Journal of Drug DeliveryVolume 2011 (2011), Article ID 980720, 13 pagesdoi:10.1155/2011/980720

Research Article

Nanoprodrugs of NSAIDs: Preparation and Characterization of Flufenamic Acid Nanoprodrugs

Bong-Seop Lee, Chi Woo Yoon, Arsen Osipov, Nuriel Moghavem, Daniel Nwachokor,  Rina Amatya, Rebekah Na, Joe L. Pantoja, Michael D. Pham, Keith L. Black, and John S. Yu

Department of Neurosurgery, Cedars-Sinai Medical Center, 8631 West Third Street, Suite 800 East, Los Angeles, CA 90048, USA

Received 16 December 2010; Revised 31 January 2011; Accepted 6 February 2011

Academic Editor: Sanyog Jain

Copyright © 2011 Bong-Seop Lee et al. Bong-Seop Lee et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

We demonstrated that hydrophobic derivatives of the nonsteroidal anti-inflammatory drug (NSAID)flufenamic acid (FA), can be formed into stable nanometer-sized prodrugs (nanoprodrugs) that inhibit the growth of glioma cells, suggesting their potential application as anticancer agent. We synthesized highly hydrophobic monomeric and dimeric prodrugs of FA via esterification and prepared nanoprodrugs using spontaneous emulsification mechanism. The nanoprodrugs were in the size range of 120 to 140 nm and physicochemically stable upon long-term storage as aqueous suspension, which is attributed to the strong hydrophobic interaction between prodrug molecules. Importantly, despite the highly hydrophobic nature and water insolubility, nanoprodrugs could be readily activated into the parent drug by porcine liver esterase, presenting a potential new strategy for novel NSAID prodrug design. The nanoprodrug inhibited the growth of U87-MG glioma cells with IC₅₀ of 20 μM, whereas FA showed IC₅₀ of 100 μM, suggesting that more efficient drug delivery was achieved with nanoprodrugs.

1. Introduction

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of drugs with analgesic, antipyretic and anti-inflammatory effects and have been widely used in the treatment of pain, fever, and inflammation. NSAIDs exert their anti-inflammatory activity through the inhibition of cyclooxygenase (COX) derived prostaglandin synthesis. COX has been recognized as the first enzyme in the formation of prostaglandin (PG) and thromboxane (TX) from arachidonic acid at the site of inflammation or after infection [1]. COX-1 isozyme is expressed constitutively in many tissues, whereas COX-2 isozyme is expressed only at the site of inflammation [2]. Recent studies have conjectured that elevated expression of COX-2 has been detected in various cancers, including colorectal, lung, breast, liver, head and neck, and brain tumors, whereas COX-1 expression was unaffected [3–5]. Several studies have also demonstrated that NSAIDs may be effective in the prevention and treatment of certain types of cancers [6–9]. The chemopreventive and antitumorigenic effects of NSAIDs are believed to be exerted through the induction of apoptosis followed by inhibition of COX-2 [10–13]. Some data also suggest a COX-2-independent mechanism because apoptosis induction by NSAIDs does not always correlate with their ability to inhibit COX-2 [14–17].

However, the major mechanism by which NSAIDs exert their anti-inflammatory activity, the inhibition of cyclooxygenase-derived prostaglandin synthesis, is also responsible for the adverse side effects, such as irritation and ulceration of the gastrointestinal (GI) mucosa [18]. It is generally believed that these GI side effects result from the combined effect of the irritation caused by the free carboxylic groups in NSAIDs and blockage of prostaglandin biosynthesis in the GI tract [19].

Prodrug strategy is widely recognized as a potential approach to overcome toxic side effects that are ascribed to the irritation caused by the free carboxylic groups in NSAIDs and blockage of prostaglandin biosynthesis in the GI tract. There have been several attempts to develop prodrugs of NSAIDs to overcome the adverse side effects as well as to improve their bioavailability by masking the carboxylic acid groups through the formation of bioreversible bonds [20–24].

The development of nanostructured biomaterials with antitumorigenic efficacy has received significant attention from the pharmaceutical industry, mainly because of their potential for precise targeting with less severe toxic side effects. Many effective anticancer therapeutics are low water soluble and must be in excessive amounts of organic cosolvents to obtain a therapeutically effective dose. This limits clinical applicability of these drugs. The formation into compact nanostructures obviates the need to use organic solvents, eliminating the interference of toxic side effects caused by cosolvents [25, 26]. In addition, by using a nanometer-sized delivery system, a significant drug loading per unit volume can be achieved, which is of crucial importance when high dosing is required.

In our effort to combine the prodrug concept and nanostructured drug/drug delivery system we demonstrated that water-insoluble prodrug compounds can be transformed into stable nanostructures obviating the need to dissolve the compounds in organic solvents. In our previous report we demonstrated six hydrophobic derivatives of NSAIDs (Figure 1) and their nanoprodrugs [27, 28]. In this study, we synthesized monomeric and dimeric prodrugs of flufenamic acid (FA, 2-[(3-trifluoromethylphenyl)amino]benzoic acid) and prepared nanoprodrugs through spontaneous emulsification of the prodrugs in acetone. Further, we demonstrated the antiproliferative effect of FA nanoprodrugs on U87GM glioma cells.

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Figure 1: Nanoprodrugs of NSAIDs. ALA: α-lipoic acid; Ind: indomethacin; Ibu: ibuprofen; Npx: naproxen; TEG: tetraethylene glycol.

2. Materials and Methods

2.1. General Procedures and Materials

Unless otherwise noted, solvents and chemicals were obtained at highest purity from Sigma-Aldrich Chemical Co. (St Louis, MO, USA) and used without further preparation. Chromatographic purification of the synthesized compounds was performed using silica gel (60 Å, 200–400 mesh). The compounds were confirmed by thin layer chromatography (TLC) silicagel plate (Merck 60 F254). Compounds containing α-lipoic acid were visualized by treatment with a solution of: 1.5 g of KMnO₄, 10 g of K₂CO₃, and 1.25 mL of 10% NaOH in 200 mL of H₂O, followed by gentle heating. The oxidized derivatives of FA were visualized under UV light. ¹H and ¹³C NMR spectra were conducted on a Varian 400 MHz spectrometer and chemical shifts (δ) were given in ppm relative to TMS. The spectra were recorded with the solvent CDCl₃ at room temperature.

2.2. High-Performance Liquid Chromatography

HPLC analysis was performed on a Merck-Hitachi analytical LaChrom D-7000 HPLC/UV detector system (Merck, Darmstadt, Germany) with a CAPCELL PAK, Type SG 120 (phenomenex, Torrance, CA, USA) C₁₈ reversed phase column (250/4.6 mm, 5 μm). The separation was performed under isocratic condition at a flow rate of 1 mL/min. The composition of the mobile phase (acetonitrile/water mixture containing 0.1% (v/v) trifluoroacetic acid) was adjusted for prodrugs and their degradation products in order to provide an appropriate retention time and separation. The detection was carried out at 254 nm.

2.3. Synthesis of FA Prodrugs

The synthesis and characterization of the monomeric derivative of α-lipoic acid (ALA) with tetraethylene glycol (TEG) (ALA-TEG-OH, Scheme 1(a)) was performed as described in [27]. The synthesis and characterization of the monomeric (Scheme 1(a)) and the dimeric (Scheme 1(b)) FA derivatives were performed as follows.

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Figure 2: ¹H NMR spectra of (a) FA-TEG-ALA, (b) FA₂TEG, and (c) flufenamic acid (FA).

Figure 3: Size of the nanoprodrugs in the absence (wo Toco) and presence (w Toco) of α-tocopherol. Light gray bar: monomeric FA-TEG-ALA; dark gray bar: dimeric FA₂TEG.

3.2. Preparation and Characterization of Nanoprodrugs of FA

The hydrophobic derivatives of FA (Schemes 1(a) and 1(b)) dissolved in acetone spontaneously formed into nanoprodrugs upon the addition into an aqueous solution containing hydrophilic surfactants by spontaneous emulsification process [27, 28, 35–38]. In this study, formulation parameters were kept the same except for the addition of α-tocopherol. In the absence of α-tocopherol the size of the nanoprodrug prepared from the dimeric FA₂TEG was significantly smaller than the size the nanoprodrug prepared from the monomeric FA-TEG-ALA, suggesting that a more compact spatial arrangement of the symmetrical dimeric derivative led to the formation of the compacter and smaller nanoprodrug.

Notably, the retention time of the dimeric FA₂TEG in RP-HPLC was almost twice as much longer than that of the monomeric FA-TEG-ALA, suggesting a higher hydrophobicity of FA₂TEG [39, 40]. It can be assumed that the size decreases with increasing hydrophobicity of the compounds, probably due to a stronger hydrophobic interaction between the molecules. In the presence of α-tocopherol the size of the FA-TEG-ALA nanoprodrug became significantly smaller when compared with the size in the absence of α-tocopherol (Figure 2).

Interestingly, practically no difference in the size was observed for the FA₂TEG nanoprodrugs in the absence and presence of α-tocopherol. This can be explained by the significant increase in the overall hydrophobicity through the addition of α-tocopherol in the case of FA-TEG-ALA, whereas it was negligible in the case of FA₂TEG, probably due to the significant initial hydrophobicity of FA₂TEG.

In the presence of α-tocopherol the difference in size between the FA₂TEG and FA-TEG-ALA nanoprodrugs became smaller, which is especially crucial when the therapeutic efficacies of the two nanoprodrugs are to be compared. This is because differences in the therapeutic efficacy can be attributed directly to the different prodrug molecules in the nanoprodrugs when the size and other components do not differ significantly from each other. Thus, the nanoprodrugs were prepared in the presence of α-tocopherol for further experiments.

To give a visualization of the nanoprodrugs, we applied the nanoparticle tracking and analysis (NTA) technique which allows direct and real-time visualization of nanoparticles in a liquid as shown in Figures 4(a) and 4(b) [41].

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(b)

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Figure 4: Image of FA-TEG-ALA nanoprodrug obtained from nanoparticle tracking analysis (NTA) (a, b) and size distribution of FA-TEG-ALA nanoprodrug as measured by (c) dynamic light scattering (Coulter N4-Plus Submicron Particle Sizer) and (d) NTA. Image (b) is a magnification of a part of the image (a).

Whereas dynamic light scattering (DLS) is an ensemble technique that tries to recover a particle size distribution from the combined signal of all particles present in the sample, nanoparticle tracking analysis (NTA) investigates the diffusion of individual particles. Thus, DLS calculates the average particle diameter by measuring fluctuation in scattering intensity, is highly affected by the presence of a few large particles, and tends to be weighted to the larger particles sizes [42]. Indeed, using DLS (Coulter N4-Plus Submicron Particle Sizer) and NTA for an identical FA-TEG-ALA nanoprodrug, the average size calculated by DLS was 126 nm, which was larger than the size calculated by NTA (97 nm) (Figures 4(c) and 4(d)). The comparison of size distribution and average size from DLS and NTA indicate that few larger nanoprodrugs (>300 nm) have significant influence on the size calculation in DLS.

The stability of the nanoprodrugs was assessed by measuring the size and contents of the intact FA prodrug molecules after 8-week storage at 4°C. In this study, the size of the nanoprodrugs remained almost unchanged (Figure 5), and the recovery yield of the prodrugs was 75% and 90% for the FA-TEG-ALA and FA₂TEG, respectively. It is believed that the nanoprodrugs from the more hydrophobic FA₂TEG formed more stable and compact nanostructures, which can be ascribed to the stronger hydrophobic assembly of FA₂TEG. This may reduce the interaction with water, and consequently decrease hydrolytic degradation and increase the structural integrity of the nanoprodrug.

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Figure 5: Long-term stability of nanoprodrugs measured by size (a) and recovery yield (b). Light gray bar: before storage; dark gray bar: after 8 weeks of storage. Monomeric nanoprodrug: FA-TEG-ALA; dimeric nanoprodrug: FA₂TEG. The results are the mean ± SD of three experiments.

3.3. Enzymatic Hydrolysis of FA Nanoprodrugs

In order to assess the enzymatic prodrug activation from the nanoprodrugs, the rate of enzymatic reconversion of the prodrugs into FA and other degradation products was investigated in vitro with porcine liver esterase. As shown in Figure 6(a), FA-TEG-ALA nanoprodrug was activated nearly completely after 5 h of incubation at 37°C, whereas no activation was observed in the FA₂TEG nanoprodrug during the same period of time. This can be attributed to the more hydrophobic nature of the dimeric FA₂TEG prodrug which makes the interaction between the molecules and enzymes more difficult. In addition, FA is bulkier than ALA, which may increase the steric hindrance towards the enzymes [43, 44]. This assumption was confirmed by the observation that the ALA was first hydrolyzed from FA-TEG-ALA followed by the breakdown of FA-TEG-OH to FA and TEG (Figures 6(c) and 7).

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Figure 6: Enzymatic hydrolysis of prodrugs FA-TEG-ALA (a, b) and FA₂TEG (c) from nanoprodrugs at 37°C. The separation was performed under isocratic condition with a 80/20 (a, c) and 50/50 (b) mixture of acetonitrile/water at a flow rate of 1 mL/min.

Figure 7: Sequence of enzymatic hydrolysis of FA prodrug FA-TEG-ALA.

3.4. Intracellular Uptake of Fluorescent-Labeled FA Nanoprodrug in U87 Glioma Cells

In order to demonstrate the cellular uptake of nanoprodrugs, we prepared fluorescent-labeled nanoprodrugs with the hydrophobic dye, coumarin 6. Due to the hydrophobic nature, the dye remained associated with the nanoprodrugs after overnight dialysis and even after the incubation in PBS buffer and cell culture medium [45]. Confocal laser scanning microscopy of U87 glioma cells treated with fluorescent-labeled nanoprodrugs showed strong internalization of the nanoprodrugs within 5 h of incubation. Both nanoprodrugs FA-TEG-ALA (Figures 8(a)–8(c)) and FA₂TEG (Figures 8(d)–8(f)) showed similar cellular uptake, whereas cells incubated in the dye-treated control medium did not show any detectable fluorescence (Figures 8(g)–8(i)).

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Figure 8: Cellular uptake of fluorescent-labeled nanoprodrugs in U87 glioma cells. Cells were incubated with FA-TEG-ALA nanoprodrug ((a)–(c)), FA₂TEG nanoprodrug ((d)–(f)) and in dye-treated medium as control ((g)–(i)). Left and middle panels show images of overlapped fluorescence of DAPI and coumarin 6, left with lower and middle with higher magnification. Right panel shows images of fluorescence of coumarin 6 only.

Some cells showed a stronger accumulation along the membrane area, while other showed more evenly distributed pattern in the cytoplasm. Interestingly, cells contained numerous tiny vesicles that were dispersed in the cytoplasmic compartment. The vesicles are probably endosomal vesicles (endosomes), suggesting that the cellular uptake occurs via endocytosis. Considering the different spatial intensity and localization of the fluorescent signals within the cells, it can be concluded that after endocytosis the nanoprodrugs escape from the endosomes to the cytoplasm and are dispersed evenly throughout the cytoplasm.

3.5. Effect of FA Nanoprodrug on Cell Proliferation

In order to evaluate the effect of FA nanoprodrugs on tumor cell growth, we studied the effect on the cell growth of U87-MG glioma cells. Glioma cells were treated with nanoprodrugs from FA-TEG-ALA and FA₂TEG, and also with FA in the concentration range of 10 to 200 μM. Cells were also treated with control nanospheres prepared from α-tocopherol only by exposure to an equimolar concentration of α-tocopherol. As shown in Figure 6, the nanoprodrug of FA-TEG-ALA completely inhibited the cell proliferation at the concentration of 50 μM, whereas the nanoprodrug of FA₂TEG inhibited only 30% at the highest concentration of 200 μM. These results were well expected because the prodrug FA₂TEG was almost inert towards chemical and enzymatic hydrolysis (Sections 3.2 and 3.3). Based on the results of this study, the stability and biodegradability of the nanoprodrugs may be adjusted to meet the needs for diverse practical applications via modification of prodrug structures. Interestingly, the inhibitory effect of the nanoprodrug FA-TEG-ALA was much higher than that of the FA (Figure 9(c)), suggesting the existence of more efficient cellular delivery mechanism for the nanoprodrug.

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Figure 9: Effect of nanoprodrug of FA-TEG-ALA (a), nanoprodrug of FA₂TEG (b), and free flufenamic acid (c) on the viability of U87-MG glioma cells.

4. Conclusion

In this study we showed that hydrophobic derivative of FA can be formed into stable nanoprodrug that is readily activated by hydrolytic enzyme and inhibits the growth of malignant cells, suggesting their potential application as anticancer agents. Nanoprodrugs of FA were prepared by spontaneous emulsification of the monomeric prodrug FA-TEG-ALA and dimeric FA₂TEG, and their antiproliferative effects were demonstrated using U87-MG glioma cells. The nanoprodrug from FA-TEG-ALA inhibited the cell growth significantly and induced cell death, whereas the nanoprodrug from FA₂TEG did not show any comparable effect on cell growth and viability. We demonstrated using fluorescent-labeded nanoprodrugs that both nanoprodrugs FA-TEG-ALA and FA₂TEG showed similar cellular uptake. Obviously, the more potent effect of the monomeric nanoprodrug is due to the higher parent drug concentration which is ascribed to the higher enzymatic activation. In addition, the FA-TEG-ALA nanoprodrug inhibited cell growth more efficiently than free FA, suggesting a delivery mechanism specific to the nanoprodrug. We are currently investigating the mechanisms of the cellular uptake and the molecular events leading to the antiproliferative effect of the FA nanoprodrug.

Acknowledgment

The authors wish to thank Mr. Duncan Griffiths (Nanosight USA) for performing nanoparticle tracking analysis (NTA) and providing many helpful comments.

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