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  1. 1. For reprint orders, please contact: R ESEARCH A RTICLE Vitamin E TPGS-emulsified poly(lactic-co- glycolic acid) nanoparticles for cardiovascular restenosis treatment Si-Shen Feng1,2,3†, Aims: Paclitaxel is one of the most effective antiproliferative agents and it has been applied Wutao Zeng1,7, in the development of drug-eluting stents. There are difficulties, however, in using paclitaxel Yean Teng Lim5,6, Lingyun Zhao1, in clinical applications owing to its poor solubility and side effects. We have synthesized Khin Yin Win1, nanoparticles of biodegradable polymers for the effective and sustainable delivery of Reida Oakley5, paclitaxel and other antiproliferative agents for restenosis treatment. Swee Hin Teoh4, Methods & results: Paclitaxel-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles were Ronald Chi Hang Lee6 & prepared by a modified solvent extraction/evaporation method with D-α-tocopheryl Shirong Pan7 polyethylene glycol 1000 succinate (TPGS) or polyvinyl alcohol (PVA) as an emulsifier. †Author for correspondence Drug-loaded nanoparticles were characterized for size and size distribution, surface 1Department of Chemical & morphology, surface charge, drug-encapsulation efficiency and in vitro drug-release kinetics. Biomolecular Engineering, National University of Cellular uptake of fluorescent nanoparticles was investigated in vitro in coronary artery Singapore, Block E5, 02–11, smooth muscle cells and in vivo in the carotid arteries of rabbits. The antiproliferative effects 4 Engineering Drive 4, of the nanoparticle formulations were assessed in vitro in close comparison with Taxol®. 117576 Singapore Tel.: +65 6516 3835; Both the PVA- and TPGS-emulsified nanoparticles have similar size and size distribution, Fax: +65 6779 1936; surface morphology and dispersion stability and showed great advantages over paclitaxel in E-mail: in vitro cellular uptake and cytotoxicity than Taxol. The TPGS-emulsified nanoparticle 2Division of Bioengineering, formulation has higher drug-encapsulation efficiency, cellular uptake and cytotoxicity than National University of Singapore, Singapore the PVA-emulsified nanoparticle formulation. IC50 in 24-h culture with coronary artery 3Nanoscience & smooth muscle cells is 748 ng/ml for paclitaxel, 708 ng/ml for PVA-emulsified nanoparticles Nanoengineering Initiative and 474 ng/ml for TPGS-emulsified nanoparticles, respectively. Conclusion: TPGS-emulsified (NUSNNI), National University of Singapore, PLGA nanoparticles have great potential for the effective and sustainable delivery of Singapore antiproliferative agents and for the development of nanoparticle-coated stents, which may 4Department of Mechanical become the third generation of cardiovascular stents. Engineering, National University of Singapore, Coronary atherosclerosis and heart attack are the polymers, for local delivery and a combination of Singapore 5Department of Surgery, leading causes of mortality in the world. The most drug therapy and devices, such as drug-eluting National University of common treatments so far include percutaneous stents; for example, the Cypher® stent, which Singapore, Singapore transluminal coronary angioplasty (PTCA) with releases sirolimus, and the TAXUS® stent, which 6Cardiac Department, or without intracoronary stents. However, releases paclitaxel. National University Hospital, Singapore 30–50% of patients experience restenosis within Paclitaxel (Taxol®) is one of the best anti- 7Division of Cardiology, 3–6 months of PTCA treatment [1]. There are two neoplastic drugs that has been found from nature Cardiovascular Medical kinds of treatment for restenosis currently: in recent decades. It has excellent therapeutic Department, The First Affiliated Hospital, mechanical treatment and drug therapy. The effects against a wide spectrum of cancers [4]. It Zhongshan University former is stenting [2]. Although popular, stenting was approved by the US FDA for ovarian cancer Medical School, Guangzhou, does not solve the problem becasue 10–15% of in 1992, for advanced breast cancer in 1994 and China the patents will suffer from restenosis again within for early-stage breast cancer in 1999. The mech- 6 months. The latter treatment type includes the anism of its anticancer effects has been inten- treatment by antiproliferative/antiplatelet/anti- sively investigated. It inhibits mitosis in tumor coagulant agents, calcium channel antagonists, cells by binding to microtubules. Paclitaxel aids inhibitors of angiotensin-converting enzyme, cor- polymerization of tubulin dimers to form micro- Keywords: cardiovascular stents, nanobiotechnology, ticosteroids or a fish-oil diet [3]. Nevertheless, drug tubules and thus stabilizes the microtubules, nanomedicine, paclitaxel, therapy is not that effective owing to the pure leading to cell death [5–7]. Paclitaxel is thus an percutaneous transluminal pharmaceutical properties and the multidrug antiproliferative drug that could be beneficial for coronary angioplasty, sirolimus resistance (MDR) effects of the antiproliferative many other diseases caused by a loss of control of agents, such as paclitaxel and sirolimus. Research cell proliferation. Among them is cardiovascular part of is thus focused on more effective drug-delivery restenosis. Due to its difficulty in clinical admin- devices, such as nanoparticles of biodegradable istration and MDR, various dosage forms of 10.2217/17435889.2.3.333 © 2007 Future Medicine Ltd ISSN 1743-5889 Nanomedicine (2007) 2(3), 333–344 333
  2. 2. RESEARCH ARTICLE – Feng, Zeng, Lim et al. paclitaxel have been under intensive investiga- become a new (the third) generation of cardiovas- tion. The dosage form used most often is Taxol, cular stents, which will solve the problems of the which is formulated in Cremophor EL. This second-generation stents – the drug-eluting stents adjuvant is responsible for serious side effects, [101]. Although successful, these drug-eluting including hypersensitivity reactions, nephro- stents have some problems, such as low drug-load- toxicity, neurotoxicity and cardiotoxicity. Some ing ability, slow and incomplete drug release, inef- of the side effects are serious, even life-threaten- ficient uptake by vascular smooth muscle cells ing [8–13]. A better dosage form, docetaxol (Taxo- (VSMCs) [17–20], late angiographic stent thrombo- tere®), was developed later. Although it achieves sis (LAST) [21] and issues of long-term safety and a higher survival rate (SR), the side effects are efficacy, which have raised the cost–effectiveness still a problem and are probably caused by the problem [22,23]. adjuvant polysobate. PLGA nanoparticle formulation of anti- Our research here has investigated the feasibil- proliferative drugs for the treatment of cardio- ity of the formulation of antiproliferative agents vascular restenosis has had a history of more than by biodegradable poly(lactic-co-glycolic acid) 10 years [24–31]. However, the reports of a PLGA (PLGA) nanoparticles, which are prepared by the nanoparticle formulation of paclitaxel for resten- solvent extraction/evaporation method by using osis treatment are few in the literature [32,33], amphiphilic poly(vinyl alcohol) (PVA) or although this drug has been used widely in drug- D-α-tocopheryl polyethylene glycol 1000 succi- eluting stents. The idea of TPGS-emulsified nate (TPGS) as an emulsifier for the treatment PLGA nanoparticles for restenosis treatment is and prevention of restenosis. The drug-loaded novel, coming from our research on TPGS used nanoparticles were then characterized by various as an effective emulsifier or as a component of the techniques, such as laser light scattering for novel PLA–TPGS copolymer in the nanoparticle nanoparticle size and size distribution, field- formulation of anticancer drugs, which resulted emission scanning electron spectroscopy in high drug EE, high cellular uptake of the (FESEM) and atomic force microscopy (AFM) nanoparticles by cancer cells, long half-life in cir- for surface morphology and zeta-potential for culation and high therapeutic effects demon- surface charge. High-performance liquid chro- strated by high area-under-the-curve (AUC) of matography (HPLC) was employed to measure the in vivo pharmacokinetic measurement [16]. the drug-encapsulation efficiency (EE) and the in vitro drug-release kinetics. Cellular uptake of Materials & methods fluorescent nanoparticles was investigated PLGA with L:G molar ratio of 50:50 and Mw of in vitro in coronary artery smooth muscle cells 40,000–75,000, PVA with Mw of (CASMCs) and in vivo in carotid arteries of rab- 30,000–70,000, fluorescence marker cou- bits, which was visualized by confocal laser scan- marin-6, phosphate-buffered saline (PBS), mini- ning spectroscopy (CLSM). The antiproliferative mum essential medium, penicillin–streptomycin effects of the nanoparticle formulations were solution, trypsin–EDTA solution, Triton® X- assessed in vitro by the MTS assay and analyzed 100, Hank’s balanced salt solution (HBSS) and with consideration of the drug-release kinetics in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth- close comparison with Taxol. oxyphenyl)-2-(4-sulfonyl)-2H-tetrazoliumn The drug-loaded PLGA nanoparticles can be (MTS) were purchased from Sigma (St Louis, used either for local delivery by balloon catheter MO, USA). Paclitaxel was purchased from Yun- or for the development of a novel type of cardio- nan Hande Biotechnology Inc (China). Taxol vascular stent – the nanoparticle-coated stent was from Bristol-Myers Squibb Caribbean Com- [101]. Why do we prefer the nanoparticle formula- pany (USA). Vitamin E D-α-tocopheryl polyeth- tion? This is because pure paclitaxel is not bio- ylene glycol 1000 succinate (vitamin E TPGS or adhesive to the cell membrane owing to its poor simply TPGS) was obtained from Eastman pharmaceutical properties, including the MDR Chemical Company (USA). Dichloromethane effects. Our pioneering research on the nano- (DCM, analytical grade) was from Merck particle formulation of paclitaxel has demon- (Darmstadt, Germany) and acetonitrile (HPLC strated that nanoparticles are more adhesive to, grade) was from Fisher Scientific (NJ, USA). and thus easier to be taken up by, cancer cells, Fetal bovine serum (FBS) was received from such as Caco-2 cells and HT-29 cells, than the Gibco (Life Technologies, AG, Switzerland). microparticle formulation and the free drug itself Ultrapure water (Millipore, Bedford, MA, USA) [14–16]. The nanoparticle-coated stents may was used throughout the experiment. 334 Nanomedicine (2007) 2(3) future science group
  3. 3. Nanoparticles of biodegradable polymers for restenosis treatment – RESEARCH ARTICLE Preparation of nanoparticles Drug EE PLGA nanoparticles loaded with paclitaxel or The amount of drug encapsulated in the nano- fluorescent marker (0.5% coumarin-6) were particles was determined in triplicates by HPLC prepared by a modified solvent extraction/evap- (Agilent LC 1100 series). 3 mg of nanoparticles oration method (single emulsion) by using PVA were dissolved in 1 ml of DCM, and 3 ml of or TPGS as the emulsifier [34,35]. In brief, 8 ml acetonitrile-water (50:50) was then added. A of dichloromethane (DCM) solution of nitrogen stream was introduced to evaporate 110 mg PLGA and 11 mg paclitaxel was added the DCM until a clear solution was obtained. drop by drop to a 120 ml aqueous phase, in The solution was put into vials to detect the which PVA 600 mg or TPGS 36 mg was paclitaxel concentration by HPLC. For HPLC added. The solution was then emulsified for analysis, a reverse phase Inertsil® ODS-3 col- 120 s using a microtip probe sonicator umn (150 x 4.6 mm i.d., pore size 5 µm, GL (XL2000, Misonix Incorporated, NY, USA) at Science, Tokyo, Japan) was used and the mobile 50 W in pulse mode. The formed oil in water phase was delivered at a rate of 1 ml/min by a (o/w) emulsion was stirred gently at room tem- pump (HP 1100 High Pressure Gradient perature (22°C) by a magnetic stirrer overnight Pump). 50 µl of sample was injected by an auto to evaporate the organic solvent. The resulting sampler (HP 1100 Autosampler) and the col- sample was collected by centrifugation umn effluent was detected at 227 nm with a (12,000 rpm, 15 min, 16°C; Eppendorf model variable wavelength detector (HP 1100 VWD). 5810R, Eppendorf, Hamburg, Germany) and The calibration curve was prepared for the washed three times with ultrapure water. The quantification of drug in the nanoparticles and product was freeze-dried (Alpha-2, Martin it was linear over the range of 50–10,000 ng/ml Christ Freeze Dryers, Germany) to obtain a with a correlation factor of r2 = 0.9999. The fine powder of nanoparticles, which was kept in measurement was performed in triplicate. The a vacuum dessicator. We did not use any cryo- drug EE was obtained as the mass ratio between protectant in the freeze-dry process because all the amount of paclitaxel incorporated in the the materials, including paclitaxel and TPGS, nanoparticles and that used in the nanoparticle are stable in lyophilization. preparation process [34,35]. Characterization of nanoparticles Surface charge Size & size distribution Zeta-potential is an indicator of surface charge, Nanoparticle size and size distribution were which determines particle stability in the dis- determined by laser light scattering with a par- persion and redispersabliity of the nano- ticle size analyzer (90 Plus, Brookhaven Insti- particles. Zeta-potential of nanoparticles was tute, Huntsville, NY, USA) at a fixed angle of determined by a zeta-potential analyzer (Zeta 90° at 25°C. In brief, the dried nanoparticles Plus, Brookhaven Instruments, Huntsville, were suspended in filtered deionized water and NY, USA) by dipping a palladium electrode in sonicated to prevent particle aggregation and the sonicated particle suspension. The mean to help form a uniform dispersion of nano- value of ten readings is reported. particles. The size distribution was given by the polydispersity index. In vitro drug-release kinetics 5 mg of the drug-loaded nanoparticles were put Surface morphology in a centrifuge tube containing 10 ml PBS Morphology of the drug-loaded nanoparticles (pH 7.4) with 0.1% tween 80. After dispersion was observed by FESEM (JSM-6700F, by a vortex mixer (S0100–230V, Labnet Interna- JEOL.LED, Japan), which requires an ion tional Inc., USA), the tube was placed in an coating with platinum by a sputter coater orbital shaker water bath at 37°C. The well- (JFC-1300, Jeol, Tokyo) for 40 s in a vacuum redispersed status of the nanoparticles for con- at a current intensity of 40 mA after preparing tinuous release measurement can be confirmed the sample on metallic studs with double-sided by the laser light-scattering measurement. At conductive tape. AFM was conducted with designated time intervals, the tube was taken out Nanoscope IIIa in the tapping mode. Before and centrifuged at 11,500 rpm for 15 min. The observation, the nanoparticles were fixed on a supernatant was removed and extracted with double-sided sticky tape that was stuck to the 5 ml DCM to determine the amount of drug standard sample stud. released inside it. The pellets were resuspended future science group 335
  4. 4. RESEARCH ARTICLE – Feng, Zeng, Lim et al. in 10 ml of fresh PBS with 0.1% tween 80 for with HBSS for 30 min, the buffer was replaced continuous release measurement. The analysis with a nanoparticle suspension (250 µg/ml in procedure was the same as described in the HBSS) and the monolayers were further incu- determination of the EE [34,35]. bated for 4 h. The monolayers were then washed three times with fresh prewarmed transport Cell culture buffer to eliminate excess nanoparticles. The In the present study, CASMCs were provided by cells were fixed with 70% ethanol and the nuclei Cambrex Bio Science Walkersville Inc, USA and were stained by propidium iodide (PI). The passages between five and ten were used. samples were mounted in the fluorescent CASMCs were cultured in Dulbecco’s modified mounting medium (Dako, CA, USA) until Eagle’s medium (DMEM) supplemented with examination was performed by the CLSM (Zeiss 20% FBS (vol/vol %) and 1% penicillin–strep- LSM 410, Germany) equipped with an imaging tomycin solution. The cells were seeded at software, Fluoview FV300. 4.3 × 104 cells/cm2 in 96-well black plates with transparent bases (Costar, IL, USA) for quantita- In vitro antiproliferative effects of tive measurement of the cellular uptake of the drug-loaded nanoparticles fluorescent nanoparticles and cytotoxicity meas- The antiproliferative effects of the paclitaxel- urement of the drug-loaded nanoparticles or on loaded PLGA nanoparticles were investigated the Lab-Tek® chambered cover glasses (Nagle in vitro by culturing CASMCs with the nano- Nunc International, Naperville, IL, USA) for particle formulation of paclitaxel in close com- confocal microscopy. The cell monolayer was parison with Taxol at the same paclitaxel cultured at 37°C in a humidified atmosphere concentration. The cell viability (survival rate) containing 5% CO2 and the medium was was determined by the MTS assay, which is a replaced every two days [34–36]. colorimetric method to determine the number of viable cells that are proliferating. It is composed Uptake of nanoparticles by CASMCs of a solution of tetrazolium, which is bioreduced Quantitative study: microplate reader analysis by metabolically active cells into a soluble forma- CASMCs were seeded in 96-well black plates zan product in the culture medium. Its absorb- and were incubated for 48 h. Cultural medium ance can be measured at 490 nm by a microplate was then replaced by transport buffer HBSS and reader (Genios, Tecan, Männedorf, Switzerland). pre-incubated at 37°C for 1 h. After equilibra- The quantity of formazan is directly proportional tion, cellular uptake of fluorescent nanoparticles to the number of living cells. After cells were was initiated by exchanging the transport seeded in a 96-well plate (Costar, IL, USA) and medium with 100 µl of the specific nanoparticle equilibrated with the DMEM medium (without suspension and incubated the cells for approxi- FBS) for 1 h, the medium was removed and the mately 1–6 h. The experiment was terminated nanoparticle suspension in DMEM with 10% by washing the cell monolayer three times with FBS was added. After incubation for a scheduled PBS to eliminate excess nanoparticles that were time, 20 µl of MTS inner salt was added to each not entrapped by the cells. The cell membrane well of a 96-well assay plate containing the sam- was permeated with Triton X-100 solution to ples in 100 µl of culture medium. The plate was expose the internalized nanoparticles for quanti- incubated for 4 h at 37°C in a humidified atmos- tative measurement. Cellular uptake of the fluo- phere containing 5% CO2 [34,35,37]. The cell rescent nanoparticles was quantified by mortality (death rate) is defined as 100% viabil- analyzing the cell lysate in a Genios microplate ity. It should be noted that it is the cell mortality, reader. Uptake was expressed as the percentage of but not the cell viability, that should be propor- the fluorescence associated with the cells versus tional to the area-under-the-curve of the drug that present in the feed solution [36]. concentration versus time. Qualitative study: CLSM Animal protocols CASMCs were seeded on Lab-Tek® chambered The animal protocol was approved by the Insti- cover glasses (Nagle Nunc International, Naper- tutional Animal Care and Use Committees ville, IL, USA) and incubated at 37°C in a 95% (IACUC, Protocol #: 802/05), Office of Life Sci- air and 5% CO2 environment. On the day of ence, National University of Singapore. Alto- the experiment, the growth medium was gether, we used ten New Zealand white rabbits replaced by HBSS (pH 7.4). After equilibration for the in vivo infusion experiment. 336 Nanomedicine (2007) 2(3) future science group
  5. 5. N an o p articles o f b iod egrad ab le p olym ers for resten osis treatm en t – R E S E A R C H A R T I C L E Table 1. The size and size distribution, drug-encapsulation efficiency and Zeta-potential of paclitaxel-loaded, PVA- or TPGS-emulsified PLGA nanoparticles. Emulsifier Size (nm) Polydispersity EE (%) Zeta-potential (mv) PVA (5.0% w/v) 257 ± 10.2 0.031 55.8 ± 4.98 -13.74 ± 1.94 TPGS (0.3 w/v) 288 ± 11.7 0.028 92.6 ± 10.0 -21.5 ± 3.57 EE: Encapsulation efficiency; PLGA: Poly(lactic-co-glycolic acid); PVA: Polyvinyl alcohol; TPGS: D-α-tocopheryl polyethylene glycol 1000 succinate. Rabbit anesthesia Statistical analysis 2.0–3.0 kg male New Zealand white rabbits were Results of the experiments are expressed as anesthetized using Ketamine/Xylazine at a dos- mean ± SD. In the nanoparticle cellular uptake age of 35 mg/kg/5 mg/kg subcutaneously fol- experiment, the student unpaired t-test was lowed by tracheal incubation and were adopted for the comparison between the PVA- maintained with 1.0–2.0 vol% isoflurance, 70% and TPGS-emulsified PLGA nanoparticles. N2O and 30 vol% oxygen. The cytotoxicity study was tested by ANOVA. Probability values of p < 0.05 and p < 0.01 Artery isolation were considered to be significant and highly Carotid arteries (averaging 3–4 cm in length) significant, respectively. were isolated with all side branches being ligated. Results & discussions Arteries injured by balloon catheter Physicochemical properties The distal vessel was punctured by a 18 G trochar. of nanoparticles The needle was withdrawn and the cannula was left Size, EE & surface charge of the in the vessel. A balloon catheter (2.5 × 10–20 mm) drug-loaded nanoparticles was introduced and advanced in a retrograde fash- The data reported in Table 1 represent the average ion into the isolated artery segment via the cannula of five measurements. The emulsifier concentra- distal in the vessel. Once positioned proximally, the tion needed to form the nanoparticles was balloon was inflated with saline to achieve visual 5.0% w/v (emulsifier weight/water phase vol- overstretch of the vessel and then withdrawn a dis- ume) for the PVA-emulsified nanoparticles and tance of 2 cm. The process was repeated two 0.3% w/v for the TPGS-emulsified nano- additional times. The catheter was then removed. particles. This means that TPGS was 16.7-times more effective than PVA as an emulsifier for use Infusion of nanoparticles in the emulsification process, that is, to make the The proximal portion was clamped by a non- same amount of nanoparticles, the required crushing vascular clamp. The nanoparticle suspen- amount of TPGS could be 16.7-times less than sion was injected at the distal end of the segment, that of PVA. A more effective recipe was also which was connected to a pressure pump via the reported [16]. This is a significant advantage of cannula. The arterial lumen was filled with the TPGS over PVA. Also from Table 1, the mean size nanoparticle suspension at 1 atm pressure for 60 s with 10% drug loading was 257 ± 10.2 nm for and the vessel was harvested and the nanoparticles the PVA-emulsified nanoparticles and flushed out by 0.9% saline water. 288 ± 11.7 nm for the TPGS-emulsified nano- particles. The light-scattering measurement of Histological examination of the particle size agrees well with that given by the nanoparticle-infused arteries Smile View software from the FESEM images. Nanoparticles loaded with the fluorescent marker The TPGS-emulsified nanoparticles achieved coumarin-6 were used in this study. Each arterial much higher EE (92.6 ± 10.0%) than the PVA- segment after nanoparticle infusion was flushed to emulsified nanoparticles (55.8 ± 4.98%). It is remove the nanoparticles not taken up by clear that TPGS has an advantage over PVA, CASMCs by 0.9% saline water and then frozen by resulting in much higher EE and, therefore, in dry ice with an OCT (Mile, Inc, Elkhart, IN, much higher drug loading in the nanoparticles. USA)-embedding compound. Cross sections of We mentioned earlier that 110 mg PLGA and 10 µm thickness were cut using a cryomicrotome 11 mg paclitaxel were added in the organic sol- and mounted on the glass slides. The slides were vent. The theoretical drug-loading ratio should observed under a confocal microscope. have been 10%. However, owing to incomplete future science group 337
  6. 6. RESEARCH ARTICLE – Feng, Zeng, Lim et al. Morphology of nanoparticles Figure 1. Scanning electron microscopy images of paclitaxel-loaded nanoparticles. Figure 1 shows the FESEM images of PVA- (Figure 1A) and TPGS-emulsified (Figure 1B) PLGA nanoparticles of 10% drug loading, A B which reveals their regular spherical shape and smooth surface without any noticeable pinholes or cracks within the instrument resolution. The size distribution of all nanoparticles was unimo- dal, with a range of 150–500 nm and a mean diameter of 200–300 nm, as confirmed by the laser light-scattering measurement. Figure 2 shows 1 µm 1 µm AFM images of paclitaxel-loaded, TPGS-emulsi- fied PLGA nanoparticles and a magnified image FESEM Images of (A) polyvinyl alcohol- or (B) D-α-tocopheryl polyethylene glycol of the nanoparticle surface, from which wrinkles 1000 succinate-emulsified, drug-loaded poly(lactic-co-glycolic acid) and a small hole can be observed. The advantage nanoparticles of 10% drug loading. of AFM is that it can reveal the true structure with much higher resolution than SEM since the encapsulation, that is, less than 100% EE, the image is obtained by direct contact or tapping of actual drug-loading ratio was modified by the the AFM tip on or over the particle surface. EE values, which is thus 5.58% for the PVA- emulsified nanoparticles and 9.26% for the In vitro drug-release kinetics TPGS-emulsified nanoparticles. The in vitro release profiles of paclitaxel from the Both types of drug-loaded nanoparticles PVA- or TPGS-emulsified PLGA nanoparticles were stable in their dispersion, possessing neg- is shown in Figure 3, from which the effect of sur- ative surface charges with high absolute values face coating on the in vitro drug-release behavior of zeta-potential (Table 1), which are can be observed. The drug-release kinetics -13.74 ± 1.94 mV for the PVA-emulsified exhibit a biphasic pattern characterized by a fast nanoparticles and -21.5 ± 3.57 mV for the initial burst during the first 5 days, followed by a TPGS-emulsified nanoparticles. The surface slow, sustained release. An initial burst during charge determines stability of the nanoparticle the first 5 days of 67.9% for the PVA-emulsified suspension and resuspensability of the nano- nanoparticles and 51.2% for the TPGS-emulsi- particles. TPGS-emulsified nanoparticles thus fied nanoparticles was followed by a first order have advantages over PVA-emulsified nano- release with a reduced rate afterwards. Approxi- particles in their suspension stability and mately 81.2% of the PVA-emulsified nano- resuspensability. This finding is in agreement particles and 66.2% of the TPGS-emulsified with that of our earlier research of nanoparti- nanoparticles were released in 30 days. Please cle formulation of paclitaxel for cancer note the release for the first 72 h is 58.6% for the treatment [37]. PVA-emulsified nanoparticles and 43.4% for the TPGS-emulsified nanoparticles. These data will Figure 2. Atomic force microscopy images of paclitaxel-loaded be used later to interpret the cellular mortality of nanoparticles. the drug formulated in the nanoparticles. It seems that the drug release from the nano- A B particles is much faster (∼1 month) than the 0.15 0.3 V release of drugs from drug-eluting stents 0.1 V (∼6 months), which represent two different 0.10 treatments of restenosis: local drug delivery and 0.0 V device plus drug; each has advantages and disad- vantages. For local drug delivery, the nanoparti- 0.05 cles are the reservoir of the drug after adsorption by the CASMCs. The 1 month (or even faster) 0.1 0 0.2 0.3 µm 0 0.05 0.10 0.15 µm drug-release period may be appropriate for the treatment. For drug-eluting stents, the stents (A) Atomic force microscopy image of paclitaxel-loaded, D-α-tocopheryl themselves are the reservoir of the drug and a polyethylene glycol 1000 succinate-emulsified poly(lactic-co-glycolic acid) longer period would result in long-term treat- nanoparticles and (B) magnified image of the nanoparticle surface. ment. Unfortunately, one of the major problems 338 Nanomedicine (2007) 2(3) future science group
  7. 7. Nanoparticles of biodegradable polymers for restenosis treatment – RESEARCH ARTICLE Figure 3. In vitro drug releases of PVA- or TPGS-emulsified, and the residues would affect the cellular paclitaxel-loaded PLGA nanoparticles of 10% drug loading. uptake measurement of the fluorescent nano- particles. The residues, however, would not affect the CLSM images because CLSM has a 90 sectioning function. Culmulative release (%) 80 70 Quantitative study 60 Figure 5 shows the effects of the incubation time 50 on the cellular uptake of the fluorescent PVA- 40 or TPGS-emulsified PLGA nanoparticles. The Vitamin E TPGS 30 -emulsified NPs nanoparticle concentration used for incubation 20 PVA-emulsified NPs with the CASMCs was 500 µg/ml. The signifi- 10 cance of the TPGS-emulsified versus PVA- 0 emulsified nanoparticles is p < 0.01. Figure 4 0 5 10 15 20 25 30 35 Time (day) demonstrates that the cellular uptake of nano- particles increased with the incubation time. Each point represents mean ± SD (n = 3). At each designated time, the TPGS-emulsified NP: Nanoparticle; PLGA: Poly(lactic-co-glycolic acid); PVA: Polyvinyl alcohol; nanoparticles could achieve much higher cellu- TPGS: D-α-tocopheryl polyethylene glycol 1000 succinate. lar uptake than the PVA-emulsified nano- particles. After incubation for 6 h, the for drug-eluting stents is that the drug coated on CASMC uptake was 38% for the TPGS-emul- the stent surface cannot be completely released. sified nanoparticles versus 21% for the An ideal solution is thus to combine the two PVA-emulsified nanoparticles. therapies, that is, to develop nanoparticle-coated Figure 6 shows the effects of the nanoparticle stents [101]. concentration on the cellular uptake of the flu- It should be pointed out that, although the orescent PVA- or TPGS-emulsified PLGA surfactant molecules are supposed to be washed nanoparticles after 4 h incubation. We can see away after formulation, incomplete washing will from this figure that the cellular uptake of result in some residues remaining on the nano- nanoparticles increased with the nanoparticle particle surface, which will affect the drug release. concentration and, at each designated nano- Moreover, the release medium also plays a deci- particle concentration of 100, 250 500 µg/ml, sive role in determining the drug-release kinetics. the TPGS-emulsified nanoparticles showed The in vivo release could thus be much faster great advantages over the PVA-emulsified than the in vitro release owing to the interactions nanoparticles in cellular uptake. For example, between the plasma proteins and the drug. This at the 500 µg/ml nanoparticle concentration, has been confirmed by the in vitro measurement the cellular uptake was 33% for the TPGS- of drug release in plasma (data not shown). emulsified nanoparticles versus 13.5% for the PVA-emulsified nanoparticles. This advantage Cellular uptake of nanoparticles is significant (p < 0.01). The quantitative Qualitative study study confirmed the results observed from the Figure 4 shows confocal microscopic images of qualitative study, showing that the TPGS- CASMCs after 4-h incubation with coumarin- emulsified nanoparticles have advantages 6-loaded, PVA- (Figure 4A) or TPGS-emulsified resulting in higher cellular internalization than (Figure 4B) PLGA nanoparticles at 37°C. The the PVA-emulsified nanoparticles. Although nuclei were stained by PI (red), and the cou- the detailed mechanism is unknown, marin-6-loaded nanoparticles (green) in the vitamin E facilitates cellular uptake of drugs. cytoplasm were visualized by overlaying images It may be concerning that the fluorescent that were obtained by fluorescein isothio- coumarin-6 markers formulated in the nano- cyanate (FITC) filter and PI filter. The images particles could leak, which may affect the result show that most of the internalized nano- of the cellular uptake measurement. To address particles are located in the cytoplasm. Some this problem, we have conducted an experi- may have penetrated into the nuclei. ment to measure the in vitro release of cou- It should be pointed out that the washing marin-6 from the nanoparticles and our results procedure may not be able to wash the showed that the leakage, in up to 24 days, was adhered nanoparticles out of the cell surface less than 5% and thus negligible [36]. future science group 339
  8. 8. RESEARCH ARTICLE – Feng, Zeng, Lim et al. Figure 4. Confocal microscopic images of coronary artery CASMCs. Data represent the mean ± SD with smooth muscle cells cultured with fluorescent nanoparticles. n = 6. There was no significant decease in the cell viability for the two types of PLGA nanoparticles compared with the control (p < 0.05), although A B the placebo PVA-emulsified PLGA nanoparticles showed a slightly larger decrease in cell viability and such a decease becomes more significant at high nanoparticle concentrations. This means that the TPGS-emulsified nanoparticles are more biocompatible than the PVA-emulsified nanoparticles. This is another advantage of TPGS versus PVA as an emulsifier. 50 µm 20 µm Figure 8 shows the effects of the drug concen- tration on CASMC viability after 72 h incuba- Confocal microscopic images of coronary artery smooth muscle cells after 4 h tion with the paclitaxel-loaded, PVA- or TPGS- incubation with coumarin-6-loaded, (A) polyvinyl alcohol- or (B) D-α-tocopheryl emulsified PLGA nanoparticle suspension ver- polyethylene glycol 1000 succinate-emulsified poly(lactic-co-glycolic acid) sus Taxol. The table in the figure shows the nanoparticles at 37°C. The nuclei were stained by propidium iodide (PI) (red), measured CASMC mortality (viability + mor- and the cellular uptake of fluorescent coumarin-6-loaded nanoparticles (green) tality = 1) as well as that after the correction in the cytoplasm were visualized by overlaying images obtained by a fluorescein isothiocyanate filter and a PI filter. The cells look unhealthy because they were made by considering the 72 h drug release being killed by the drug-loaded nanoparticles. found from the drug-release profiles (Figure 3). The data represent mean ± SD of n = 6. The In vitro antiproliferative effects of significance of the TPGS-emulsified nanoparti- drug-loaded nanoparticles cles versus PVA-emulsified nanoparticles is We first tested the cytotoxicity of the placebo p < 0.01 at 25 ng/ml drug concentration and PVA- or TPGS-emulsified PLGA nanoparticles, p < 0.05 at 250 and 500 ng/ml drug concentra- that is, the nanoparticles with no drug encapsu- tions. From the table it can be seen that the via- lated. Figure 7 show the cytotoxicity of the placebo bility (the percentage of the CASMCs that PVA- or TPGS-emulsified PLGA nanoparticles at survived) after 72 h culture at 25 ng/ml paclit- nanoparticle concentrations of 2.5, 25 and axel concentration is 80.5% for Taxol, 79.1% 100 µg/ml after 72 h incubation with the for the PVA-emulsified nanoparticle formula- tion and 78.2% for the TPGS-emulsified nano- particle formulation. The mortality (the Figure 5. Effects of the incubation time on the cellular percentage of the CASMCs killed) after 72 h uptake of the fluorescent PVA- or TPGS-emulsified culture at 25 ng/ml paclitaxel concentration is PLGA nanoparticles. thus 19.5% for Taxol, 20.9% for the PVA- emulsified nanoparticle formulation and 21.8% for the TPGS-emulsified nanoparticle formula- 50 PVA tion, which means that the PVA- and the TPGS CASMC uptake of nanoparticles (%) 40 TPGS-emulsified nanoparticle formulations of paclitaxel have 1.07- and 1.12-times higher 30 antiproliferative effects than the Taxol after 72 h 20 treatment. Such advantages of the nanoparticle formulations versus the free drug should have 10 been even more significant if the sustainable drug-release manner of the nanoparticle formula- 0 1 2 4 6 tion were further considered [16]. The drug release Incubation time (h) from the nanoparticles for the first 72 h was found to be 58.6% for the PVA-emulsified nano- The nanoparticle concentration was 500 µg/ml. Each point represents particles and 43.4% for the TPGS-emulsified mean ± SD (n = 4). The significance of the TPGS-emulsified versus nanoparticles, respectively (Figure 3). Moreover, PVA-emulsified nanoparticles is p < 0.01. the drug release is from 0% at t = 0 to 58.6 or CASMC: Coronary artery smooth muscle cell; PLGA: Poly(lactic-co-glycolic acid); PVA: Polyvinyl alcohol; TPGS: D-α-tocopheryl polyethylene glycol 1000 43.4% when Taxol was 100% immediately avail- succinate. able to the cells. The corrected mortality after 72 h culture at 25 ng/ml paclitaxel concentration 340 Nanomedicine (2007) 2(3) future science group
  9. 9. Nanoparticles of biodegradable polymers for restenosis treatment – RESEARCH ARTICLE Figure 6. Effects of the nanoparticle Another way to evaluate the antiproliferative concentration on the cellular uptake of effectiveness of the drug in the various formula- the fluorescent PVA- or TPGS-emulsified tions is to measure their IC50, which is defined PLGA nanoparticles after as the drug concentration needed to kill 50% of 4 h incubation. the CASMCs at a given period, say in 24 h. This can be obtained by finding the inter- section of the viability versus the drug concen- 40 PVA tration curve with a horizontal line of viability Cell uptake (%) TPGS at 50%. By extrapolation, we can find from 30 Figure 8 that the IC50 in 24 h would be 748 ng/ml for Taxol, 708 ng/ml for the PVA- 20 emulsified nanoparticle formulation and 10 474 ng/ml for the TPGS-emulsified nano- particle formulation, which implies that the 0 PVA-emulsified nanoparticle formulation is 100 250 500 Nanoparticle concentration (µg/ml) 5.35% more effective than Taxol and the TPGS-emulsified nanoparticle formulation is The significance of the TPGS-emulsified 36.6% more effective than Taxol and 33.1% nanoparticles versus PVA-emulsified is p < 0.01. more effective than the PVA-emulsified nano- PLGA: Poly(lactic-co-glycolic acid); PVA: Polyvinyl alcohol; TPGS: D-α-tocopheryl polyethylene glycol particle formulation in 24 h treatment. Consid- 1000 succinate. ering the sustainable-release manner of the nanoparticle formulations, their advantage over should thus be 0.209/0.586/0.5 = 0.713 for the the free drug should be even greater. If we used PVA-emulsified nanoparticle formulation and the corrected data in Figure 8, the IC50 would 0.218/0.434/0.5 = 1.005 for the TPGS-emulsi- have been 748 ng/ml for Taxol, 209 ng/ml for fied nanoparticle formulation, which means that the PVA- and the TPGS-emulsified nano- Figure 7. Cytotoxicity of the placebo particle formulations of paclitaxel should actu- PVA- or TPGS-emulsified PLGA ally have 3.66- and 5.15-times higher nanoparticles (with no drug antiproliferative effects than Taxol after the encapsulated inside the nanoparticles) 72 h treatment. at various nanoparticle concentrations As can be seen from the table in Figure 8, the after 72 h incubation with CASMCs. difference in the measured mortality of the CASMCs after 72 h culture with the PVA- or PVA TPGS-emulsified PLGA nanoparticles at the 120 TPGS same 25, 250, 500 ng/ml paclitaxel concentra- Percentage of control tions is not significant before corrected by drug 100 release, which is 20.9, 33.9 and 38.1% for the 80 PVA-emulsified nanoparticles versus 21.8, 60 38.8 and 48.7% for the TPGS-emulsified 40 nanoparticles. Nevertheless, the cellular uptake of the nanoparticles after 6 h culture was 20 found before to be 21% for the PVA-emulsi- 0 2.5 25 100 fied nanoparticles compared with 38% for the Nanoparticle concentration (µg/ml) TPGS-emulsified nanoparticles (Figure 5). These two results seem to conflict. A fair expla- Data represent mean ± SD with n = 6. There were nation, however, can be found from the drug- no significant changes in cell viability between PLGA nanoparticles and the control (p < 0.05), release kinetics. The 72 h drug release is 55% although the PVA-emulsified PLGA nanoparticles for the PVA-emulsified nanoparticles, which is showed a slight decrease in cell viability at high much higher than the 24% for the TPGS- nanoparticle concentration. emulsified nanoparticles. The effects of the CASMC: Coronary artery smooth muscle cell; higher cellular uptake of the TPGS-emulsified PLGA: Poly(lactic-co-glycolic acid); PVA: Polyvinyl nanoparticles might have been balanced by alcohol; TPGS: D-α-tocopheryl polyethylene glycol that of the lower drug-release rate. 1000 succinate. future science group 341
  10. 10. RESEARCH ARTICLE – Feng, Zeng, Lim et al. Figure 8. Effects of the drug concentration on CASMC viability of the fluorescent nanoparticles from these fig- after 72 h incubation with the paclitaxel-loaded, PVA- or ures: Figure 9A is the control, Figure 9B is the TPGS-emulsified PLGA nanoparticle suspension versus Taxol®. PVA-emulsified PLGA nanoparticles, Figure 9C is the TPGS-emulsified nanoparticles and Figure 9D is the TPGS-emulsified nanoparticles Taxol® at 100-times higher resolution. We can see little 100 PVA fluorescence in the control carotid artery wall CASMC cell viability (%) TPGS (Figure 9A). After the fluorescent nanoparticle 80 infusion, fluorescence could be clearly observed in the carotid arteries walls (Figure 9B). The 60 TPGS-emulsified nanoparticles showed advan- tages in cellular uptake compared with the 40 PVA-emulsified nanoparticles (Figure 9C & D). As mentioned previously, the infusion time of 20 the fluorescent nanoparticle suspension in the arteries was 60 s. Such a short period was 0 applied to address the concern of retention of Mortality (%) 25 ng/ml 250 ng/ml 500 ng/ml the nanoparticles by the arteries in actual prac- (1) Taxol 19.5 31.7 36.3 tice of local delivery by catheter. It is clear that (2) PVA 20.9 33.9 38.1 nanoparticle-coated stents could have advan- (3) TPGS 21.8 38.6 49.7 tages compared with local delivery, which could (2)/(1) 1.07 1.07 1.05 result in higher nanoparticle retention. This (3)/(1) 1.12 1.22 1.37 should be further investigated. (4) 72 h 58.6% for PVA-emulsified nanoparticles drug release 43.4 % for TPGS-emulsified nanoparticles Discussion & future perspective (5) PVA (Corr) 71.3 115.6 130.1 (6) TPGS(Corr) 229.0 Although our in vivo experiment showed effec- 100.5 177.9 (5)/(1) tive internalization of the paclitaxel-loaded, 3.66 3.65 3.58 (6)/(1) 5.15 5.61 6.31 TPGS-emulsified PLGA nanoparticles, further experiments are needed to show the advantages The attached table shows the measured CASMC mortality (viability + mortality of the nanoparticle formulation versus the origi- = 1) as well as that after the correction made by considering the 72 h drug nal drug in resulting in better therapeutic release found from the drug-release profiles (Figure 3). The data represent effects. This means that an in vivo restenosis mean ± SD of n = 6. The significance of the TPGS-emulsified nanoparticles model should be developed by balloon inflation versus PVA-emulsified nanoparticles is p < 0.01 at 25 ng/ml drug injury, which should then be treated by the nan- concentration and p < 0.05 at 250 and 500 ng/ml drug concentration. oparticle formulation of paclitaxel in close com- CASMC: Coronary artery smooth muscle cell; PLGA: Poly(lactic-co-glycolic parison with Taxol. We shall continue this acid); PVA: Polyvinyl alcohol; TPGS: D-α-tocopheryl polyethylene glycol 1000 succinate. research as soon as possible. Although the above research showed that the nanoparticle formulation of antiproliferative the PVA-emulsified nanoparticle formulation agents could have advantages versus the original and 160 ng/ml for the TPGS-emulsified drug for cardiovascular restenosis treatment and nanoparticle formulation. that the TPGS-emulsified PLGA nanoparticles These in vitro experiments, of course, are just may have even better effects than the traditional a preliminary evaluation of toxicity or therapeu- PVA-emulsified PLGA nanoparticles, it is still tic activity of the nanoparticle formulation. Fur- unclear whether the MDR effects are involved in ther in vivo study will determine if the the CASMC treatment by paclitaxel, that is, formulation can be used for clinical trials before whether CASMCs are rich in multidrug pump it can become a commercial product. proteins (P-glycoproteins). Paclitaxel-eluting stents are effective in reducing restenosis and one Arterial uptake of nanoparticles could argue that the current issue of late stent Figure 9 shows confocal microscopic images of thrombosis could be related to a continued signif- cross sections of the carotid arteries of rabbits icant reduction in smooth muscle cell prolifera- that were injured by balloon catheter and then tion as well as endothelial coverage of the stent infused by the fluorescent nanoparticle suspen- struts, certainly not lack of efficacy of the drug sion. We can observe the carotid arterial uptake delivery. From this point of view, the nanoparticle 342 Nanomedicine (2007) 2(3) future science group
  11. 11. Nanoparticles of biodegradable polymers for restenosis treatment – RESEARCH ARTICLE Figure 9. Confocal microscopic images of the uptake of the formulation may be more useful for local drug drug-loaded nanoparticles by carotid arteries of rabbits. delivery for the treatment of cardiovascular restenosis. Further investigations are needed. A B Conclusion We synthesized PVA- and TPGS-emulsified PLGA nanoparticles to formulate antiprolifera- tive agents with paclitaxel as a model drug for the treatment and prevention of cardiovascular reste- nosis. We found that the nanoparticle formula- tions of paclitaxel can achieve much higher cellular uptake and much better in vitro anti- proliferative effects than Taxol. The emulsifier 150 µm 150 µm used in the nanoparticle preparation process plays a key role in determining the drug EE, C D drug-release kinetics, cellular uptake and thus antiproliferative effectiveness of the formulated drug. The TPGS-emulsified nanoparticles have great advantages versus the PVA-emulsified nanoparticles for local delivery of antiprolifera- tive drugs, which can also be used in developing nanoparticle-coated stents. Acknowledgements 150 µm 20 µm This research is supported by research grants (A) Control. (B) Polyvinyl alcohol-emulsified poly(lactic-co-glycolic acid) R-397–000–014–112 (SS Feng: PI), National University nanoparticles. (C) D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS)- of Singapore (NUS). The authors are grateful of the review- emulsified nanoparticles. (D) TPGS-emulsified nanoparticles (magnification ers for their thoughtful comments, without which this paper 100×). could not have reached its current status. Executive summary • Paclitaxel is one of the most effective antiproliferative agents and has been used in drug-eluting stents; however, owing to its undesired physicochemical and pharmaceutical properties, it has difficulties in formulation and delivery. Nanoparticles of biodegradable polymers can help to solve these problems. • In this study, we prepared paclitaxel-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles by a modified solvent extraction/evaporation method with D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS or simply TPGS) as an emulsifier, which was meant to have advantages versus those prepared by traditional emulsifiers, such as polyvinyl alcohol (PVA). • Cellular uptake of fluorescent nanoparticles can be visualized and measured in vitro in coronary artery smooth muscle cells (CASMCs) and in vivo in carotid arteries of rabbits. Both showed excellent effects of the TPGS-emulsified nanoparticles. • The TPGS-emulsified nanoparticles had a higher drug-encapsulation efficiency, cellular uptake and cytotoxicity than PVA- emulsified nanoparticle formulations. The IC50 in 24 h culture with CASMCs is only 474 ng/ml for the TPGS-emulsified nanoparticles in comparison with 708 ng/ml for the PVA-emulsified nanoparticles and 748 ng/ml for Taxol®, respectively. • TPGS-emulsified PLGA nanoparticles are of great potential for the effective and sustainable delivery of antiproliferative agents and for the development of nanoparticle-coated stents, which may become the third generation of cardiovascular stents. Bibliography 3. Herrman JP, Hermans WR, Vos J, agent from Taxus brevifolia. J. Am. Chem. 1. Popma JJ, Califf RM, Topol EJ: Clinical Serruys PW: Pharmacological approaches Soc. 93, 2325–2327 (1971). trials of restenosis after coronary angioplasty. to the prevention of restenosis following 5. Lopes NM, Adams EG, Pitts TW, Circulation 84, 1426–1436 (1991). angioplasty – the search for the holy-grail. Bhuyan BK: Cell kill kinetics and cell 2. Wilensky RL, March KL, Grudus-Pislo I, Drugs 46, 18–52 (1993). cycle effects of Taxol on human and Spuedy AJ, Hathaway DR: Methods and 4. Wani MC, Taylor HL, Wall ME, hamster ovarian cell line. Cancer devices for local-drug delivery in coronary Coggon P, McPhail AT: Plant antitumor Chemother. Pharmacol. 32, 235–242 and peripheral arteries. Trends Cardiovasc. agents. VI. The isolation and structure of (1993). Med. 3, 163–170 (1993). taxol, a novel antileukemic and antitumor future science group 343
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