- Email: [email protected]
Preparation and in vitro Studies of Stealth PEGylated PLGA Nanoparticles as Carriers for Arsenic Trioxide*
Chin. J . Chem. Eng., 15(6) 795-801
(2007)
Preparation and in vitro Studies of Stealth PEGylated PLGA Nanoparticles as Carriers for Arsenic Trioxide* WANG Zhiqing(3 & %)", LIU Wei(3q X)",**, XU Huibi(f&fig)band YANG Xiangliang
B)"
(#I# College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China Department of Chemistry, Huazhong University of Science and Technology, Wuhan 430074, China
a
Abstract The aim of this study was to prepare arsenic trioxide (AT0)-loaded stealth PEGylated PLGA nanoparticles (PEG-PLGA-NPs) and to assess the merits of PEG-PLGA-NPs as drug carriers for AT0 delivery. PEG-PLGA copolymer was synthesized with methoxypolyethyleneglycol (M,=5000), D, L-lactide, and glycolide by the ring-opening polymerization method. Amorphous AT0 was transformed into cubic crystal form to increase its solubility in the organic solvent. ATO-loaded PEG-PLGA-NPs were prepared by the modified spontaneous emulsification solvent diffusion (SESD) method, and the main experimental factors influencing the characteristics of nanoparticles were investigated, to optimize the preparation. To confirm the escape of PEG-PLGA-NPs from phagocytosis by phagocytes, PEG-PLGA-NPs labeled rhodamine B uptake by murine peritoneal macrophages (MPM) were analyzed by flow cytometry. The results showed that the physicochemical characteristics of PEG-PLGA-NPs were affected by the type and concentration of the emulsifiers, polymer concentration, and drug concentration. ATO-loaded PEG-PLGA-NPs, with particle size of 120.8nm, zeta potential of - 10.73mV, encapsulation efficiency of 73.6%, and drug loading of 1.36%, were prepared under optimal conditions. The images of transmission electron microscopy (TEM) indicated that the optimized nanoparticles were near spherical and without aggregation or adhesion. The release experiments in vitro showed the AT0 release from PEG-PLGA-NPs exhibited consequently sustained release for more than 26d, which was in accordance with Higuchi equation. The uptake of PEG-PLGA-NPs by MPM was found to decrease markedly compared to PLGA-NPs. The experimental results showed that PEG-PLGA-NPs were potential nano drug delivery carriers for ATO. Keywords arsenic trioxide, PEGylated PLGA nanoparticles, ring-opening polymerization, spontaneous emulsification solvent diffusion method, in vitro drug release, phagocytic uptake
1 INTRODUCTION Arsenic trioxide (As203, ATO) has been an effective therapeutic agent for acute promyelocytic leukemia (APL) in clinics. The mechanisms of action are associated with the induction of apoptosis and differentiation[11. Recently, in vitro studies have revealed that clinically achievable concentrations of AT0 can trigger apoptosis of leukemia and lymphoma cells as well as some solid tumor cells, including neuroblastoma and gastric, transitional, renal, esophageal, prostate, colorectal, and hepatocellular cancers or cell lines[2]. This suggests that ATO-induced apoptosis may also be seen in a variety of tumor models. However, the activity of AT0 against solid tumors in vivo has not been as effective as against APL, because the dosages of AT0 required to exert these effects are much higher than those required to inhibit hematologic malignancies[3]. Severe toxic reactions, including flaccid paralysis, oncogenicity, cardiac toxicity, and renal failure, have been observed under high dosage of ATO[4,5]. A previous pharmacokmetic study has shown that plasma arsenic eliminates rapidly and the elimination half-life of AT0 in plasma is very short after intravenous administration[6]. It is known that 95% to 97% of blood arsenic is bound to hemoglobin and can be distributed rapidly into some tissues and organs[3,7], which may be the reason that AT0 is not suitable for application in solid tumors. To overcome the problems, a new drug delivery
system is needed that can control drug delivery and extend drug circulation time in blood. Over the past decades, various polymeric nanoparticles have been used in drug delivery research, as they can effectively deliver drugs to the target site and increase therapeutic benefit[8]. However, these conventional particulate drug carriers can be rapidly cleared by the cells of the mononuclear phagocytes system (MPS) [8-121. At present, many studies have highlighted the development of stealth nanoparticles as drug carriers, which can avoid, or at least reduce the uptake by phagocytes and prolong the time of drugs, with effective concentration, in blood circulation[9,lo]. Polyethyleneglycol (PEG), a modified biodegradable polymer, is one of the most popular materials to prepare stealth nanoparticles[ 113. Nanoparticles prepared from PEG modified poly (D, L-lactide-co-glycolide) (PEG-PLGA) have been investigated because of their controlled release, biodegradability, and biocompatibility[121. In the PEG-PLGA copolymers, PEG has been found to be a particularly effective steric stabilizer, probably because of its high hydrophilicity, chain flexibility, electrical neutrality, and absence of functional groups, which prevent interactions with biological components, and reduce complement activation and interaction of the uptake of nanoparticles by MPS in vivo[ 13,141. As a result, the PEG-PLGA nanoparticles exhibit prolonged blood circulation time after intravenous administration in experimental animals. Moreover,
Received 2007-01-03, accepted 2007-07-26.
* Supported by the Special Funds for Major State Basic Research Program of China (973 Program. No. 2007CB935800) National High Icchnology Research and Development Program of China (863 Program. No.2004AA215 162). ** To whom correspondence should be addressed. E-mail: [email protected]
and the
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some studies have also proved that PEGylated PLGA nanoparticles (PEG-PLGA-NPs) bear a targeted delivery characteristic to cancer cells[ 151. In this study, PEG-PLGA was synthesized by ring-opening polymerization. ATO-loaded PEG-PLGA-NPs were prepared by the modified spontaneous emulsification solvent diffusion (SESD) method. The effect of the type of emulsifiers, emulsifier concentration, polymer concentration, and drug concentration on the physicochemical characteristics of ATO-loaded PEG-PLGA-NPs was investigated. In addition, the in vitro release of AT0 from optimized nanoparticles was measured by atomic fluorescence spectrometry, and the uptake of nanoparticles by murine peritoneal macrophages (MPM) was assessed.
2 MATERIALS AND METHODS 2.1 Materials AT0 (As203) was purchased from Aldrich (USA). D, L-lactide and glycolide were purchased from Tianyuan Biomaterials Co. Ltd (China). Methoxypolyethyleneglycol (MePEG, M , = 5000), polyvinyl alcohol (PVA) (M,=25000), stannous octoate, and rhodamine B were obtained from Sigma Chemical Co. Ltd (USA). Poloxamer 188 was supplied by China Pharmaceutical University (China). Tween 80 (T-80) was obtained from Tianjin Bodi Chemical Company (China). Male Kuming mice (20g 2g) were obtained from the Laboratory Animal Center, Tongji Medical College, Huazhong University of Science & Technology (China). 2.2 Synthesis of PEG-PLGA copolymer PEG-PLGA copolymer was synthesized by the ring-opening polymerization under vacuum using stannous octoate as a catalyst[l6]. D, L-lactide and glycolide (molar ratio 1 : 1) and MePEG were introduced into a bottle-neck flask. Stannous octoate (0.5%, by mass), used as a catalyst, was dissolved in hexane and added to the reaction mixture. The feed was degassed through vacuumlnitrogen cycles and applied to the molten mixture at 135°C. The flask was sealed under vacuum. Then, the polymerization reaction was carried out at 180°C for 5h under vacuum. The synthesized copolymer was recovered by dissolving in dichloromethane followed by precipitation in ice-cold diethyl ether thrice. The precipitated copolymer was filtered out and dried under vacuum at 40°C for 24h.
2.3 Physicochemical characteristics of the copolymer 'H NMR spectrum was recorded in CDC13 on a Bruker spectrometer (Germany) operating at 5OOMHz. Chemical shifts (6) were given using tetramethylsilane (TMS) as an internal reference. Fourier transform infrared (FI'IR) spectrum was obtained from a neat film, cast from the chloroform copolymer solution between KBr tablets, with 1700 spectrometer (Perktn-Elmer, USA). Gel permeation chromatography (GPC) was performed in tetrahydrofuran with a Waters 510 December, 2007
chromatographic instrument (USA). Molecular weight (M,) and molecular weight distribution (MJM,,) of the copolymers were calculated using polystyrene as the standard.
2.4 Transformation of AT0 crystal form Amorphous AT0 was transformed into cubic crystal form according to previous methods[ 171. Briefly, amorphous AT0 was dissolved in 4mol.L- NaOH solution. SO2 gas, generated in a round bottle from NaHS03 and high concentration H2S04, was passed into the solution for 5min under continuous stirring at room temperature. Then, the precipitate of the cubic AT0 crystal was separated from the solution. 2.5 Preparation of PEG-PLGA-NPs and PLGANPs Nanoparticles were prepared by SESD method[ 181, with modifications. First, PEG-PLGA or PLGA and drug (AT0 or rhodamine B) were dissolved in the organic solvent mixture consisting of acetonitrile/ethanol (1 1, by volume). Then, the mixture solution was slowly dropped into the emulsifier solution under moderate stirring at room temperature. Finally, the organic phases were eliminated under reduced pressure at 40°C. The produced nanoparticles were collected by centrifugation (15000r.min- ') at 4 "C and washed with ultra pure water, thrice, to remove the emulsifier. The obtained nanoparticle suspension was freeze-dried and kept in a desiccator. 2.6 Physicochemical characteristics of the nanoparticles Particle size and zeta potential of nanoparticles were measured by photon correlation spectroscopy (PCS) using a Nano-ZS90 laser particle analyzer (Malvern Instruments, UK) at a wavelength of 633nm and a scattering angle of 90". Samples were diluted with ultra pure water for PCS measurement and a solution containing sodium chloride, to adjust the conductivity to SOpS.cm-' for zeta potential measurement. All measurements were performed at room temperature. The surface morphology of nanoparticles was observed by Tecnai G2 20 transmission electron microscope (FEI Co., Netherlands). Samples for TEM observation were redispersed in ultra pure water, and dropped on copper grids stained with phosphotungstic acid solution (2%) and dried in air at room temperature before being loaded on the microscope. 2.7 Determination of the drug loading capacity of PEG-PLGA-NPs lOmg of freeze-dried A?-loaded PEG-PLGA-NPs were dissolved in 4mol.L- NaOH soluFion. The dispersion was centrifugated (15000r.min- ) at 4°C. Arsenite in clear solution was diluted, and then determined by the hydride generating atomic fluorescence spectrometer (AFSa230, Beijing Haiguang Corp. China). The measurements were performed at 197.3nm (the
Preparation and in vitro Studies of Stealth PEGylated PLGA Nanoparticles as Carriers for Arsenic Trioxide
resonance wavelengths of arsenic). The hydride generation was performed with 2.0% N a B b in O.lmol.L-' NaOH and lmol.L-' HC1. Argon was used as the carrier gas at a flow rate of 0.25~-min-'. The entrapment efficiency was obtained as the ratio of the amount of AT0 incorporated in nanoparticles to the total amount of AT0 used. Drug content was calculated as the ratio of the mass of drug in nanoparticles to the total initial mass amount of the polymer. Drug loading capacity represented the ratio of the mass of the drug in the nanoparticles to the total initial mass amount of the polymer.
2.8 In vitro release studies of ATO-loaded PEGPLGA-NPs 5mg of ATO-loaded PEG-PLGA-NPs were enclosed in dialysis bags (cellulose membrane, M, cut-off 12000, Sigma) and incubated in 500ml of PBS (pH 7.4) under gentle stirring at 37°C. At predetermined time intervals, supernatants (300~1)were taken and ultrafiltrated with millipore. The precipitated particles were resuspended in fresh solution and returned to the incubation medium. The ultrafiltrated solution containing released AT0 was then analyzed by atomic fluorescence spectroscopy to determine the percentage release of the drug from the nanoparticles. 2.9 In vitro uptake of nanoparticles by MPM MPM were routinely cultured in monolayers in complete culture medium at 37°C and 5% CO2. For phagocytosis quantification, the cells were incubated for 24h at 37°C in a 24-well culture plate and then washed, to remove the nonadherent cells. Adherent cells were further incubated in a completed medium. The cell number was adjusted to 5 X lo5 cells in each well. After 24h of incubation, 2 0 0 ~ 1of nanoparticles labeled rhodamine B suspension were added and iycubated for 75min. After centrifugation (1000r.min- , lOmin), the cells were separated from the free nanoparticles and washed with PBS thrice. Finally, the fluorescent intensity of the nanoparticle-phagocytosed cells was analyzed by a Beckman Coulter flow cytometer (USA). 2.10 Statistical analysis The experiments were repeated thrice and the results were expressed as mean +standard deviation (S.D.) Statistical analysis was done using two-tailed Student's t-test. In all cases, P<0.05 was considered statistically significant.
3 RESULTS AND DISCUSSION 3.1 Physicochemical characteristics of the copolymer The basic chemical structure of the PEG-PLGA and PLGA copolymer was confirmed by using the 1 H NMR and FTIR spectrum. The results of the spectra were consistent with the structures of copolymers (figures not shown). M, and M,/M, of the copolymers determined by GPC are shown in Table 1.
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Table 1 M, and MJM, of PLGA and PEG-PLGA copolymer Copolymer PLGA PEG-PLGA
MW 15000
16500
MwJMn 2. I5 2.08
3.2 Transformation of AT0 crystal form and improvement of solubility in ethanol AT0 possesses three types of crystal forms, namely the amorphism, cube, and monocline forms. Amorphous AT0 is slightly soluble in water, but undissolvable in ethanol, whereas, AT0 in the monoclinic or cubic crystal form can be dissolved in ethanol. However, amorphous AT0 is usually sold in the market. Thus, to increase the dissolvability of AT0 in ethanol, a chemical method was employed to transform the amorphous AT0 into a cubic crystal form. After transformation, the dissolvability of AT0 in ethanol increased remarkably. Table 2 shows the dissolvability of amorphous AT0 and cubic crystal form of AT0 in ethanol. Table 2 Dissolvability of arnorphisrn and cubic crystal form of AT0 in ethanol Crystal form of AT0 amorphism cube
Dissolvability X 1.12 1050
mgm-'
3.3 Physicochemicalcharacteristics of ATO-loaded PEG-PLGA-NPs 3.3.1 Effect of the type of emulsifiers During the preparation of nanoparticles, an emulsifier was added to stabilize the nanoparticles. However, it could also influence the properties of the nanoparticles formed[ 19,201. Previous studies suggested that ideal stealth nanoparticles should possess a smaller size (<200nm), low zeta potential values, and good stereo-flexibility on their surface[21]. In this study, three types of nonionic emulsifiers, Poloxamer 188, Tween 80, and PVA, having different hydrophilic-lipophilic balance (HLB), were used to prepare ATO-loaded PEG-PLGA-NPs. From Table 3, it was found that at the emulsifier concentration of 0.5%, the particle size was the lowest, moreover, the entrapment efficiency and drug loading was the highest when Tween 80 was employed. Based on a previous study, smaller size was liable to avoid the uptake of mononuclear phagocytes system (MPS) and prolong the circulation time of nanoparticles in the blood stream in vivo, Tween 80 was used in latter experiments. 3.3.2 Effect of the emulsifier Concentration The effect of the emulsifier concentration on the characterization of ATO-loaded PEG-PLGA-NPs was investigated (Table 4). Table 4 shows that particle size was increased with Tween 80 concentration from 0.2% to 1.5%. The largest particle size was observed at a concentration of 1.5%, resulting probably from the higher viscosity of the aqueous phase and consequently larger droplets Chin. J. Ch. E. 15(6) 795 (2007)
Chin. J. Ch. E. (Vol. 15, No. 6)
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Table 3 Effect of several emulsifiers on PEG-PLGA-NPscharacteristics Emulsifiers
Particle size, nm
Polydispersity index
PVA
177.15 18.6'
0.208 f0.023'
Poloxamer 188
148.3f9.4"
Tween 80
120.8f8.2
-
~
Zeta potential, mV Entrapment efficiency, % Drug loading, % - 11.73 f4.12
49.0f 2.8'
0.9 1f0.09"
0.107k0.025'
-9.25 f 2 . 7 4
62.8 f1.2"
1.16kO.04'
0.113 f0.006
-10.73f1.81
73.6f 1.9
~-
~
1.36f0.06 -
~
Note: The preparation parameters: PEG-PLGA concentration (0.40mgm-'), organic solvent mixture (lOml), aqueous phase (0.5% emulsifier, 60ml), drug concentration (0.l0mgm-I). The results were expressed as mean fS.D., n=3. 0 P<0.05 compared with Tween 80. 0 P(O.01 compared with Tween 80.
Emulsifier, %
Table 4 Effect of T-80 concentration on PEG-PLGA-NPs characteristics Particle size, nm Polydispersity index Zeta potential, mV Entrapment efficiency, %
Drug loading, %
0.2
115.1f7.7°
0.161 f0.009
- 12.20f1.22
65.1f2.6'
1.20+_0.08'
0.5
120.8f8.2 136.95 10.5O
0.1 13f0.006 0.178 f0.006
- 10.73f 1.81
1.o
-9.4552.53
73.6f 1.9 60.3 f1.2'
1.11+0.04'
1.5
147.1f9.9"
0.146f0.003
-8.145 1.80
55.1 f1.7'
1.02f0.05'
1.36f0.06
Note: For preparation conditions see Table 3. The results were also expressed as mean fS.D., n = 3 . 0 P<0.05, compared with 0.5% Tween 80.
within the emulsion[22]. Negative zeta potential decreased with an increase in the concentration of Tween 80, which might be attributed to the nonionic properties of Tween 80 covering the surface of nanoparticles [23]. In addition, high emulsifier concentration would lead to a drop in entrapment efficiency and drug loading of PEG-PLGA-NPs. 3.3.3 Effect of the polymer concentration The effect of the polymer concentration on ATO-loaded PEG-PLGA-NPs characteristics was shown in Table 5. As shown in Table 5 , particle size, entrapment efficiency, and drug loading increased constantly with an increase in copolymer concentration. The increase
in particle size was because of an increase in viscosity of the organic phase, when a higher concentration of copolymer was employed, which would hamper the acetonitrile/ethanol spontaneous diffusion toward aqueous solution. At the same time, high copolymer concentration in the organic phase resulted in an increase in the entrapment efficiency and drug loading. Therefore, appropriate copolymer concentration was important to ensure an improvement in entrapment efficiency and drug loading of PEG-PLGA-NPs. 3.3.4 Efect of the A T 0 concentration The effect of AT0 concentration on the characteristics of PEG-PLGA-NPs is shown in Table 6. As displayed in Table 6, higher AT0 concentra-
Table 5 Effect of polymer concentration on PEG-PLGA-NPscharacteristics Copolymer, rng.rn1-l
Particle size, nm
Polydispersity index
Zeta potential, mV
Entrapment efficiency, % Drug loading, %
2.0
100.25 7.7'
0.091 f0.009
- 14.55f2.50'
54.4f 1.5'
0.99f0.06'
4.0
120.8f8.2
0.113k0.006
- 10.73f 1.81
73.6f 1.9
1.36f0.06 1.40f0.04
75.8f 1.3 6.0 150.3f 15.6' 0.099f0.011 -9.135 1.24 1.49 f0.07' 80.7 f2.2' 8.0 178.4f 22.4' 0.098f0.009 -8.91 f1.51 Note: Aqueous phase (0.5%emulsifier), for the other preparation conditions see Table 3. The results were expressed as mean f S . D . , n=3. 0 P<0.05, compared with 4.0mgm-l PEG-PLGA in organic phase.
Table 6 Effect of drug concentration on PEG-PLGA-NPs characteristics Drug, mg.rn1-l
Particle size, nm
Polydispersity index
Zeta potential, mV
Entrapment efficiency, %
Drug loading, %
0.05
112.21f6.70°
0.101 f0.009
0.10
120.76k8.2
0.113 f0.006
- 10.05 f 1.60
76.4 f1.5
0.7 1 f0.05'
- 10.73f1.81
73.6f1.9
1.36 f0.06
0.20
137f 13.60'
0.099 f0.01 1
-11.04f 1.86
40.9 f1.6'
1.5 1 f0.07'
-10.91f2.04 0.30 168.4f22.41° 0.098 f0.009 Note: For the preparation conditions see Table 3. The results were also expressed as mean 0 P<0.05 compared with O.lOmgm-' ATO. 0 P(O.01 compared with 0.10mgm-l ATO.
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30.7 f2.2' fS.D., n=3.
1.76fO.12O
Preparation and in v h o Studies of Stealth PEGylated PLGA Nanoparticles as Carriers for Arsenic Trioxide
tion induced larger particle size, but the zeta potential had no significant change, which could be from the result of cubic AT0 without residue charge. Although the AT0 concentration increased from 0.05 to 0.30mg.ml- I , the entrapment efficiency decreased from (76.4h 1.5)% to (30.7 h 2.2)%. The results demonstrated that when the AT0 concentration reached a saturation limit, the drug loading capacity of PEG-PLGA-NPs would keep constant. 3.3.5 Optimal ATO-loaded PEG-PLGA-NPs On the basis of these results, it is concluded that physicochemical characteristics of ATO-loaded PEG-PLGA-NPs could be controlled by altering several experimental conditions, such as, the type of emulsifiers, concentration of emulsifiers, polymers, and ATO. Furthermore, the preparation parameters were important effectors, which affected the loading capacity of PEG-PLGA-NPs. The optimal formulation was established based on the defined factors, with particle size, zeta potential, entrapment efficiency, and drug loading efficiency. The final ATO-loaded nanospheres were prepared by an orthogonal manufacture protocol using a certain type of emulsifier, emulsifier concentration, PEG-PLGA concentration, and AT0 concentration as factors (data not shown). The optimal conditions for preparation of ATO-loaded PEG-PLGA-NPs are as follows: (1) Tween 80 is the most proper emulsifier for preparing ATO-loaded PEG-PLGA-NPs. (2) The concentration of the emulsifier is 0.5%. (3) The concentration of the copolymer in the organic phase is 4.0mgm-l. (4) The volume ratio of the organic phase and the aqueous phase is 1 : 6 (10ml:60ml). ( 5 ) The concentration of drug (ATO) is 0.10mg.ml- . The morphology and particle size of the optimized ATO-loaded PEG-PLGA-NPs examined by TEM are shown in Fig. 1.
Figure 1 TEM image of ATO-loaded PEG-PLGA-NPs(A) and a single ATO-loaded PEG-PLGA-NPs(B)
The TEM images indicated that the optimized nanoparticles were spherical or near spherical in shape without any aggregation or adhesion, and the particle size was around 120nm. Zeta potential was - 10.73mV. For the stability investigations, ATO-loaded PEG-PLGA-NPs suspensions were stored at 4°C. The particle size and zeta potential did not change signifi-
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cantly over a storage period of four weeks. Therefore, the nanoparticle system was highly stable for a month.
3.4 In vitro release behavior of ATO-loaded PEGPLGA-NPs The drug release behavior of PEG-PLGA-NPs was shown in Fig.2.
s d
-p
e’
100
80
e,
60
-z 40 5 20 i c
2
0
0
time, d
Figure 2 In vifro release behavior of ATO-loaded PEG-PLGA-NPs [The results were expressed as mean i~S.D. (n=3)]
Figure 2 shows that the drug release behavior of PEG-PLGA-NPs displayed a typical sustained fashion. The obtained release data for PEG-PLGA-NPs within 26 days was fitted into a Higuchi equation [Drug Release (%)=6.3979t’”+3.1529, f=0.9518]. The sustained release would mainly depend on drug diffusion and copolymer matrix erosion, which were slower processes. Moreover, the release results demonstrated that the PEG segment played an important role in adjusting the drug release rate. This could be valuable for preparing drug controlled release systems to satisfy clinical demands[23].
3.5 In vitro uptake of nanoparticles by MPM AT0 could not be directly used to test the uptake of MPM because arsenic would induce apoptosis of normal mononuclear cells[24]. Rhodamine B was thus used as a model fluorescent drug to study the cellular uptake of macrophages. To confirm the escape of PEG-PLGA-NPs from phagocytosis by MPM compared with PLGA-NPs, PEG-PLGA-NPs, and PLGA-NPs, labeled rhodamine B was prepared under optimal conditions, and the uptake of nanoparticles by MPM were analyzed by flow cytometry. As shown in Fig.3 the uptake percent of PLGA-NPs was about 62%, which was about 2.6-folds greater than that of PEG-PLGA-NPs (24%, Tween 80 was removed) and 3.7-folds greater than that of PEG-PLGA-NPs (17%, with Tween 80 as emulsifier). The result indicated that the nanoparticles modified with PEG could significantly reduce the uptake by MPM, which was in accord with the results reported previously[10-121. The depressed uptake of stealth nanoparticles might depend on the presence of PEG chains on the surface of nanoparticles, which form a hydrophilic cloud and decrease the zeta potential of nanoparticles, which prevent interactions with biological components, reduce complement activation, and interaction of the uptake of nanoparticles by MPS Chin. J. Ch. E. 15(6) 795 (2007)
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4
5
6
Figure 3 Uptake of PEG-PLGA-NPs and PLGA-NPs by MPM [Results were expressed as a percentage of nanoparticles labeled rhodamine B obtained from the cells for flow cytometry (mean f S . D . , n=3)]
in viva[ 10,141. In addition, the nonionic emulsifier could also cover the surface of the nanoparticles to reduce the zeta potentia1[25,26]. Therefore, the uptake of PEG-PLGA-NPs with Tween 80 as emulsifier was further decreased.
4 CONCLUSIONS In the present investigation, biodegradable PEG-PLGA copolymer was synthesized by the ring-opening polymerization method, and the ATO-loaded PEG-PLGA-NPs were prepared by the SESD method. The physicochemical characteristics of ATO-loaded PEG-PLGA-NPs were affected by the type of emulsifiers, emulsifier concentration, polymer concentration, and drug concentration. ATO-loaded PEG-PLGA-NPs with particle size of 120.8nm, zeta potential of - 10.73mV, encapsulation efficiency of 73.6%, and drug loading of 1.36% were prepared under optimal conditions. PEG-PLGA-NPs exhibited a sustained in vitro release that lasted for more than 26 days, which fit the Higuchi equation. The uptake of PEG-PLGA-NPs by MPM was significantly lower than that of PLGA-NPs. These experimental results indicated that PEG-PLGA-NPs could be used to develop drug carriers for ATO. More detailed and exhaustive investigations are needed to fully realize the potential of these drug carriers. The bio-distribution, cellular toxicity, and pharmacokinetics of ATO-loaded PEG-PLGA-NPs in cells and animal models are currently under investigation in this laboratory.
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Murakami, H., Kobayashi, M., Takeuchi, H., Kawashima, Y., “Further application of a modified spontaneous emulsification solvent diffusion method to various types of PLGA and PLA polymers for preparation of nanoparticles”, Powder Technol., 107( 1/2), 137-143(2000). Feng, S.S., Huang, G., “Effects of emulsifiers on the controlled release of paclitaxel [Taxol(R)] from nanospheres of biodegradable polymers”, J. Control. Release, 71( I), 53-69(200 1 ). Hans, M.L., Lowman, A.M., “Biodegradable nanoparticles for drug delivery and targeting”, Curr. Opin. Solid State Mat Sci., 6(4), 319-327(2002). Zambaux, M.F., Bonneaux, F., Gref, R., Maincent, P., Dellacherie, E., Alonso, M.J., Labrude, P., Vigneron, C., “lnfluence of experimental parameters on the characteristics of poly (lactic acid) nanoparticles prepared by a double emulsion method”, J. Control. Release, 50( 1-3), 3 1-40( 1998). Douglas, S.J., Illum, L., Davis, S.S., “Particle size and size distribution of poly (butyl 2-cyanoacrylate) nanoparticles (11) lnfluence of stabilizers”, J. Colloid. Interfnce
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Chin. J. Ch. E. 15(6) 795 (2007)
(2007)
Preparation and in vitro Studies of Stealth PEGylated PLGA Nanoparticles as Carriers for Arsenic Trioxide* WANG Zhiqing(3 & %)", LIU Wei(3q X)",**, XU Huibi(f&fig)band YANG Xiangliang
B)"
(#I# College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China Department of Chemistry, Huazhong University of Science and Technology, Wuhan 430074, China
a
Abstract The aim of this study was to prepare arsenic trioxide (AT0)-loaded stealth PEGylated PLGA nanoparticles (PEG-PLGA-NPs) and to assess the merits of PEG-PLGA-NPs as drug carriers for AT0 delivery. PEG-PLGA copolymer was synthesized with methoxypolyethyleneglycol (M,=5000), D, L-lactide, and glycolide by the ring-opening polymerization method. Amorphous AT0 was transformed into cubic crystal form to increase its solubility in the organic solvent. ATO-loaded PEG-PLGA-NPs were prepared by the modified spontaneous emulsification solvent diffusion (SESD) method, and the main experimental factors influencing the characteristics of nanoparticles were investigated, to optimize the preparation. To confirm the escape of PEG-PLGA-NPs from phagocytosis by phagocytes, PEG-PLGA-NPs labeled rhodamine B uptake by murine peritoneal macrophages (MPM) were analyzed by flow cytometry. The results showed that the physicochemical characteristics of PEG-PLGA-NPs were affected by the type and concentration of the emulsifiers, polymer concentration, and drug concentration. ATO-loaded PEG-PLGA-NPs, with particle size of 120.8nm, zeta potential of - 10.73mV, encapsulation efficiency of 73.6%, and drug loading of 1.36%, were prepared under optimal conditions. The images of transmission electron microscopy (TEM) indicated that the optimized nanoparticles were near spherical and without aggregation or adhesion. The release experiments in vitro showed the AT0 release from PEG-PLGA-NPs exhibited consequently sustained release for more than 26d, which was in accordance with Higuchi equation. The uptake of PEG-PLGA-NPs by MPM was found to decrease markedly compared to PLGA-NPs. The experimental results showed that PEG-PLGA-NPs were potential nano drug delivery carriers for ATO. Keywords arsenic trioxide, PEGylated PLGA nanoparticles, ring-opening polymerization, spontaneous emulsification solvent diffusion method, in vitro drug release, phagocytic uptake
1 INTRODUCTION Arsenic trioxide (As203, ATO) has been an effective therapeutic agent for acute promyelocytic leukemia (APL) in clinics. The mechanisms of action are associated with the induction of apoptosis and differentiation[11. Recently, in vitro studies have revealed that clinically achievable concentrations of AT0 can trigger apoptosis of leukemia and lymphoma cells as well as some solid tumor cells, including neuroblastoma and gastric, transitional, renal, esophageal, prostate, colorectal, and hepatocellular cancers or cell lines[2]. This suggests that ATO-induced apoptosis may also be seen in a variety of tumor models. However, the activity of AT0 against solid tumors in vivo has not been as effective as against APL, because the dosages of AT0 required to exert these effects are much higher than those required to inhibit hematologic malignancies[3]. Severe toxic reactions, including flaccid paralysis, oncogenicity, cardiac toxicity, and renal failure, have been observed under high dosage of ATO[4,5]. A previous pharmacokmetic study has shown that plasma arsenic eliminates rapidly and the elimination half-life of AT0 in plasma is very short after intravenous administration[6]. It is known that 95% to 97% of blood arsenic is bound to hemoglobin and can be distributed rapidly into some tissues and organs[3,7], which may be the reason that AT0 is not suitable for application in solid tumors. To overcome the problems, a new drug delivery
system is needed that can control drug delivery and extend drug circulation time in blood. Over the past decades, various polymeric nanoparticles have been used in drug delivery research, as they can effectively deliver drugs to the target site and increase therapeutic benefit[8]. However, these conventional particulate drug carriers can be rapidly cleared by the cells of the mononuclear phagocytes system (MPS) [8-121. At present, many studies have highlighted the development of stealth nanoparticles as drug carriers, which can avoid, or at least reduce the uptake by phagocytes and prolong the time of drugs, with effective concentration, in blood circulation[9,lo]. Polyethyleneglycol (PEG), a modified biodegradable polymer, is one of the most popular materials to prepare stealth nanoparticles[ 113. Nanoparticles prepared from PEG modified poly (D, L-lactide-co-glycolide) (PEG-PLGA) have been investigated because of their controlled release, biodegradability, and biocompatibility[121. In the PEG-PLGA copolymers, PEG has been found to be a particularly effective steric stabilizer, probably because of its high hydrophilicity, chain flexibility, electrical neutrality, and absence of functional groups, which prevent interactions with biological components, and reduce complement activation and interaction of the uptake of nanoparticles by MPS in vivo[ 13,141. As a result, the PEG-PLGA nanoparticles exhibit prolonged blood circulation time after intravenous administration in experimental animals. Moreover,
Received 2007-01-03, accepted 2007-07-26.
* Supported by the Special Funds for Major State Basic Research Program of China (973 Program. No. 2007CB935800) National High Icchnology Research and Development Program of China (863 Program. No.2004AA215 162). ** To whom correspondence should be addressed. E-mail: [email protected]
and the
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Chin. J. Ch. E. (Vol. 15, No. 6 )
some studies have also proved that PEGylated PLGA nanoparticles (PEG-PLGA-NPs) bear a targeted delivery characteristic to cancer cells[ 151. In this study, PEG-PLGA was synthesized by ring-opening polymerization. ATO-loaded PEG-PLGA-NPs were prepared by the modified spontaneous emulsification solvent diffusion (SESD) method. The effect of the type of emulsifiers, emulsifier concentration, polymer concentration, and drug concentration on the physicochemical characteristics of ATO-loaded PEG-PLGA-NPs was investigated. In addition, the in vitro release of AT0 from optimized nanoparticles was measured by atomic fluorescence spectrometry, and the uptake of nanoparticles by murine peritoneal macrophages (MPM) was assessed.
2 MATERIALS AND METHODS 2.1 Materials AT0 (As203) was purchased from Aldrich (USA). D, L-lactide and glycolide were purchased from Tianyuan Biomaterials Co. Ltd (China). Methoxypolyethyleneglycol (MePEG, M , = 5000), polyvinyl alcohol (PVA) (M,=25000), stannous octoate, and rhodamine B were obtained from Sigma Chemical Co. Ltd (USA). Poloxamer 188 was supplied by China Pharmaceutical University (China). Tween 80 (T-80) was obtained from Tianjin Bodi Chemical Company (China). Male Kuming mice (20g 2g) were obtained from the Laboratory Animal Center, Tongji Medical College, Huazhong University of Science & Technology (China). 2.2 Synthesis of PEG-PLGA copolymer PEG-PLGA copolymer was synthesized by the ring-opening polymerization under vacuum using stannous octoate as a catalyst[l6]. D, L-lactide and glycolide (molar ratio 1 : 1) and MePEG were introduced into a bottle-neck flask. Stannous octoate (0.5%, by mass), used as a catalyst, was dissolved in hexane and added to the reaction mixture. The feed was degassed through vacuumlnitrogen cycles and applied to the molten mixture at 135°C. The flask was sealed under vacuum. Then, the polymerization reaction was carried out at 180°C for 5h under vacuum. The synthesized copolymer was recovered by dissolving in dichloromethane followed by precipitation in ice-cold diethyl ether thrice. The precipitated copolymer was filtered out and dried under vacuum at 40°C for 24h.
2.3 Physicochemical characteristics of the copolymer 'H NMR spectrum was recorded in CDC13 on a Bruker spectrometer (Germany) operating at 5OOMHz. Chemical shifts (6) were given using tetramethylsilane (TMS) as an internal reference. Fourier transform infrared (FI'IR) spectrum was obtained from a neat film, cast from the chloroform copolymer solution between KBr tablets, with 1700 spectrometer (Perktn-Elmer, USA). Gel permeation chromatography (GPC) was performed in tetrahydrofuran with a Waters 510 December, 2007
chromatographic instrument (USA). Molecular weight (M,) and molecular weight distribution (MJM,,) of the copolymers were calculated using polystyrene as the standard.
2.4 Transformation of AT0 crystal form Amorphous AT0 was transformed into cubic crystal form according to previous methods[ 171. Briefly, amorphous AT0 was dissolved in 4mol.L- NaOH solution. SO2 gas, generated in a round bottle from NaHS03 and high concentration H2S04, was passed into the solution for 5min under continuous stirring at room temperature. Then, the precipitate of the cubic AT0 crystal was separated from the solution. 2.5 Preparation of PEG-PLGA-NPs and PLGANPs Nanoparticles were prepared by SESD method[ 181, with modifications. First, PEG-PLGA or PLGA and drug (AT0 or rhodamine B) were dissolved in the organic solvent mixture consisting of acetonitrile/ethanol (1 1, by volume). Then, the mixture solution was slowly dropped into the emulsifier solution under moderate stirring at room temperature. Finally, the organic phases were eliminated under reduced pressure at 40°C. The produced nanoparticles were collected by centrifugation (15000r.min- ') at 4 "C and washed with ultra pure water, thrice, to remove the emulsifier. The obtained nanoparticle suspension was freeze-dried and kept in a desiccator. 2.6 Physicochemical characteristics of the nanoparticles Particle size and zeta potential of nanoparticles were measured by photon correlation spectroscopy (PCS) using a Nano-ZS90 laser particle analyzer (Malvern Instruments, UK) at a wavelength of 633nm and a scattering angle of 90". Samples were diluted with ultra pure water for PCS measurement and a solution containing sodium chloride, to adjust the conductivity to SOpS.cm-' for zeta potential measurement. All measurements were performed at room temperature. The surface morphology of nanoparticles was observed by Tecnai G2 20 transmission electron microscope (FEI Co., Netherlands). Samples for TEM observation were redispersed in ultra pure water, and dropped on copper grids stained with phosphotungstic acid solution (2%) and dried in air at room temperature before being loaded on the microscope. 2.7 Determination of the drug loading capacity of PEG-PLGA-NPs lOmg of freeze-dried A?-loaded PEG-PLGA-NPs were dissolved in 4mol.L- NaOH soluFion. The dispersion was centrifugated (15000r.min- ) at 4°C. Arsenite in clear solution was diluted, and then determined by the hydride generating atomic fluorescence spectrometer (AFSa230, Beijing Haiguang Corp. China). The measurements were performed at 197.3nm (the
Preparation and in vitro Studies of Stealth PEGylated PLGA Nanoparticles as Carriers for Arsenic Trioxide
resonance wavelengths of arsenic). The hydride generation was performed with 2.0% N a B b in O.lmol.L-' NaOH and lmol.L-' HC1. Argon was used as the carrier gas at a flow rate of 0.25~-min-'. The entrapment efficiency was obtained as the ratio of the amount of AT0 incorporated in nanoparticles to the total amount of AT0 used. Drug content was calculated as the ratio of the mass of drug in nanoparticles to the total initial mass amount of the polymer. Drug loading capacity represented the ratio of the mass of the drug in the nanoparticles to the total initial mass amount of the polymer.
2.8 In vitro release studies of ATO-loaded PEGPLGA-NPs 5mg of ATO-loaded PEG-PLGA-NPs were enclosed in dialysis bags (cellulose membrane, M, cut-off 12000, Sigma) and incubated in 500ml of PBS (pH 7.4) under gentle stirring at 37°C. At predetermined time intervals, supernatants (300~1)were taken and ultrafiltrated with millipore. The precipitated particles were resuspended in fresh solution and returned to the incubation medium. The ultrafiltrated solution containing released AT0 was then analyzed by atomic fluorescence spectroscopy to determine the percentage release of the drug from the nanoparticles. 2.9 In vitro uptake of nanoparticles by MPM MPM were routinely cultured in monolayers in complete culture medium at 37°C and 5% CO2. For phagocytosis quantification, the cells were incubated for 24h at 37°C in a 24-well culture plate and then washed, to remove the nonadherent cells. Adherent cells were further incubated in a completed medium. The cell number was adjusted to 5 X lo5 cells in each well. After 24h of incubation, 2 0 0 ~ 1of nanoparticles labeled rhodamine B suspension were added and iycubated for 75min. After centrifugation (1000r.min- , lOmin), the cells were separated from the free nanoparticles and washed with PBS thrice. Finally, the fluorescent intensity of the nanoparticle-phagocytosed cells was analyzed by a Beckman Coulter flow cytometer (USA). 2.10 Statistical analysis The experiments were repeated thrice and the results were expressed as mean +standard deviation (S.D.) Statistical analysis was done using two-tailed Student's t-test. In all cases, P<0.05 was considered statistically significant.
3 RESULTS AND DISCUSSION 3.1 Physicochemical characteristics of the copolymer The basic chemical structure of the PEG-PLGA and PLGA copolymer was confirmed by using the 1 H NMR and FTIR spectrum. The results of the spectra were consistent with the structures of copolymers (figures not shown). M, and M,/M, of the copolymers determined by GPC are shown in Table 1.
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Table 1 M, and MJM, of PLGA and PEG-PLGA copolymer Copolymer PLGA PEG-PLGA
MW 15000
16500
MwJMn 2. I5 2.08
3.2 Transformation of AT0 crystal form and improvement of solubility in ethanol AT0 possesses three types of crystal forms, namely the amorphism, cube, and monocline forms. Amorphous AT0 is slightly soluble in water, but undissolvable in ethanol, whereas, AT0 in the monoclinic or cubic crystal form can be dissolved in ethanol. However, amorphous AT0 is usually sold in the market. Thus, to increase the dissolvability of AT0 in ethanol, a chemical method was employed to transform the amorphous AT0 into a cubic crystal form. After transformation, the dissolvability of AT0 in ethanol increased remarkably. Table 2 shows the dissolvability of amorphous AT0 and cubic crystal form of AT0 in ethanol. Table 2 Dissolvability of arnorphisrn and cubic crystal form of AT0 in ethanol Crystal form of AT0 amorphism cube
Dissolvability X 1.12 1050
mgm-'
3.3 Physicochemicalcharacteristics of ATO-loaded PEG-PLGA-NPs 3.3.1 Effect of the type of emulsifiers During the preparation of nanoparticles, an emulsifier was added to stabilize the nanoparticles. However, it could also influence the properties of the nanoparticles formed[ 19,201. Previous studies suggested that ideal stealth nanoparticles should possess a smaller size (<200nm), low zeta potential values, and good stereo-flexibility on their surface[21]. In this study, three types of nonionic emulsifiers, Poloxamer 188, Tween 80, and PVA, having different hydrophilic-lipophilic balance (HLB), were used to prepare ATO-loaded PEG-PLGA-NPs. From Table 3, it was found that at the emulsifier concentration of 0.5%, the particle size was the lowest, moreover, the entrapment efficiency and drug loading was the highest when Tween 80 was employed. Based on a previous study, smaller size was liable to avoid the uptake of mononuclear phagocytes system (MPS) and prolong the circulation time of nanoparticles in the blood stream in vivo, Tween 80 was used in latter experiments. 3.3.2 Effect of the emulsifier Concentration The effect of the emulsifier concentration on the characterization of ATO-loaded PEG-PLGA-NPs was investigated (Table 4). Table 4 shows that particle size was increased with Tween 80 concentration from 0.2% to 1.5%. The largest particle size was observed at a concentration of 1.5%, resulting probably from the higher viscosity of the aqueous phase and consequently larger droplets Chin. J. Ch. E. 15(6) 795 (2007)
Chin. J. Ch. E. (Vol. 15, No. 6)
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Table 3 Effect of several emulsifiers on PEG-PLGA-NPscharacteristics Emulsifiers
Particle size, nm
Polydispersity index
PVA
177.15 18.6'
0.208 f0.023'
Poloxamer 188
148.3f9.4"
Tween 80
120.8f8.2
-
~
Zeta potential, mV Entrapment efficiency, % Drug loading, % - 11.73 f4.12
49.0f 2.8'
0.9 1f0.09"
0.107k0.025'
-9.25 f 2 . 7 4
62.8 f1.2"
1.16kO.04'
0.113 f0.006
-10.73f1.81
73.6f 1.9
~-
~
1.36f0.06 -
~
Note: The preparation parameters: PEG-PLGA concentration (0.40mgm-'), organic solvent mixture (lOml), aqueous phase (0.5% emulsifier, 60ml), drug concentration (0.l0mgm-I). The results were expressed as mean fS.D., n=3. 0 P<0.05 compared with Tween 80. 0 P(O.01 compared with Tween 80.
Emulsifier, %
Table 4 Effect of T-80 concentration on PEG-PLGA-NPs characteristics Particle size, nm Polydispersity index Zeta potential, mV Entrapment efficiency, %
Drug loading, %
0.2
115.1f7.7°
0.161 f0.009
- 12.20f1.22
65.1f2.6'
1.20+_0.08'
0.5
120.8f8.2 136.95 10.5O
0.1 13f0.006 0.178 f0.006
- 10.73f 1.81
1.o
-9.4552.53
73.6f 1.9 60.3 f1.2'
1.11+0.04'
1.5
147.1f9.9"
0.146f0.003
-8.145 1.80
55.1 f1.7'
1.02f0.05'
1.36f0.06
Note: For preparation conditions see Table 3. The results were also expressed as mean fS.D., n = 3 . 0 P<0.05, compared with 0.5% Tween 80.
within the emulsion[22]. Negative zeta potential decreased with an increase in the concentration of Tween 80, which might be attributed to the nonionic properties of Tween 80 covering the surface of nanoparticles [23]. In addition, high emulsifier concentration would lead to a drop in entrapment efficiency and drug loading of PEG-PLGA-NPs. 3.3.3 Effect of the polymer concentration The effect of the polymer concentration on ATO-loaded PEG-PLGA-NPs characteristics was shown in Table 5. As shown in Table 5 , particle size, entrapment efficiency, and drug loading increased constantly with an increase in copolymer concentration. The increase
in particle size was because of an increase in viscosity of the organic phase, when a higher concentration of copolymer was employed, which would hamper the acetonitrile/ethanol spontaneous diffusion toward aqueous solution. At the same time, high copolymer concentration in the organic phase resulted in an increase in the entrapment efficiency and drug loading. Therefore, appropriate copolymer concentration was important to ensure an improvement in entrapment efficiency and drug loading of PEG-PLGA-NPs. 3.3.4 Efect of the A T 0 concentration The effect of AT0 concentration on the characteristics of PEG-PLGA-NPs is shown in Table 6. As displayed in Table 6, higher AT0 concentra-
Table 5 Effect of polymer concentration on PEG-PLGA-NPscharacteristics Copolymer, rng.rn1-l
Particle size, nm
Polydispersity index
Zeta potential, mV
Entrapment efficiency, % Drug loading, %
2.0
100.25 7.7'
0.091 f0.009
- 14.55f2.50'
54.4f 1.5'
0.99f0.06'
4.0
120.8f8.2
0.113k0.006
- 10.73f 1.81
73.6f 1.9
1.36f0.06 1.40f0.04
75.8f 1.3 6.0 150.3f 15.6' 0.099f0.011 -9.135 1.24 1.49 f0.07' 80.7 f2.2' 8.0 178.4f 22.4' 0.098f0.009 -8.91 f1.51 Note: Aqueous phase (0.5%emulsifier), for the other preparation conditions see Table 3. The results were expressed as mean f S . D . , n=3. 0 P<0.05, compared with 4.0mgm-l PEG-PLGA in organic phase.
Table 6 Effect of drug concentration on PEG-PLGA-NPs characteristics Drug, mg.rn1-l
Particle size, nm
Polydispersity index
Zeta potential, mV
Entrapment efficiency, %
Drug loading, %
0.05
112.21f6.70°
0.101 f0.009
0.10
120.76k8.2
0.113 f0.006
- 10.05 f 1.60
76.4 f1.5
0.7 1 f0.05'
- 10.73f1.81
73.6f1.9
1.36 f0.06
0.20
137f 13.60'
0.099 f0.01 1
-11.04f 1.86
40.9 f1.6'
1.5 1 f0.07'
-10.91f2.04 0.30 168.4f22.41° 0.098 f0.009 Note: For the preparation conditions see Table 3. The results were also expressed as mean 0 P<0.05 compared with O.lOmgm-' ATO. 0 P(O.01 compared with 0.10mgm-l ATO.
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30.7 f2.2' fS.D., n=3.
1.76fO.12O
Preparation and in v h o Studies of Stealth PEGylated PLGA Nanoparticles as Carriers for Arsenic Trioxide
tion induced larger particle size, but the zeta potential had no significant change, which could be from the result of cubic AT0 without residue charge. Although the AT0 concentration increased from 0.05 to 0.30mg.ml- I , the entrapment efficiency decreased from (76.4h 1.5)% to (30.7 h 2.2)%. The results demonstrated that when the AT0 concentration reached a saturation limit, the drug loading capacity of PEG-PLGA-NPs would keep constant. 3.3.5 Optimal ATO-loaded PEG-PLGA-NPs On the basis of these results, it is concluded that physicochemical characteristics of ATO-loaded PEG-PLGA-NPs could be controlled by altering several experimental conditions, such as, the type of emulsifiers, concentration of emulsifiers, polymers, and ATO. Furthermore, the preparation parameters were important effectors, which affected the loading capacity of PEG-PLGA-NPs. The optimal formulation was established based on the defined factors, with particle size, zeta potential, entrapment efficiency, and drug loading efficiency. The final ATO-loaded nanospheres were prepared by an orthogonal manufacture protocol using a certain type of emulsifier, emulsifier concentration, PEG-PLGA concentration, and AT0 concentration as factors (data not shown). The optimal conditions for preparation of ATO-loaded PEG-PLGA-NPs are as follows: (1) Tween 80 is the most proper emulsifier for preparing ATO-loaded PEG-PLGA-NPs. (2) The concentration of the emulsifier is 0.5%. (3) The concentration of the copolymer in the organic phase is 4.0mgm-l. (4) The volume ratio of the organic phase and the aqueous phase is 1 : 6 (10ml:60ml). ( 5 ) The concentration of drug (ATO) is 0.10mg.ml- . The morphology and particle size of the optimized ATO-loaded PEG-PLGA-NPs examined by TEM are shown in Fig. 1.
Figure 1 TEM image of ATO-loaded PEG-PLGA-NPs(A) and a single ATO-loaded PEG-PLGA-NPs(B)
The TEM images indicated that the optimized nanoparticles were spherical or near spherical in shape without any aggregation or adhesion, and the particle size was around 120nm. Zeta potential was - 10.73mV. For the stability investigations, ATO-loaded PEG-PLGA-NPs suspensions were stored at 4°C. The particle size and zeta potential did not change signifi-
799
cantly over a storage period of four weeks. Therefore, the nanoparticle system was highly stable for a month.
3.4 In vitro release behavior of ATO-loaded PEGPLGA-NPs The drug release behavior of PEG-PLGA-NPs was shown in Fig.2.
s d
-p
e’
100
80
e,
60
-z 40 5 20 i c
2
0
0
time, d
Figure 2 In vifro release behavior of ATO-loaded PEG-PLGA-NPs [The results were expressed as mean i~S.D. (n=3)]
Figure 2 shows that the drug release behavior of PEG-PLGA-NPs displayed a typical sustained fashion. The obtained release data for PEG-PLGA-NPs within 26 days was fitted into a Higuchi equation [Drug Release (%)=6.3979t’”+3.1529, f=0.9518]. The sustained release would mainly depend on drug diffusion and copolymer matrix erosion, which were slower processes. Moreover, the release results demonstrated that the PEG segment played an important role in adjusting the drug release rate. This could be valuable for preparing drug controlled release systems to satisfy clinical demands[23].
3.5 In vitro uptake of nanoparticles by MPM AT0 could not be directly used to test the uptake of MPM because arsenic would induce apoptosis of normal mononuclear cells[24]. Rhodamine B was thus used as a model fluorescent drug to study the cellular uptake of macrophages. To confirm the escape of PEG-PLGA-NPs from phagocytosis by MPM compared with PLGA-NPs, PEG-PLGA-NPs, and PLGA-NPs, labeled rhodamine B was prepared under optimal conditions, and the uptake of nanoparticles by MPM were analyzed by flow cytometry. As shown in Fig.3 the uptake percent of PLGA-NPs was about 62%, which was about 2.6-folds greater than that of PEG-PLGA-NPs (24%, Tween 80 was removed) and 3.7-folds greater than that of PEG-PLGA-NPs (17%, with Tween 80 as emulsifier). The result indicated that the nanoparticles modified with PEG could significantly reduce the uptake by MPM, which was in accord with the results reported previously[10-121. The depressed uptake of stealth nanoparticles might depend on the presence of PEG chains on the surface of nanoparticles, which form a hydrophilic cloud and decrease the zeta potential of nanoparticles, which prevent interactions with biological components, reduce complement activation, and interaction of the uptake of nanoparticles by MPS Chin. J. Ch. E. 15(6) 795 (2007)
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Figure 3 Uptake of PEG-PLGA-NPs and PLGA-NPs by MPM [Results were expressed as a percentage of nanoparticles labeled rhodamine B obtained from the cells for flow cytometry (mean f S . D . , n=3)]
in viva[ 10,141. In addition, the nonionic emulsifier could also cover the surface of the nanoparticles to reduce the zeta potentia1[25,26]. Therefore, the uptake of PEG-PLGA-NPs with Tween 80 as emulsifier was further decreased.
4 CONCLUSIONS In the present investigation, biodegradable PEG-PLGA copolymer was synthesized by the ring-opening polymerization method, and the ATO-loaded PEG-PLGA-NPs were prepared by the SESD method. The physicochemical characteristics of ATO-loaded PEG-PLGA-NPs were affected by the type of emulsifiers, emulsifier concentration, polymer concentration, and drug concentration. ATO-loaded PEG-PLGA-NPs with particle size of 120.8nm, zeta potential of - 10.73mV, encapsulation efficiency of 73.6%, and drug loading of 1.36% were prepared under optimal conditions. PEG-PLGA-NPs exhibited a sustained in vitro release that lasted for more than 26 days, which fit the Higuchi equation. The uptake of PEG-PLGA-NPs by MPM was significantly lower than that of PLGA-NPs. These experimental results indicated that PEG-PLGA-NPs could be used to develop drug carriers for ATO. More detailed and exhaustive investigations are needed to fully realize the potential of these drug carriers. The bio-distribution, cellular toxicity, and pharmacokinetics of ATO-loaded PEG-PLGA-NPs in cells and animal models are currently under investigation in this laboratory.
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