the dye was sufficiently stable inside the micro/nanoparticles to resist endolysosomal destruction that are acidic in nature. These properties ensure that the coumarin-6 will not dye the cells instead the fluorescence images that we viewed under inverted microscope was actually the viable encapsulated coumarin-6 inside the particles. In addition, to overcome any possibility that coumarin-6 was not homogenously loaded in the particles with different modifiers, standard curves were prepared from each formulation separately as shown in Figure 1 to normalise the baseline. Surface modification of the microparticles has a great influence on the cellular uptake as discussed in different studies (Tabata and Ikada, 1988a, 1988b; Rudt et al., 1993; Rudt and Mu¨ller, 1993a). In particular, hydrophobicity of the particles possesses an important role in determining the cell uptake of microparticles (Tabata and Ikada, 1988a; Andrianov and Payne, 1998). Tabata and Ikada (1988b) prepared microparticles of less than 2 mm size from L-lactic acid, DL-lactic acid, or glycolic acid homopolymers and copolymers of different molecular weights (2800–13 000 Da) and monomer compositions (lactide/glycolide ratio from 0 to 100). The phagocytosis of the microspheres was studied by mouse peritoneal macrophages in cell culture system. It was found that the chemical nature of starting polymers did not affect the extent of phagocytosis. In fact, contact angle measurement revealed that the prepared polymers were similar in their hydrophobicity wherein the contact angles were in the narrow range (66.5–72.5 degrees). However, the phagocytosis of the microspheres was reduced upon pre-coating with BSA, PVA, dextran, carboxymethyl cellulose (CMC), and polyvinyl alcohol (PVP). Rudt et al. (1993) studied the in vitro phagocytosis of varying size, surface-modified polystyrene particles (85, 480, 1030 and 3190 nm) in cultures of human granulocytes. Particles surface was modified by the adsorption of poloxamine polymers and Antarox surfactants (coating). It was found that uptake decreased with increasing adsorption layer thickness, length of the polyethylene oxide chain in the molecule and surface hydrophilicity of coated particles for both poloxamine and Antarox. In addition, Antarox surfactants that possessed a more hydrophobic anchor part (nonylphenol) for adsorption on the particle surface than poloxamine (polypropylene oxide) proved less effective in reducing phagocytosis. Moreover, in a similar study, Rudt and Mu¨ller (1993a) used human granulocytes to study the phagocytosis of 3190 nm polystyrene particles after the surface was modified by the adsorption of poloxamer block copolymers (coating). Poloxamers varied in the molecular structure: the molecular weight and the lengths of the polyethylene oxide (EO)
and the polypropylene oxide (PO) chains. It was found that poloxamer polymers with short EO and PO chains were less effective in reducing phagocytosis whereas poloxamers with the largest hydrophobic anchor parts (PO centre block) in combination with the longest EO chains proved to be most efficient in preventing phagocytosis (poloxamers 338 and 407). The observed reductions in in vitro phagocytic uptake were attributed to particle properties related to the thickness of the adsorbed layers, steric stabilisation, and reduction in surface hydrophobicity. In the present study, in the descending order of hydrophobicity, the microparticle modified with Tween 80 (HLB ¼ 15.0)5Triton X100 (HLB ¼ 13.5)5Span 80 (HLB ¼ 4.3) based on HLB. Whereas for NSO that does not have HLB value, and yet mainly consists of fatty acids that are hydrophobic in nature, we had classify hydrophobicity of NSO to be comparable to Span 80 based on observation that both of the microparticles exhibited similar difficulty to be re-suspended in water in contrast to Tween 80 or Triton X100 modified microparticles. Based on Figure 4(A), there was no significant difference in the cell uptake between NSO and Span 80 modified microparticles at the same concentration; p ¼ 1.000 for the concentration 1 mg/ml, p ¼ 0.998 for 2.5 mg/ml and p ¼ 1.000 for 5 mg/ml. At 1 mg/ml concentration, the uptake of microparticle modified with Tween-80 was significantly (p ¼ 0.017) higher than Span 80 but insignificantly different from Triton X100 (p ¼ 1.000) and NSO (p ¼ 0.170). In addition, no significant difference was observed between Triton X100 and NSO at 1 mg/ml concentration. On the other hand, at 2.5 mg/ml concentration, the uptake of microparticles modified with Tween 80 was significantly higher than Span 80 and NSO but not Triton X100 (p ¼ 0.311). It appeared that Triton X100 did not show any significant difference from any of the other three formulations at 2.5 mg/ml concentration. Furthermore, at the highest concentration tested (5 mg/ml), Tween 80 modified microparticles exhibited the highest uptake efficiency than the others followed by Triton X100 and Span 80. The results imply that the relatively hydrophobic microparticles (modified with Span 80 and NSO) showed similar cellular uptake regardless of concentrations whereas for relatively hydrophilic microparticles (modified with Tween 80 and Triton X100), a different uptake efficiency profile was observed. At low concentration, these relatively hydrophilic microparticles were taken up more efficiently than the relatively hydrophobic ones but not significantly different particularly from NSO modified microparticle. Addition of more microparticles (intermediate concentration) caused significantly higher uptake of the most hydrophilic microparticle (modified with Tween 80), than the
DOI: 10.3109/02652048.2014.898709
Cell uptake of Nigella sativa oil-PLGA microparticle
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Figure 4. Quantification of cellular uptake of microparticles by PC-12 cells. (A) Effect of different surfactants and NSO on the cellular uptake of the particles at the same concentration. (B) Effect of different concentrations of each microparticle formulation on cellular uptake. Asterisk (*) indicates significant difference (p50.05).
hydrophobic microparticles (modified with Span 80 and NSO) but it appeared that no significant different when comparing the uptake with the relatively less hydrophilic microparticles (modified with Triton X100). The uptake of Tween 80 modified microparticle continues to be dramatically increased at high concentration while Triton X100 modified microparticles exhibited less dramatic increase in uptake. Both particles however attained significantly higher uptake than relatively hydrophobic microparticles. These results clearly indicate that the uptake efficiency by PC-12 cells appear to favour relatively hydrophilic microparticles. With regard to the effect of concentration, the cell uptake of microparticles modified with NSO, Span 80, and Triton X100 did not show any significant difference in the tested range of concentrations (Figure 4B). In contrast, the cell uptake of microparticles modified with Tween 80 at 5 mg/ml concentration was significantly higher than its corresponding 1 and 2.5 mg/ml concentrations whereas no significant difference was observed between 1 and 2.5 mg concentrations (p ¼ 0.377). This finding shows that only Tween 80 modified microparticles displayed a concentration-dependent cell uptake in the concentration range used in this study.
To highlight, our findings are with disagreement with several studies, which reported that the hydrophobic microparticles were more readily taken up by the cell lines of interest than the hydrophilic ones (Tabata and Ikada, 1988b; Eldridge et al., 1990; Rudt et al., 1993; Rudt and Mu¨ller, 1993a). This incongruence may be due to the difference in the types of cell lines used in each study. As aforementioned, majority of uptake studies used white blood cells or epithelial cells to study the cell uptake of microparticles. For instance, mouse peritoneal macrophages were used by Tabata and Ikada (1988b), human granulocytes by Rudt and Mu¨ller (1993a), Rudt and Mu¨ller (1993b), and Rudt et al. (1993), gut-associated lymphoid tissues by Eldridge et al. (1990) and Caco-2 Cells by Desai et al. (1997). In the present study, neuron-like PC-12 cells which served as a model for neurorelated diseases were used. The uptake was higher for the more hydrophilic microparticles especially at high concentrations than the hydrophobic particles. In particular, Tween 80 as a surface modifier was the strongest in promoting the cell uptake. Indeed, cell uptake of the nanoparticles and microparticles may follow different mechanisms depending on the particle size, surface chemistry and cell types (Hu et al., 2009). Not many data
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Figure 5. Cellular uptake of NSO and Tween 80 microparticles by PC-12 cell line as seen by fluorescence inverted microscope. The cells were incubated with coumarin-6 containing microparticles for 4 h then washed with ice-cold PBS before viewing. (A) Untreated PC-12 cell line (phase contrast image), (B) PC-12 cell line treated with NSO microparticles (merged phase contrast image), (C) PC-12 cell line treated with Tween 80 microparticles (merged phase contrast image), (D) PC-12 cell line treated with Tween 80 microparticles (fluorescence phase contrast image for the same field). Note for online version: Different colour appeared in B and C actually due to different tone of the same colour, i.e. orange whereby in the B it appeared darker than in the C. The arrow labelled with ‘‘1’’ shows the mitotic cells while ‘‘2’’ shows the differentiated cells. Boxes were drawn to surround individual cell to aid clarity in understanding the images with arrow ‘‘3’’ pointing to the nucleus and arrow ‘‘4’’ pointing to the cytoplasm containing the microparticles.
available about the mechanism of the uptake of microparticles into cell lines other than white blood cells or epithelial cells. However, phagocytosis is the commonly reported mechanism for microparticles (Desai et al., 1997; Foster et al., 2001; Thiele et al., 2001; Yoshida and Babensee, 2006; Hu et al., 2009). Here, it could be that the phagocytosis route with non-specific interactions between the microparticles and cells that governed the uptake of the modified microparticles. Previous study by Tahara et al. (2010) reported that the uptake of Tween 80 modified nanospheres by A549 cells was enhanced by an unknown specific endocytic pathway. However, it is not clear if similar mechanism had taken place here. It seems that microparticles modified with Triton X100, Span 80 and NSO had reached its saturation of cell uptake even at the lowest concentration tested. The saturation of cell uptake of microparticles or nanoparticles had been reported before (Davda and Labhasetwar, 2002; Qaddoumi et al., 2004). On the other hand, the uptake of Tween 80 microparticles was still increasing at the higher concentrations. This indicates that Tween 80 microparticle may utilise a specific pathway for endocytosis. Due to unavailability of confocal microscope, we had proceeded with capturing the images using the inverted fluorescence microscope which had been employed by other study of cellular uptake. The study had even derived some quantitative data using such instrument (Rivolta et al., 2011). The use of fluorescence microscopy alone in cell uptake studies were also reported in other studies (Zauner et al., 2001; Patel et al., 2012).
It is clearly depicted by Figure 5 that all the cells appeared healthy post microparticles loading throughout the incubation time of 4 h. The images from fluorescence microscope revealed that NSO microparticles were successfully internalised into PC-12 cells. It seems that the cell uptake of NSO microparticles occurred preferentially during mitosis (Figure 5). The dividing cells appear smaller and rounded while the differentiated cells appear elongated and longer. This finding may be related to the known anticancer effect of NSO and N. sativa extracts (Ait Mbarek et al., 2007; Randhawa and Alghamdi, 2011). On the other hand, the cell uptake of Tween 80 microparticles seems to take place during various stages of the cell cycle based on the homogenous distribution of the fluorescence images (Figure 5C). These results confirmed that the cell uptake had taken place for NSO microparticles by PC-12 cells. They also suggest that Tween 80 may be served as a useful co-modifier to the NSO-PLGA microparticles in facilitating cell uptake for maximum cell loading.
Conclusion The results revealed that a non-scavenging cell, the neuron-like PC-12 cells was able to take up PLGA microparticles modified with NSO and various surfactants at varying efficiency. Selective uptake of NSO microparticles during mitotic stage suggest potential use in targeted drug delivery particularly envisaged for neurodegenerative diseases. Unlike the uptake by white blood
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cells or epithelial cells, the uptake efficiency of PC-12 cells favoured relatively hydrophilic microparticles, which may be the case for other non-scavenging cells, whereby the use of Tween 20 as uptake enhancer is highly purported.
Declaration of interest This work was funded by Ministry of Science, Technology & Innovation of Malaysia (MOSTI) (Grant ID: 02-01-08-SF0101).
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Cellular uptake of Nigella sativa oil-PLGA microparticle by PC-12 cell line Abd Almonem Doolaanea1, Nur ‘Izzati Mansor1, Nurul Hafizah Mohd Nor1, and Farahidah Mohamed1,2,3 1
Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University Malaysia (IIUM), Kuantan, Malaysia, International Institute of Halal Research & Training (INHART), Kulliyyah of Engineering, Kuala Lumpur, Malaysia, and 3IKOP Sdn. Bhd., Pilot Plant Pharmaceutical Manufacturing, Faculty of Pharmacy, IIUM, Kuantan, Malaysia
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Abstract
Keywords
The aim of this study is to investigate the cell uptake of Nigella sativa oil (NSO)-PLGA microparticle by neuron-like PC-12 cells in comparison to surfactants; hydrophilic (Tween 80 & Triton X100) and hydrophobic (Span 80). Solvent evaporation was used to precisely control the size, zeta potential and morphology of the particle. The results revealed varying efficiencies of the cell uptake by PC-12 cells, which may be partially attributed to the surface hydrophobicity of the microparticles. Interestingly, the uptake efficiency of PC-12 cells was higher with the more hydrophilic microparticle. NSO microparticle showed evidence of being preferably internalised by mitotic cells. Tween 80 microparticle showed the highest cell uptake efficiency with a concentration-dependent pattern suggesting its use as uptake enhancer for nonscavenging cells. In conclusion, PC-12 cells can take up NSO-PLGA microparticle which may have potential in the treatment of neurodegenerative disease.
Hydrophobicity, microparticles, neurodegenerative, Nigella sativa oil, PLGA, surfactant, uptake
Introduction Neurodegenerative diseases include a variety of conditions characterised by the loss of neuronal activities among which the Alzheimer’s and Parkinson’s diseases are the most common neurodegenerative diseases worldwide (Sahni et al., 2011). It was reported that more than 24.3 million people were affected by Alzheimer’s disease with at least one new case reported every 7 s. In turn, it is one of the most severe progressive burden in the disease management globally (Ferri et al., 2006). In addition, dementia cases were predicted to increase up to 71% and 300% in the developed countries and developing countries, respectively, by 2040 (Ferri et al., 2006). The blood–brain barrier (BBB) is presented by the cerebrovascular endothelium sealed with tight junctions. Extra structures are present like supportive cells such as pericytes, astrocyte endfeet, and a discontinuous basal membrane (or basal lamina). The BBB is the primary impediment for the delivery of large or hydrophilic substances into the brain (Krol, 2012). It was reported that a sustained-release of drug was achievable by direct delivery to the brain interstitium using a biodegradable polymer as the micro-carrier while the fate of the drug is associated with the rate of local binding and internalisation (Garcia-Garcia et al., 2005). The superiority of the micro-carrier over other delivery strategies is notably due to its size, which easily allows direct implantation of the micro-carrier containing drugs by stereotaxy in the discrete functional areas of the brain without damaging the surrounding
Address for correspondence: Farahidah Mohamed, Department of Pharmaceutical Technology, Faculty of Pharmacy, International Islamic University Malaysia (IIUM), Kuantan, Malaysia, Malaysia. Tel: +60133299016. Fax: 609 571 6775. E-mail: [email protected]
History Received 4 September 2013 Revised 16 January 2014 Accepted 10 February 2014 Published online 2 April 2014
tissue. Besides, this implantation was more convenient than the insertion of catheters and pumps via open surgery with feasible multiple and repeat dosing (Patel et al., 2009). The GliadelÕ , a polymer-mediated implant drug delivery system, delivering antineoplastic agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU; also known as carmustine) to the brain tumours, represents the first successful drug developed from this microencapsulation technology. It was also the first new treatment procedure approved by the FDA for malignant gliomas (Wang et al., 2002). There are numerous compounds of plant origin that exhibit neuro-protective and neuro-regenerative effects. These include Nigella sativa, curcumin, catechins and resveratrol (Sahni et al., 2011). Curcumin is one of the most extensively studied phytochemicals for its benefits in neuro-degenerative diseases (Dikshit et al., 2006; Yang et al., 2008; Zhao et al., 2011). Whereas, N. sativa, an annual herbaceous plant belonging to the Ranuculacea family had received greater attention than curcumin since it is an ancient herb and an established ethno-medicine. Mature seeds of N. sativa have been consumed for edible and medicinal purposes. The seeds and their oil/extracts have been documented to have multiple medical benefits, amongst them neuroregeneration is of great concern. In a review, Paarakh (2010) reported that N. sativa was demonstrated to have antitumour, antidiabetic, antidepressant, anti-inflammatory, antioxidant, anticonvulsant, antinociceptive and anxiolytic properties. It was predicted that the list of its therapeutic effects is endless. It has been shown that N. sativa volatile oil caused morphological improvement on the degenerative neurons in the frontal cortex and brain stem following chronic toluene exposure in rats (Kanter, 2008). In a separate study, Azzubaidi et al. (2012) demonstrated that N. sativa fixed oil produced a noticeable spatial cognitive preservation in rats when challenged with chronic cerebral hypoperfusion.
Cell uptake of Nigella sativa oil-PLGA microparticle
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DOI: 10.3109/02652048.2014.898709
Microencapsulation of the active substances has been proven to enhance efficacy of therapeutic agent of interest by providing a sustained release coupled with potential of targeting to the desired site. While several researches have reported on curcumin microencapsulation, researches investigating microencapsulation of N. sativa oil (NSO) are profoundly lacking particularly those intended for brain delivery. Similarly, several studies have been conducted on the cell uptake by white blood cells or epithelial cells whereas studies using other cell types are still lacking. Therefore, this study aimed to investigate the cell uptake of poly (lactic-co-glycolic acid) (PLGA) microparticles modified with NSO and other surfactants (Span 80, Tween 80 and Triton X100) by neuron-like cell line, i.e. PC-12 cells. These microparticles are intended to be used for the treatment of neurodegenerative diseases. PLGA was used as the encapsulating polymer because of its well-known biocompatibility and biodegradability (Bala et al., 2004). Coumarin-6 was used as a fluorescent probe to quantify the cell uptake and to aid visualisation of the microparticles in the cell culture. PC-12 cell line was established in 1976 as a useful model system for neurobiological and neurochemical studies (Greene and Tischler, 1976). In several studies, PC-12 cell line has served as an in-vitro model for neurodegenerative diseases like Alzheimer’s disease (Shearman et al., 1994; Ge and Lahiri, 2002; Leutz et al., 2002), Parkinson’s disease (Elkon et al., 2001; Ryu et al., 2002) and Huntington’s disease (Peters et al., 2002). It was also used as a model for neurotoxicological studies (Shafer and Atchison, 1991), as an ischemic tolerance model (Hillion et al., 2005) and for cell signalling studies (Vaudry et al., 2002).
Materials PLGA5004 [lactic to glycolic acid ratio is 50:50 and the intrinsic viscosity (IV) is 0.4 dl/g] was obtained from PURAC (Gorinchem, Netherland). Polyvinyl alcohol (PVA) was obtained from BDH Chemicals (Poole, England). Dichloromethane (DCM) and acetone (AC) were purchased from Sigma-Aldrich (Steinheim, Germany). Span 80, Tween 80 and Triton X100 were supplied by Merck (Hohen-brunn, Germany). Nigella sativa oil, manufactured by cold-press was obtained from Hemani (Karachi, Pakistan). All other chemicals used in this study were of analytical grade unless otherwise stated. Fabrication of microparticle Microparticles were prepared by a modified double emulsionsolvent evaporation method (Ismail et al., 2012) according to the formulation as shown in Table 1. The oil phase was prepared by dissolving PLGA in a mixture of DCM and acetone followed by the addition of coumarin-6 and either NSO or the respective surfactants as surface modifiers to incur either hydrophobic or hydrophilic properties to the microparticles. The primary W/O Table 1. Formulation of the PLGA microparticles loaded with N. sativa oil and different surfactants.
Internal aqueous phase External aqueous phase and hardening
emulsion was obtained by homogenising the internal aqueous phase consisted of distilled water with the oil phase at 14 000 rpm for 1 min using IKAÕ T10 basic homogeniser (IKA Werke GmbH and Co., Staufen, KG, Germany). The primary emulsion was directly transferred to an aqueous PVA solution and homogenised at 14 000 rpm for 3 min to yield the secondary W/O/W emulsion. The latter was stirred for 2 h to evaporate the solvents while hardening the microparticles. Finally, the microparticles were collected by centrifugation at 5000 g for 3 min, washed three times with distilled water then lyophilised. Characterisation of the microparticles Particle size analysis Particle size distribution of the microparticles was measured by laser diffraction using Laser Particle Size Analyser BT-9300H (Dandong Bettersize Instruments, Dandong, China) and expressed as volume median diameter (D 50%). The span value was used to express particle size distribution (polydispersity) and calculated as the difference in particle diameters at 90% (D 90%) and 10% (D 10%) cumulative volume, divided by the D 50%. Surface morphology Microparticles were sprinkled onto aluminium stubs, pre-pasted with carbon adhesive tapes. Samples were sputter-coated with gold-palladium and viewed using Carl Zeiss EvoÕ 50 Scanning Electron Microscope (Oberkochen, Germany). Zeta potential analysis
Materials and methods
Oil phase
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PLGA5004 Coumarin-6 Dichloromethane Acetone Formulation-1: Span 80 Formulation-2: Tween 80 Formulation-3: Triton X100 Formulation-4: NSO H2O Polyvinyl alcohol 3%
100 mg 100 mg 1 ml 1 ml 22 ml
200 ml 22 ml
Zeta potentials for the microparticles in different pH values ranging from 3.0 to 9.0 were obtained by Zetasizer Nano Z (Malvern Instruments Ltd, Malvern, Worcestershire, UK) equipped with a Malvern autotitrator MPT-2. Briefly, about 1 mg of microparticles were suspended in 10 ml deionised water then the suspension was titrated with either 0.25 M HCl or 0.25 M NaOH to the desired pH starting from pH 3.0 to pH 9.0. Cell culture and cellular uptake measurement PC-12 cell line (American Type Tissue Culture, ATCC) was grown in 12-well plates supplied with Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO) and 10% FBS until 80% confluency. For each formulation, microparticles were suspended in serum-free DMEM and added to the wells at different concentrations (1, 2.5 and 5 mg microparticles per 1 ml medium). After incubation for 4 h at 37 C and 5% CO2, the cells were washed three times with icecold phosphate buffered saline (PBS) and the images were captured with a Nikon Eclipse Ti fluorescence microscope. After that, cellular uptake was quantified and normalised to total cell protein according to a modified method previously described by Davda and Labhasetwar (2002), as the following: Total cell protein First, a standard curve for a protein was constructed using a series of accurately prepared standard solutions of bovine serum albumin (BSA) (CalBiochem, San Diego, CA). The quantification was based on Bradford method (Bradford, 1976) using Bioquant reagent (Merck) according to the manufacturer’s instructions. From every standard solution, 10 ml were added to 100 ml of Bioquant reagent in 96-well plate and after 2 min the absorbance was measured at 595 nm using Tecan Infinite 200 microplate reader (Tecan Austria GmbH, Gro¨dig, Austria). The standard curve exhibited linearity in the tested range of BSA concentrations (0.01–0.1 mg/ml) with R2 ¼ 0.981. The cells in each well were lysed by adding 200 ml Promega cell culture lysis buffer
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(CCLR) (Promega, Madison, WI). Total cell protein in each well was quantified by adding 10 ml of the lysate to 100 ml of Bioquant reagent prior to measuring the absorbance at 595 nm as mentioned above.
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Quantification of the cellular uptake efficiency The fluorescent dye coumarin-6 was encapsulated in the microparticles and then extracted from each well following appropriate incubation time. To achieve that, the remaining lysate and the cell debris in each well were lyophilised, then 1 ml of methanol was added and incubated at 37 C for 24 h at 100 rpm using rotary incubator shaker (Innova 4000; New Brunswick Scientific Inc., Edison, NJ). A volume of 200 ml of the coumarin-6 that had dissolved into methanol was transferred to a black 96-well plate and the measurement of the fluorescence intensity was carried out using a fluorescence spectrophotometer (Perkin Elmer Corp., Norwalk, CT) at 450 nm excitation and 505 nm emission. To correlate the fluorescence intensity to the amount of microparticles, a standard curve for the fluorescence from the microparticles was obtained by adding known amounts of the microparticles containing coumarin-6 to 80% confluent PC-12 cells. After 4 h incubation time, the cells were lysed and lyophilised. Coumarin-6 was extracted with methanol and the fluorescence intensity from every known microparticle-cell mixture was quantified as mentioned above. Standard curves obtained from these known mixtures were shown in Figure 1. To overcome the possibility of encapsulating different amounts of coumarin-6 in the different formulations, a separate standard curve was obtained for each formulation. Finally, the cellular uptake efficiency was represented as the mass of microparticles (mg) relative to the mass of total cell protein (mg). Statistical analysis All data were reported as mean of triplicates ± standard deviation. One-way ANOVA with 95% confidence interval (p50.05) followed by Tukey post-test was applied to acquire
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significant differences between variables using SPSS (ver. 18) software (Chicago, IL).
Results and discussion Several studies reported the effect of the particle size on the cellular uptake by different cell lines (Tabata and Ikada, 1988a; Rudt and Mu¨ller, 1992, 1993b; Desai et al., 1997; Champion et al., 2008; Patel et al., 2012). The optimal particle size for cell uptake seems to be cell type-dependent but this has been paradoxically reported in the literature. Tabata and Ikada (1988a) found that the maximal phagocytosis of polystyrene and phenylated polyacrolein microspheres by mouse peritoneal macrophages occurred for the particle size of range 1.0–2.0 mm. Similar size range (2–3 mm) was found to be an optimal range for polystyrene microspheres to be phagocytosed by rat alveolar macrophages (Champion et al., 2008). In contrast, Foged et al. (2005) studied the uptake of 0.04–15 mm polystyrene particle by human dendritic cells and found that particles of diameters 0.5 mm and below were optimal for the cell uptake. Desai et al. (1997) used intestinal epithelial Caco-2 cells to study the uptake of PLGA microparticles of mean diameters 0.1 mm, 1 mm, and 10 mm containing bovine serum albumin as a model protein and 6-coumarin as a fluorescent marker. It was found that the small diameter microparticles (0.1 mm) had significantly greater uptake compared to larger diameter microparticles. These discrepancies may be due to the different cell lines employed in each study and also different types of polymers used in the microparticle fabrication. It also suggests that different uptake mechanisms were involved in different cell types. To date, most of the microparticle uptake studies used white blood cells (mostly macrophages and dendritic cell) and epithelial cells. The reasons behind this may include the trend in developing vaccine delivery using microparticle (Eldridge et al., 1990; O’Hagan, 1998) in addition to the role of macrophages (and other scavenging cells) in clearing the microparticles that need to be delineated to avoid rapid clearance (Juliano, 1988). In addition, the importance of rapid uptake by epithelial cells which were
Figure 1. Standard curves of the fluorescence intensity against microparticle concentrations. Known amounts of microparticles containing coumarin-6 were added to 80% confluent PC-12 cells in 12-well plates. After 4 h incubation, the cells were lysed, lyophilised then the coumarin-6 was extracted with methanol. The fluorescence intensity was measured at 450 nm excitation and 505 nm emissions. A: Span 80Span 80-loaded microparticles, B: Tween 80-loaded microparticles, C: Triton X100-loaded microparticles, D: NSO-loaded microparticles.
Cell uptake of Nigella sativa oil-PLGA microparticle
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DOI: 10.3109/02652048.2014.898709
desirable for therapeutic microparticles delivered through oral or pulmonary delivery (Desai et al., 1997; Edwards et al., 1998) were also prevalent. In this study, PC-12 cells were used as the model cell line in view of the microparticles’ usage as micro-carrier for therapeutics intended for the treatment or prevention of neurodegenerative diseases, and as such the testing on a neuron-like cell line is highly relevant. More importantly, we had successfully demonstrated that our modified method of microparticle preparation was able to precisely control the particle size to yield similar size range of particles regardless of type of surfactants. This was important to eliminate the influence of the particle size and hence investigation of other factors can be further evaluated. We fabricated all the four formulations to have similar particle size (D 50% was in the a narrow range from 3.82 ± 0.07 mm to 4.01 ± 0.10 mm) with no significant difference among all the formulations except for the difference between Span 80 microparticles and Triton X100 microparticles at D 10% and D 50% (p ¼ 0.020 and p ¼ 0.022, respectively). The polydispersity index was less than 0.5 with no significant difference (Table 2). Moreover, the four microparticle formulations exhibited smooth surface morphology (Figure 2) and comparable zeta-potential
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values in the range from pH 3.0 to 9.0 except for a slightly lower zeta-potential value for Span 80Span 80 (at pH &7.0, Span 80 microparticles displayed 13.2 mV compared to 5.5 mV for NSO microparticles) (Figure 3). Similar microparticles’ characteristics will eliminate the influence of zeta potential (Foged et al., 2005; He et al., 2010) on the cellular uptake efficiency. By controlling aforementioned characteristics, this study was able to focus on the influence of surface modification by means of surfactant types on the cell uptake of the microparticles. Coumarin-6 was employed here due to the previous studies (Desai et al., 1997; Qaddoumi et al., 2004; Tahara et al., 2010) that reported its reliable characteristic suitable for cellular and tissue uptake study of PLGA micro/nanoparticles. A recently published study (Panyam et al., 2003) on Coumarin-6 demonstrated that it did not cause acute toxicity and for that reason we did not perform the cell viability assay. The coumarin-6 was found stable inside the PLGA micro/nanoparticle post-encapsulation and upon contact with the cellular media will not instantly be released prior to being taken up by the micro/nanoparticles (Panyam et al., 2003). The study also reported that less than 0.5% of the dye was released when exposed to either physiological pH buffer or lipid environment mimicking cell membrane or even
Table 2. Particle size and polydispersity index (span value) of the microparticles fabricated with different surfactants and NSO. Formulation
D 10% (mm)
D 50% (mm)
D 90% (mm)
Span value
Span 80 microparticles Tween 80 microparticles Triton X100 microparticles NSO microparticles
3.02 ± 0.11 3.05 ± 0.10 3.16 ± 0.03* 3.12 ± 0.02
3.82 ± 0.07 3.84 ± 0.06 4.01 ± 0.10* 3.95 ± 0.16
4.72 ± 0.03 4.72 ± 0.03 5.02 ± 0.22 4.95 ± 0.50
0.44 ± 0.03 0.44 ± 0.03 0.46 ± 0.04 0.46 ± 0.11
Note: (*) indicates significant difference (p50.05) compared to Span 80 microparticles.
Figure 2. Scanning electron microscope images for PLGA microparticles fabricated with NSO and other surfactants. All scale bars are equivalent to 10 mm.
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Figure 3. Zeta-potential for PLGA microparticles fabricated with NSO and different ?surfactants. The measurements were carried out by suspending the microparticles in distilled water followed by titrating with either 0.25 M HCl or 0.25 M NaOH for corresponding pH 3.0 to 9.0.
acidic pH. The latter pH is useful in indicating that the dye was sufficiently stable inside the micro/nanoparticles to resist endolysosomal destruction that are acidic in nature. These properties ensure that the coumarin-6 will not dye the cells instead the fluorescence images that we viewed under inverted microscope was actually the viable encapsulated coumarin-6 inside the particles. In addition, to overcome any possibility that coumarin-6 was not homogenously loaded in the particles with different modifiers, standard curves were prepared from each formulation separately as shown in Figure 1 to normalise the baseline. Surface modification of the microparticles has a great influence on the cellular uptake as discussed in different studies (Tabata and Ikada, 1988a, 1988b; Rudt et al., 1993; Rudt and Mu¨ller, 1993a). In particular, hydrophobicity of the particles possesses an important role in determining the cell uptake of microparticles (Tabata and Ikada, 1988a; Andrianov and Payne, 1998). Tabata and Ikada (1988b) prepared microparticles of less than 2 mm size from L-lactic acid, DL-lactic acid, or glycolic acid homopolymers and copolymers of different molecular weights (2800–13 000 Da) and monomer compositions (lactide/glycolide ratio from 0 to 100). The phagocytosis of the microspheres was studied by mouse peritoneal macrophages in cell culture system. It was found that the chemical nature of starting polymers did not affect the extent of phagocytosis. In fact, contact angle measurement revealed that the prepared polymers were similar in their hydrophobicity wherein the contact angles were in the narrow range (66.5–72.5 degrees). However, the phagocytosis of the microspheres was reduced upon pre-coating with BSA, PVA, dextran, carboxymethyl cellulose (CMC), and polyvinyl alcohol (PVP). Rudt et al. (1993) studied the in vitro phagocytosis of varying size, surface-modified polystyrene particles (85, 480, 1030 and 3190 nm) in cultures of human granulocytes. Particles surface was modified by the adsorption of poloxamine polymers and Antarox surfactants (coating). It was found that uptake decreased with increasing adsorption layer thickness, length of the polyethylene oxide chain in the molecule and surface hydrophilicity of coated particles for both poloxamine and Antarox. In addition, Antarox surfactants that possessed a more hydrophobic anchor part (nonylphenol) for adsorption on the particle surface than poloxamine (polypropylene oxide) proved less effective in reducing phagocytosis. Moreover, in a similar study, Rudt and Mu¨ller (1993a) used human granulocytes to study the phagocytosis of 3190 nm polystyrene particles after the surface was modified by the adsorption of poloxamer block copolymers (coating). Poloxamers varied in the molecular structure: the molecular weight and the lengths of the polyethylene oxide (EO)
and the polypropylene oxide (PO) chains. It was found that poloxamer polymers with short EO and PO chains were less effective in reducing phagocytosis whereas poloxamers with the largest hydrophobic anchor parts (PO centre block) in combination with the longest EO chains proved to be most efficient in preventing phagocytosis (poloxamers 338 and 407). The observed reductions in in vitro phagocytic uptake were attributed to particle properties related to the thickness of the adsorbed layers, steric stabilisation, and reduction in surface hydrophobicity. In the present study, in the descending order of hydrophobicity, the microparticle modified with Tween 80 (HLB ¼ 15.0)5Triton X100 (HLB ¼ 13.5)5Span 80 (HLB ¼ 4.3) based on HLB. Whereas for NSO that does not have HLB value, and yet mainly consists of fatty acids that are hydrophobic in nature, we had classify hydrophobicity of NSO to be comparable to Span 80 based on observation that both of the microparticles exhibited similar difficulty to be re-suspended in water in contrast to Tween 80 or Triton X100 modified microparticles. Based on Figure 4(A), there was no significant difference in the cell uptake between NSO and Span 80 modified microparticles at the same concentration; p ¼ 1.000 for the concentration 1 mg/ml, p ¼ 0.998 for 2.5 mg/ml and p ¼ 1.000 for 5 mg/ml. At 1 mg/ml concentration, the uptake of microparticle modified with Tween-80 was significantly (p ¼ 0.017) higher than Span 80 but insignificantly different from Triton X100 (p ¼ 1.000) and NSO (p ¼ 0.170). In addition, no significant difference was observed between Triton X100 and NSO at 1 mg/ml concentration. On the other hand, at 2.5 mg/ml concentration, the uptake of microparticles modified with Tween 80 was significantly higher than Span 80 and NSO but not Triton X100 (p ¼ 0.311). It appeared that Triton X100 did not show any significant difference from any of the other three formulations at 2.5 mg/ml concentration. Furthermore, at the highest concentration tested (5 mg/ml), Tween 80 modified microparticles exhibited the highest uptake efficiency than the others followed by Triton X100 and Span 80. The results imply that the relatively hydrophobic microparticles (modified with Span 80 and NSO) showed similar cellular uptake regardless of concentrations whereas for relatively hydrophilic microparticles (modified with Tween 80 and Triton X100), a different uptake efficiency profile was observed. At low concentration, these relatively hydrophilic microparticles were taken up more efficiently than the relatively hydrophobic ones but not significantly different particularly from NSO modified microparticle. Addition of more microparticles (intermediate concentration) caused significantly higher uptake of the most hydrophilic microparticle (modified with Tween 80), than the
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Figure 4. Quantification of cellular uptake of microparticles by PC-12 cells. (A) Effect of different surfactants and NSO on the cellular uptake of the particles at the same concentration. (B) Effect of different concentrations of each microparticle formulation on cellular uptake. Asterisk (*) indicates significant difference (p50.05).
hydrophobic microparticles (modified with Span 80 and NSO) but it appeared that no significant different when comparing the uptake with the relatively less hydrophilic microparticles (modified with Triton X100). The uptake of Tween 80 modified microparticle continues to be dramatically increased at high concentration while Triton X100 modified microparticles exhibited less dramatic increase in uptake. Both particles however attained significantly higher uptake than relatively hydrophobic microparticles. These results clearly indicate that the uptake efficiency by PC-12 cells appear to favour relatively hydrophilic microparticles. With regard to the effect of concentration, the cell uptake of microparticles modified with NSO, Span 80, and Triton X100 did not show any significant difference in the tested range of concentrations (Figure 4B). In contrast, the cell uptake of microparticles modified with Tween 80 at 5 mg/ml concentration was significantly higher than its corresponding 1 and 2.5 mg/ml concentrations whereas no significant difference was observed between 1 and 2.5 mg concentrations (p ¼ 0.377). This finding shows that only Tween 80 modified microparticles displayed a concentration-dependent cell uptake in the concentration range used in this study.
To highlight, our findings are with disagreement with several studies, which reported that the hydrophobic microparticles were more readily taken up by the cell lines of interest than the hydrophilic ones (Tabata and Ikada, 1988b; Eldridge et al., 1990; Rudt et al., 1993; Rudt and Mu¨ller, 1993a). This incongruence may be due to the difference in the types of cell lines used in each study. As aforementioned, majority of uptake studies used white blood cells or epithelial cells to study the cell uptake of microparticles. For instance, mouse peritoneal macrophages were used by Tabata and Ikada (1988b), human granulocytes by Rudt and Mu¨ller (1993a), Rudt and Mu¨ller (1993b), and Rudt et al. (1993), gut-associated lymphoid tissues by Eldridge et al. (1990) and Caco-2 Cells by Desai et al. (1997). In the present study, neuron-like PC-12 cells which served as a model for neurorelated diseases were used. The uptake was higher for the more hydrophilic microparticles especially at high concentrations than the hydrophobic particles. In particular, Tween 80 as a surface modifier was the strongest in promoting the cell uptake. Indeed, cell uptake of the nanoparticles and microparticles may follow different mechanisms depending on the particle size, surface chemistry and cell types (Hu et al., 2009). Not many data
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Figure 5. Cellular uptake of NSO and Tween 80 microparticles by PC-12 cell line as seen by fluorescence inverted microscope. The cells were incubated with coumarin-6 containing microparticles for 4 h then washed with ice-cold PBS before viewing. (A) Untreated PC-12 cell line (phase contrast image), (B) PC-12 cell line treated with NSO microparticles (merged phase contrast image), (C) PC-12 cell line treated with Tween 80 microparticles (merged phase contrast image), (D) PC-12 cell line treated with Tween 80 microparticles (fluorescence phase contrast image for the same field). Note for online version: Different colour appeared in B and C actually due to different tone of the same colour, i.e. orange whereby in the B it appeared darker than in the C. The arrow labelled with ‘‘1’’ shows the mitotic cells while ‘‘2’’ shows the differentiated cells. Boxes were drawn to surround individual cell to aid clarity in understanding the images with arrow ‘‘3’’ pointing to the nucleus and arrow ‘‘4’’ pointing to the cytoplasm containing the microparticles.
available about the mechanism of the uptake of microparticles into cell lines other than white blood cells or epithelial cells. However, phagocytosis is the commonly reported mechanism for microparticles (Desai et al., 1997; Foster et al., 2001; Thiele et al., 2001; Yoshida and Babensee, 2006; Hu et al., 2009). Here, it could be that the phagocytosis route with non-specific interactions between the microparticles and cells that governed the uptake of the modified microparticles. Previous study by Tahara et al. (2010) reported that the uptake of Tween 80 modified nanospheres by A549 cells was enhanced by an unknown specific endocytic pathway. However, it is not clear if similar mechanism had taken place here. It seems that microparticles modified with Triton X100, Span 80 and NSO had reached its saturation of cell uptake even at the lowest concentration tested. The saturation of cell uptake of microparticles or nanoparticles had been reported before (Davda and Labhasetwar, 2002; Qaddoumi et al., 2004). On the other hand, the uptake of Tween 80 microparticles was still increasing at the higher concentrations. This indicates that Tween 80 microparticle may utilise a specific pathway for endocytosis. Due to unavailability of confocal microscope, we had proceeded with capturing the images using the inverted fluorescence microscope which had been employed by other study of cellular uptake. The study had even derived some quantitative data using such instrument (Rivolta et al., 2011). The use of fluorescence microscopy alone in cell uptake studies were also reported in other studies (Zauner et al., 2001; Patel et al., 2012).
It is clearly depicted by Figure 5 that all the cells appeared healthy post microparticles loading throughout the incubation time of 4 h. The images from fluorescence microscope revealed that NSO microparticles were successfully internalised into PC-12 cells. It seems that the cell uptake of NSO microparticles occurred preferentially during mitosis (Figure 5). The dividing cells appear smaller and rounded while the differentiated cells appear elongated and longer. This finding may be related to the known anticancer effect of NSO and N. sativa extracts (Ait Mbarek et al., 2007; Randhawa and Alghamdi, 2011). On the other hand, the cell uptake of Tween 80 microparticles seems to take place during various stages of the cell cycle based on the homogenous distribution of the fluorescence images (Figure 5C). These results confirmed that the cell uptake had taken place for NSO microparticles by PC-12 cells. They also suggest that Tween 80 may be served as a useful co-modifier to the NSO-PLGA microparticles in facilitating cell uptake for maximum cell loading.
Conclusion The results revealed that a non-scavenging cell, the neuron-like PC-12 cells was able to take up PLGA microparticles modified with NSO and various surfactants at varying efficiency. Selective uptake of NSO microparticles during mitotic stage suggest potential use in targeted drug delivery particularly envisaged for neurodegenerative diseases. Unlike the uptake by white blood
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cells or epithelial cells, the uptake efficiency of PC-12 cells favoured relatively hydrophilic microparticles, which may be the case for other non-scavenging cells, whereby the use of Tween 20 as uptake enhancer is highly purported.
Declaration of interest This work was funded by Ministry of Science, Technology & Innovation of Malaysia (MOSTI) (Grant ID: 02-01-08-SF0101).
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