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One-pot facile preparation of PEG-modified PLGA nanoparticles: Effects of PEG and PLGA on release properties of the particles
Colloids and Surfaces A: Physicochem. Eng. Aspects 469 (2015) 66–72
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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
One-pot facile preparation of PEG-modified PLGA nanoparticles: Effects of PEG and PLGA on release properties of the particles Nao Yoneki, Taku Takami, Tomoki Ito, Ryosuke Anzai, Kengo Fukuda, Keita Kinoshita, Seiichi Sonotaki, Yoshihiko Murakami ∗ Department of Organic and Polymer Materials Chemistry, Faculty of Engineering, Tokyo University of Agriculture and Technology, Japan
h i g h l i g h t s
g r a p h i c a l
• A block copolymer-assisted emulsifi-
We investigated the effects of preparation conditions and structural properties of both PEG–PLA block copolymers and PLGA on the properties of PEG-modified PLGA nanoparticles via a block copolymerassisted emulsification/evaporation method.
cation was applied to nanoparticles preparations. • The nanoparticles with a diameter of 200 nm were obtained under optimized conditions. • The block copolymers greatly affected the release properties of nanoparticles.
a r t i c l e
i n f o
Article history: Received 16 November 2014 Received in revised form 1 January 2015 Accepted 4 January 2015 Available online 15 January 2015 Keywords: Nanoparticle Surface modification Poly(ethylene glycol) (PEG) Poly(lactide-co-glycolide) (PLGA) Drug delivery system
a b s t r a c t We show here a one-pot facile preparation of poly(ethylene glycol) (PEG)-modified poly(lactideco-glycolide) (PLGA) nanoparticles by means of a novel “block copolymer-assisted” emulsification/evaporation method. In the present article, we clarified the effects of preparation conditions and the structural properties of both polymeric modifiers and particle-forming hydrophobic polymers on the properties of the PEG-modified PLGA nanoparticles. We first clarified that the concentration of PLGA, the volume ratio of an organic solvent to a PVA solution, and a stirring rate were major factors affecting the diameter of the nanoparticles. We successfully prepared the nanoparticles, with a diameter of approximately 200 nm under the optimized conditions. The additive amount and the composition of PEG–PLA block copolymers affected the surface charge of the nanoparticles. Finally, we clarified that the compositions and molecular weights of the block copolymers greatly affected the release properties of PEG-modified nanoparticles. © 2015 Elsevier B.V. All rights reserved.
1. Introduction A drug delivery system (DDS) is a time- and site-specific system whereby drugs are delivered to a target site, consequently
∗ Corresponding author at: Department of Organic and Polymer Materials Chemistry, Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Tel.: +81 42 388 7387; fax: +81 42 388 7387. E-mail address: [email protected] (Y. Murakami). http://dx.doi.org/10.1016/j.colsurfa.2015.01.011 0927-7757/© 2015 Elsevier B.V. All rights reserved.
a b s t r a c t
resulting in both a reduction of side effects and an enhancement of the drugs’ pharmaceutical effects. Important factors for regulating DDS efficiency are an enhancement of absorption, a controlled release, and a targeting of drugs. Drug targeting is an important technique for achieving effective chemotherapy for cancers. Successful targeting requires the selection of suitable drug carriers including liposomes [1–3], micelles [4–9], and nano/microparticles [10,11]. Liposomes are suitable for encapsulating hydrophobic drugs, whereas polymeric micelles are suitable for encapsulating hydrophilic drugs. Although both of the carriers have been
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actively developed, the preparation of liposomes is sometimes difficult, whereas polymeric micelles exhibit a lack of drug preservation. By contrast, nano/microparticles can stably encapsulate both hydrophilic and hydrophobic drugs by both being prepared from stable emulsions where drugs dissolve in the emulsion droplet and blocking the invasion of water molecules into the particles. For effective administration into blood, it is important to control the diameter, surface properties, and drug preservation of drug carriers. Drug carriers are easily entrapped by a reticuloendothelial system (RES), because the carriers are recognized as foreign substances in blood vessels. Because the increased hydrophobicity of the carriers’ surface makes the carriers prone to easy entrapment by an RES, much attention has been paid to the development of effective surface-modification methods. Polyethylene glycol (PEG) has been a well known biocompatible modifier of drug carriers [12,13] and protein particles [14–18], because the presence of PEG both renders the carriers unrecognizable to phagocytes and reduces the aggregation of the carriers. However, a conjugation of PEG to carrier surface is difficult, because conjugation chemistry needs to be optimized for each polymer–particle combination [19]. Furthermore, poly(lactide-co-glycolide) (PLGA) and polylactide (PLA) have been used commonly in the preparation of particles [20–22], because the polymers are biocompatible and exhibit easily controllable degradation behaviors. However, PLGA and PLA particles have a reputation as being materials for which surface modification is difficult, because PLGA and PLA have almost no reactive groups in their molecules. In addition, although some investigations concerning surface modifications by electrostatic coupling reactions have been reported [23], the resulting interactions between a given particle and modifiers remain so weak as to be inapplicable to drug carriers. In this regard, we have proposed a novel one-pot facile technique for preparing surface-modified nano/microparticles to address the above problems [24-26]. Fig. 1 shows the proposed scheme for preparing the surface-modified particles via a “block copolymer-assisted” emulsification/evaporation method. First, o/w emulsion is prepared by the addition of an organic solvent dissolving both hydrophobic polymers and amphiphilic block copolymers into an aqueous solution containing emulsion stabilizers. In the first step, amphiphilic block copolymers present on the emulsion surface help stabilize the emulsion. In the next step, the hydrophobic polymers and the hydrophobic part of the amphiphilic block polymers gradually get tangled during the organic solvent’s evaporation from o/w emulsion. Consequently, the hydrophilic polymer chains are introduced on the surface of the finally resulting particles. The surface properties and drug-release behaviors of particles are controllable by changes to the molecular compositions, molecular weights, concentrations and mixed ratios of both hydrophobic polymers and amphiphilic block copolymers. We have succeeded in preparing nanoparticles for intravenous drug delivery [24] and microparticles for pulmonary drug delivery by means of the newly developed method [25,26]. However, in the former case, the diameter of the obtained particles was large (approximately 600–900 nm), because a membrane emulsification technique was used for the preparation of the particles. Furthermore, the drugrelease properties of the surface-modified particles still require clarification. The present paper describes two important topics: an effort to prepare PEG-modified nanoparticles (of which diameter is approximately 200 nm) by means of a high-speed homogenization (instead of membrane emulsification) based on our novel emulsification/evaporation techniques; and the compound-release behaviors of the resulting PEG-modified nanoparticles. This is the first report concerning the effects of “physically” combined surfacemodifiers on the release properties of the resulting polymeric nanoparticles.
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Fig. 1. The scheme of the novel block polymer-assisted emulsification/evaporation technique for preparing surface-modified nanoparticles. The hydrophobic polymers and the hydrophobic parts of amphiphilic block polymers gradually got tangled as the organic solvent evaporated from the o/w emulsion.
2. Materials and methods 2.1. Materials Ethylene oxide (Sumitomo Seika Chemicals Co., Ltd., Osaka, Japan) was purified by distilling it with CaH2 . dl-Lactide (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was recrystallized twice from ethyl acetate. 2-Methoxyethanol was distilled with sodium under reduced pressure. Potassium naphthalene was obtained by mixing potassium and naphthalene in anhydrous tetrahydrofuran (THF) for 18 h. PLGA7520, PLGA7510, PLGA7505, PLGA5020 and PLGA5005 (PLGA hijk represents PLGA whose composition ratio of lactic acid and molecular weight is hi and jk kg/mol, respectively) were purchased from Wako Pure Chemical Industries (Osaka, Japan) and stored in a freezer prior to use. Poly(vinyl alcohol) (PVA, the degree of polymerization: 500, saponification degree: 86–90 mol%) as an emulsion stabilizer was purchased from Wako Pure Chemical Industries and used without further purification. Rhodamine B was purchased from Wako Pure Chemical Industries. All the other reagents were of analytical grade and were used without further purification.
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tape and coating a thin platinum film (approximately 5 nm in thickness) on the sample under a reduced pressure with an MSP-1S ion coater (Vacuum Device Inc., Ibaraki, Japan). The volume-averaged diameter and the zeta potential of the particles were determined by dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, UK). The zeta potentials were determined under the condition that the concentration of NaCl was 1 × 10−4 M. 2.4. Release of hydrophobic compounds from surface-modified and surface-unmodified PLGA nanoparticles
Fig. 2. The scheme of the synthesis of a methoxy-terminated poly(ethylene glycol)–poly(lactic acid) (PEG–PLA) block copolymers by means of employing ringopening polymerization from ethylene oxide and dl-lactide.
2.2. Synthesis of methoxy-terminated poly(ethylene glycol)–poly(lactic acid) (PEG–PLA) block copolymers A methoxy-terminated poly(ethylene glycol)–poly(lactic acid) (PEG–PLA) block copolymer was synthesized by ring-opening polymerization of both ethylene oxide and dl-lactide in THF according to the previously reported method with slight modifications [24–30], as shown in Fig. 2. First, 2-methoxyethanol (0.3–1.0 mmol) and potassium naphthalene (1 molar equivalent of 2-methoxyethanol) were mixed in THF for 1 h. The purified ethylene oxide (80–131 mmol) was added to the obtained potassium 2-methoxyethoxide solution (total volume: 50 ml). After the solution was stirred for 48 h, THF solution of purified dl-lactide (17–70 mmol) was added to the solution. After the reaction, the resulting block copolymer was precipitated into cold 2-propanol, stored in a freezer for 12 h, centrifuged at 10,500 rpm (SS-1500, Sakuma Seisakusho Co., Ltd., Tokyo, Japan), and lyophilized in benzene. The average molecular weight of the obtained block copolymer was determined by the use of gel permeation chromatography (GPC) (system: LC2000plus, JEOL Ltd., Tokyo, Japan; column: TSKgel G3000HHR , TOSOH, Tokyo, Japan; eluent: DMF in the presence of 10 mM LiBr, flow: 1 ml/min; column temperature: 40 ◦ C) and 1 H NMR (AL-300, 300 MHz, JEOL Ltd., Tokyo, Japan). 2.3. Preparation and characterization of surface-modified and surface-unmodified PLGA particles The surface-modified PLGA particles were prepared according to an emulsification/evaporation technique by means of homogenization of the emulsion solution, as shown in Fig. 1. PLGA7510 (0.06–1.25 w/v%), and PEG–PLA block copolymers were dissolved in a dichloromethane–toluene mixed solvent where the molar ratio of PEG–PLA/PLGA was 1–5. PVA as an emulsion stabilizer was dissolved in pure Milli-Q water (0.2–2 w/v%). The organic solvent was added to the PVA solution where the volume ratio of the organic solvent/the PVA solution was 0.25–2, and subsequently, an o/w emulsion was formed by homogenization (5000–20,000 rpm, 2–10 min). The emulsion was then stirred at 150 rpm overnight at room temperature with a propeller type impeller so that the organic solvent from the o/w emulsion droplets would evaporate. Finally, we obtained purified surface-modified particles by centrifuging the solution at 7500 rpm and washing the particles with pure Milli-Q water three times. Surface-unmodified PLGA particles were prepared by means of the same method except that PEG was absent. The surface morphology of the surface-modified and surfaceunmodified PLGA particles was observed by means of a scanning electron microscope (SEM, VE-9800, KEYENCE Co., Ltd., Osaka, Japan). We prepared the specimens for SEM observation by mounting the sample on an aluminum plate with double-sided adhesive
We prepared rhodamine B-loaded nanoparticles by dissolving rhodamine B (0.01 w/v%) in the organic solvent in the preparation stage of the o/w emulsion. The obtained nanoparticles were dispersed in water at room temperature. At specific time intervals, the particles were collected by centrifugation at 7500 rpm, the amount of released compounds in the supernatant solution was determined by spectrofluorophotometer (ex : 500 nm, em : 576 nm, FP-6500, Jasco Inc., Tokyo, Japan), and the particles were redispersed. 3. Results and discussion 3.1. Synthesis and characterization of the PEG–PLA block copolymer The obtained PEG–PLA block polymers were characterized by 1 H NMR and GPC. The 1 H NMR spectra of the PEG–PLA block copolymer shown in Fig. 3 indicate that the block copolymers were synthesized successfully (the peak at around 1.8 ppm corresponds presumably to some impurities). Table 1 summarizes the results of the synthesis of the PEG–PLA block polymers A–E with different compositions. The number–average molecular weights, Mn , of the PEG and PLA units were determined on the basis of GPC and 1 H NMR, respectively. It was confirmed that the obtained Mn of PEG and PLA were 2650–9380 and 3260–7150, and the distribution of Mw /Mn was narrow. 3.2. Effects of preparation conditions on the diameter of nanoparticles The passive targeting of drug carriers is commonly based on the enhanced permeability and retention (EPR) effect wherein molecules of a specific size (approximately 20–200 nm) accumulate selectively in solid tumors so that the molecules exhibit therapeutic
Fig. 3. Typical 1 H NMR spectra of a PEG–PLA block copolymer.
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Table 1 Characterization of synthesized methoxy-terminated poly(ethylene glycol)-block-poly(lactic acid) (PEG–PLA) block copolymer. Code
A B C D E
PEG (including methoxy terminus)
PLA
PEG–PLA (including methoxy terminus)
Mn
Mw /Mn
Mn
Mn
4690 2650 4160 7840 9380
1.10 1.05 1.05 1.07 1.05
4490 4440 7150 3730 3260
effects for a long period [31]. In this study, we evaluated the effects of preparation conditions (concentration of an emulsion stabilizer (PVA) and a particle-forming hydrophobic polymer (PLGA), a molar ratio of PEG–PLA to PLGA, a volume ratio of the organic solvent to an aqueous PVA solution, a stirring time, and a stirring rate) on the diameter of the PEG-modified nanoparticles. Fig. 4 shows the effects of preparation conditions on the diameter of the PEG-modified particles, which was determined by means of DLS. PVA acts as an emulsion stabilizer by adsorbing the surface of the emulsion and by consequently making its droplets stably disperse in a continuous phase. However, it was confirmed that the emulsion solution foamed as the concentration of PVA increased. The foam prevented the successful formation of uniformly dispersed droplets of o/w emulsions. As a result, the diameter of the obtained nanoparticles increased as the concentration of PVA increased, as shown in Fig. 4a. The phenomenon was observed when PVA concentration was over 0.3 w/v%. In general, the presence of foam reduces emulsion stability and consequently makes resulting nanoparticles heterogeneous. By contrast, the emulsion was unstable when the concentration of PVA was zero, suggesting that PVA had an important role in enhancing the stability of o/w emulsion. The concentration of PVA was thus optimized to be 0.3 w/v%. Next, the concentration of polymers (particle-forming hydrophobic polymers and surface modifiers) was evaluated. In general, the diameter of the obtained nanoparticles becomes small when the concentration of particle-forming polymers decreases [32]. In our case, we also confirmed the same phenomena, as shown in Fig. 4b. However, the decrease in the concentration of PLGA will reduce the productivity of the resulting nanoparticles. Furthermore, in some cases, we observed large particles or aggregate (with a diameter over 300 nm) when PLGA concentration was over 0.2 w/v%. The concentration of PLGA was thus optimized to be
9180 7090 11,310 11,600 12,600
Mw /Mn 1.19 1.16 1.13 1.07 1.06
0.125 w/v%. It was noteworthy that the concentration of PEG–PLA block copolymers had almost no influence on the diameter of the resulting nanoparticles, as shown in Fig. 4c, presumably because the concentration of PEG–PLA block copolymer affected mainly the density of PEG that was introduced on the particle surface. In addition to chemical compounds present in emulsion systems, the volume ratio of a dispersed phase to a continuous phase is an important factor governing the diameter of emulsion droplets. In our case, the diameter of the nanoparticles decreased as the volume ratio of organic solvent to PVA solution decreased, as shown in Fig. 4d. The diameter of the particles became constant below the ratio of 0.5. Furthermore, the decrease in the amount of an organic solvent where the polymeric compounds have been dissolved will generally reduce the productivity of the resulting nanoparticles. Thus, the volume ratio of organic solvent to PVA solution was optimized to be 0.5. In general, when an emulsion is prepared, stirring conditions are important factors affecting the diameter of the nanoparticles. In our study, the diameter of nanoparticle decreased while the stirring rate increased or the stirring time increased, as shown in Fig. 4e and f, because the increased stirring rate and stirring time made emulsion droplets small. As the stirring time increased, the temperature of the resulting emulsion increased and consequently decreased the stability of the emulsion. The stirring rate and stirring time were thus optimized to be 20,000 rpm and 5 min, respectively. Furthermore, in our recent study, we found that emulsions exhibited the highest stability when the density of a dispersed phase (an organic solvent) was adjusted to be 1.00 g/cm3 , which is the same as the density of a continuous phase (an aqueous solution), because the differences in the densities of the two phases play a critical role in regulating the stability of emulsions [24]. Finally, the nanoparticles having a diameter of approximately 200 nm and a narrow size distribution were successfully prepared
Fig. 4. The effects of preparation conditions on the diameter of the PEG-modified particles that was determined by means of DLS (arrows refer to optimized conditions).
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Fig. 5. SEM image of nanoparticles obtained under optimized conditions.
with high reproducibility, as shown in Fig. 5 (DLS-based diameter of the particles were 214 ± 11 nm), under the preceding optimal conditions (the concentration of PVA: 0.3 w/v%; the concentration of PLGA: 0.125 w/v%; the molar ratio of PEG–PLA to PLGA: 1; the volume ratio of an organic solvent to a PVA solution: 0.5; the stirring rate: 20,000 rpm; and the stirring time: 5 min) in the presence of a dichloromethane–toluene mixture having a density of 1.00 g/cm3 as a dispersed phase. 3.3. The surface properties of surface-modified PLGA nanoparticles Controlling the surface properties of nanoparticles was important for their successful performance as drug carriers. We observed the surface morphology of surface-modified PLGA nanoparticles by means of SEM. The nanoparticles exhibited a spherical shape and a diameter of about 200 nm. The results of our SEM-image observation support the preceding DLS-based results, wherein the nanoparticles having a diameter of 200 nm were successfully prepared with high reproducibility. Furthermore, we evaluated the surface property of the nanoparticles by using zeta potential, an important factor for the regulation of the stability of the nanoparticles. In the preparation stage of the nanoparticles, PLGA and the hydrophobic part of the amphiphilic PEG–PLA block copolymer gradually got tangled as the organic solvent evaporated from the o/w emulsion. Consequently, the PEG chain, which was introduced on the surface of the finally obtained nanoparticles, would improve the low intravital stability of the particles, because the PEG chain both prevents nanoparticles from contacting RES and reduces their nonspecific interactions with blood vessels. Thus, it is important to control the surface charge of the nanoparticles by suitably selecting the composition and the additive amount of the PEG–PLA block copolymers. Fig. 6 shows the zeta potential of the PEG-modified PLGA nanoparticles, which were prepared from PLGA and PEG–PLA with different compositions. The surface of the surface-unmodified PLGA nanoparticles had a negative charge, approximately −24 mV, which was attributable to the carboxyl groups of PLGA present on the particle surface. We found that a PLA chain length of PEG–PLA block copolymers hardly affected the surface charge of the nanoparticles (by the comparison of the effects that block copolymers A and C had), whereas an increased PEG chain length of PEG–PLA block copolymers increased surface charge (by the comparison of the effect that block copolymers A, D and E had). The differences between effects of PLA and PEG were attributed to the fact that PLA and PEG chains of PEG–PLA block copolymers were present inside and on the surface of PLGA particles. Fig. 6 shows that, as the molar
Fig. 6. The zeta potentials of the PEG-modified PLGA nanoparticles prepared from PLGA and PEG–PLA with different compositions.
ratio of PEG–PLA to PLGA increased, the surface electric charge of the particle increased or decreased, and then reached saturated values when the molar ratio of PEG–PLA to PLGA was over 2. These phenomena are presumably due to two possible factors: (1) in general, it is difficult to introduce all added PEG–PLA block copolymers at the surface of emulsion droplets when the emulsions form, and (2) in general, the PEG density on particles’ surfaces hardly changes when PEG–PLA block copolymers are added because the resulting nanoparticles grow significantly in size. Moreover, we found that the zeta potential of the PEG-modified nanoparticles decreased as the molar ratio of PEG–PLA to PLGA increased in cases where the block copolymer B was used, because the PEG chain length was too short to be present at the interface of emulsions. 3.4. Release of hydrophobic compounds from surface-modified and surface-unmodified PLGA nanoparticles It is important to prepare nanoparticles exhibiting desired drugrelease properties for the design of drug carriers that circulate in blood vessels suitably and result in successful drug effects at target sites. In this study, thus, we have evaluated the effect of nanoparticles’ preparation conditions on the nanoparticles’ drug-release properties. Fig. 7 shows an effect of the preparation conditions on the drugrelease properties of PEG-unmodified and PEG-modified PLGA particles. The preparation conditions include factors such as the hydrophobicity of inclusions and both the compositions and molecular weights of both hydrophobic polymers and block copolymers. In all the evaluations, the diameter and dispersion of the nanoparticles did not change during the experiment of the release. The average diameters of the particles used were almost the same (218 ± 6 nm). Therefore, at least, it was no effect of the particles’ diameter on the drug-release properties shown in Fig. 7. For the PEG-unmodified nanoparticles, the molecular weight of PLGA affected the release profile of rhodamine B. As shown in Fig. 7a, the release of rhodamine B was repressed as the length of the PLGA chain increased because of an enhanced entanglement of particle-forming polymers. The effect of particle-forming polymers on the release properties of the particles was consistent with previously reported results concerning self-assembling nanoparticles of PLGA-grafted pullulan [33], PEG-grafted PLA nanoparticles [34], and PLGA nanoparticles [35]. For the PEG-modified nanoparticles, we found that compositions of amphiphilic block copolymers were attributable to the
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Fig. 7. The effect of the preparation conditions on the drug-release properties of (a) PEG-unmodified and (b–d) PEG-modified PLGA nanoparticles. The block copolymers used were block copolymers E, A, and C for (b), (c), and (d), respectively.
release profiles of rhodamine B from PEG-modified nanoparticles, as shown in Fig. 7b–d. The release results shown in Fig. 7b–d were obtained by the use of PEG-modified PLGA nanoparticles that were prepared in the presence of PEG–PLA block copolymer E, A and C, respectively. In all the evaluations, the molar ratio of PEG–PLA block copolymers to PLGA was fixed at 2 during preparation of the nanoparticles. By comparing the results concerning PEG-unmodified and PEGmodified PLGA nanoparticles where a PEG chain length was long (Fig. 7a and b), the release of rhodamine B from the PEG-modified PLGA nanoparticles was reduced when PLGA5005 and PLGA7505 with molecular weights of 5000 were used as particle-forming polymers. Basically, the release from the nanoparticles prepared from PLGA with a low molecular weight is fast, because the particle-forming PLGA chains entangle partially and sparsely. However, because the presence of PEG–PLA block polymers made both emulsion droplets more stable and polymeric compounds more highly packed, the resulting PEG-modified nanoparticles exhibited reduced drug-release properties. In addition, the presence of PEG chains and simultaneously formed hydration layers possibly reduced the drug-release properties of the particles. When PLGA5020 and PLGA7520 (PLGAs with high molecular weights) were used, the release of rhodamine B from the resulting particles was slow intrinsically, because the PLGA chains entangled densely. Thus, we observed almost no effect of the presence of block copolymers on the change in the particles’ release properties. By contrast, we conducted a comparison between the results concerning PEG-unmodified PLGA nanoparticles and PEG-modified PLGA nanoparticles where a PEG chain length was short (Fig. 7a and c): the PEG–PLA block polymer had almost no effect on the change in the particles’ release properties. These results shown in Fig. 7a–d exemplify an important aspect of PEG present on the surface of the particles for the regulation of their release properties. We compared the results for PEG-unmodified PLGA nanoparticles with the results for PEG-modified PLGA nanoparticles where a PLA chain length was long (Fig. 7a and d). We found that the release from the PEG-modified PLGA nanoparticles was reduced when
PLGA5005 and PLGA7505 (with molecular weights of 5000) served as particle-forming polymers, presumably because of the formation of densely entangled PLGA–PLA layers. We observed almost no effect of the presence of block copolymers on the change in the particles’ release properties when PLGA5020 and PLGA7520 were used as particle-forming polymers, because the polymers with a high molecular weight were sufficiently entangled to incorporate compounds effectively even though no block copolymers coexisted in the preparation stage of emulsions. 4. Conclusions In the present article, we have used “block copolymer-assisted” emulsification/evaporation method to investigate the effects that preparation conditions and structural properties of both PEG–PLA block copolymers and PLGA might have on the properties of PEG-modified nanoparticles. We investigated several preparation conditions: concentrations of an emulsion stabilizer (PVA), concentration of a particle-forming hydrophobic polymer (PLGA), a molar ratio of PEG–PLA to PLGA, a volume ratio of an organic solvent to aqueous PVA solution, a stirring time, and a stirring rate. The results of our DLS measurements and SEM observations show that the concentration of PLGA, the volume ratio of an organic solvent to a PVA solution, and the stirring rate are major factors affecting the diameter of the nanoparticles. We successfully prepared the nanoparticles, with a diameter of approximately 200 nm under optimal conditions (the concentration of PVA: 0.3 w/v%; the concentration of PLGA: 0.125 w/v%; the molar ratio of PEG–PLA to PLGA: 1; the volume ratio of an organic solvent to a PVA solution: 0.5; the stirring rate: 20,000 rpm; and the stirring time: 5 min) in the presence of a dichloromethane–toluene mixture having a density of 1.00 g/cm3 as a dispersed phase. In addition, the additive amount and the composition of PEG–PLA block copolymers affected the surface charge of the nanoparticles. Finally, we clarified that the compositions and molecular weights of the block copolymers greatly affected the release properties of PEG-modified nanoparticles. For fully characterizing PEG-modified nanoparticles, in vivo
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release studies with hydrophobic drugs are necessary in future investigations. For designing drug carriers that circulate in blood, an attractive technique involves surface modification of hydrophobic particles with hydrophilic polymers. However, the conventional chemical conjugation of polymers at the surface of particles needs to be optimized for each polymer–particle combination. Further, some biodegradable and biocompatible particles have been known to involve difficult surface modification, because polymers as starting materials have almost no reactive groups in their molecules in such a case. We expect that our novel preparation method (a “block copolymer-assisted” emulsification/evaporation method) will enable efficient fabrication of surface-modified nanoparticles that can be useful in drug delivery systems. Acknowledgments This study was funded, in part, by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT, 24560238) and the Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization (NEDO, 06A05001a) of Japan. References [1] Y. Ikehara, N. Shiuchi, S. Kabata-Ikehara, H. Nakanichi, N. Yokoyama, H. Takagi, T. Nagata, Y. Koide, K. Kuzushima, T. Takahashi, K. Tsujimura, K. Kojima, Effective induction of anti-tumor immune responses with oligomannose-coated liposome targeting to intraperintoneal phagocytic cells, Cancer Lett. 260 (2008) 137–145. [2] M.G. Bonicelli, L. Giansanti, M. Ierino, G. Mancini, Interaction of cationic liposomes with cell membrane models, J. Colloid Interface Sci. 355 (2011) 1–8. [3] T. Guan, Y. Miao, L. Xu, S. Yang, J. Wang, H. He, X. Tang, C. Cai, H. Xu, Injectable nimodipine-loaded nanoliposomes: preparation, lyophilization and characteristics, Int. J. Pharm. 410 (2011) 180–187. [4] K. Kataoka, A. Harada, Y. Nagasaki, Block copolymer micelles for drug delivery: design, characterization and biological significance, Adv. Drug Deliv. Rev. 47 (2001) 113–131. [5] U. Kedar, P. Phutane, S. Shidhaye, V. Kadam, Advances in polymeric micelles for drug delivery and tumor targeting, Nanomedicine 6 (2010) 714–729. [6] M. Nishihara, Y. Murakami, T. Shinoda, J. Yamamoto, M. Yokoyama, Synthesis and characterization of temperature-responsive amphiphilic block copolymer containing liquid crystalline unit, Chem. Lett. 37 (2008) 1214–1215. [7] Y. Uchida, Y. Murakami, Trilayered polymeric micelle: a newly-developed macromolecular assembly that can incorporate hydrophilic compounds, Colloids Surf. B: Biointerfaces 79 (2010) 198–204. [8] Y. Uchida, Y. Murakami, Successful preferential formation of the novel macromolecular assembly – trilayered polymeric micelle – that can incorporate hydrophilic compounds: the optimization of factors affecting its formation from amphiphilic block copolymers, Colloids Surf. B: Biointerfaces 84 (2011) 346–353. [9] Y. Uchida, K. Fukuda, Y. Murakami, The hydrogel containing a novel vesicle-like soft crosslinker, a trilayered polymeric micelle, shows characteristic rheological properties, J. Polym. Sci. B: Polym. Phys. 51 (2013) 124–131. [10] M.L. Manca, G. Loy, M. Zaru, A.M. Fadda, S.G. Antimisiaris, Release of rifampicin from chitosan, PLGA and chitosan-coated PLGA microparticles, Colloids Surf. B: Biointerfaces 67 (2008) 166–170. [11] G. Gasparini, S.R. Kosvintsev, M.T. Stillwell, R.G. Holdich, Preparation and characterization of PLGA particles for subcutaneous controlled drug release by membrane emulsification, Colloids Surf. B: Biointerfaces 61 (2008) 199–207. [12] H.S. Han, J. Lee, H.R. Kim, S.Y. Chae, M. Kim, G. Saravanakumar, H.Y. Yoon, D.G. You, H. Ko, K. Kim, I.C. Kwon, J.C. Park, J.H. Park, Robust PEGylated hyaluronic acid nanoparticles as the carrier of doxorubicin: mineralization and its effect on tumor targetability in vivo, J. Controlled Release 168 (2010) 105–114.
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Hirata, Characterization of polymer-enzyme complex as a novel biocatalyst for nonaqueous enzymology, J. Mol. Catal. B: Enzym. 22 (2003) 79–88. [18] Y. Murakami, A. Hirata, Enzymatic synthesis of peptides – review, SeibutsuKogaku Kais. 76 (1998) 238–254. [19] J. Park, P.M. Fong, J. Lu, K.S. Russell, C.J. Booth, W.M. Saltzman, T.M. Fahmy, PEGylated PLGA nanoparticles for the improved delivery of doxorubicin, Nanomedicine 5 (2009) 410–418. [20] A.N.F. Versypt, D.W. Pack, R.D. Braatz, Mathematical modeling of drug delivery from autocatalytically degradable PLGA microspheres – a review, J. Controlled Release 165 (2013) 29–37. [21] F. Ito, Y. Uchida, Y. Murakami, Facile technique for preparing organic-inorganic composite particles: monodisperse poly(lactide-co-glycolide) (PLGA) particles having silica nanoparticles on the surface, Colloids Surf. A: Physicochem. Eng. Aspects 361 (2010) 109–117. [22] S.I. Thamake, S.L. Raut, Z. Gryczynski, A.P. Ranjan, J.K. Vishwanatha, Alendronate coated poly-lactic-co-glycolic acid (PLGA) nanoparticles for active targeting of metastatic breast cancer, Biomaterials 29 (2012) 7164–7173. [23] S. Fischer, C. Foerg, S. Ellenberger, H.P. Merkle, B. Gander, One-step preparation of polyelectrolyte-coated PLGA microparticles and their functionalization with model ligands, J. Controlled Release 111 (2006) 135–144. [24] Y. Kanakubo, F. Ito, Y. Murakami, Novel one-pot facile technique for preparing nanoparticles modified with hydrophilic polymers on the surface via block polymer-assisted emulsification/evaporation process, Colloids Surf. B: Biointerfaces 78 (2010) 85–91. [25] T. Takami, Y. Murakami, Development of PEG-PLA/PLGA microparticles for pulmonary drug delivery prepared by a novel emulsification technique assisted with amphiphilic block copolymers, Colloids Surf. B: Biointerfaces 87 (2011) 433–438. [26] T. Takami, Y. Murakami, Unexpected and successful one-step formation of porous polymeric particles only by mixing organic solvent and water under low-energy-input conditions, Langmuir 30 (2014) 3329–3336. [27] Y. Murakami, M. Yokoyama, H. Nishida, Y. Tomizawa, H. Kurosawa, A simple hemostasis model for the quantitative evaluation of hydrogel-based local hemostatic biomaterials on tissue surface, Colloids Surf. B: Biointerfaces 65 (2008) 186–189. [28] Y. Murakami, M. Yokoyama, H. Nishida, Y. Tomizawa, H. Kurosawa, In vivo and in vitro evaluation of gelation and hemostatic properties of a novel tissueadhesive hydrogel containing a crosslinkable polymeric micelle, J. Biomed. Mater. Res. B: Appl. Biomater. 91B (2009) 102–108. [29] H. Moroishi, C. Yoshida, Y. Murakami, A free-standing, sheet-shaped, hydrophobic biomaterial containing polymeric micelles formed from poly(ethylene glycol)-poly(lactic acid) block copolymer for possible incorporation/release of hydrophilic compounds, Colloids Surf. B: Biointerfaces 102 (2013) 597–603. [30] T. Ito, C. Yoshida, Y. Murakami, Design of novel sheet-shaped chitosan hydrogel for wound healing: a hybrid biomaterial consisting of both PEG-grafted chitosan and crosslinkable polymeric micelles acting as drug containers, Mater. Sci. Eng. C 33 (2013) 3697–3703. [31] H. Maeda, J. Sawa, Y. Matsumura, K. Hori, Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review, J. Controlled Release 65 (2000) 271–284. [32] M.D. Shau, M.F. Shih, C.C. Lin, I.C. Chuang, W.C. Hung, W.E. Hennink, J.K. Cherng, A one-step process in preparation of cationic nanoparticles with poly(lactideco-glycolide)-containing polyethlenimine gives efficient gene delivery, Eur. J. Pharma. Sci. 46 (2012) 522–529. [33] Y.-I. Jeong, H.-S. Na, J.-S. Oh, K.-C. Choi, C.-E. Song, H.-C. Lee, Adriamycin release from self-assembling nanospheres of poly(DL-lactide-co-glycolide)grafted pullulan, Int. J. Pharma. 322 (2006) 154–160. [34] S. Essa, J.M. 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Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
One-pot facile preparation of PEG-modified PLGA nanoparticles: Effects of PEG and PLGA on release properties of the particles Nao Yoneki, Taku Takami, Tomoki Ito, Ryosuke Anzai, Kengo Fukuda, Keita Kinoshita, Seiichi Sonotaki, Yoshihiko Murakami ∗ Department of Organic and Polymer Materials Chemistry, Faculty of Engineering, Tokyo University of Agriculture and Technology, Japan
h i g h l i g h t s
g r a p h i c a l
• A block copolymer-assisted emulsifi-
We investigated the effects of preparation conditions and structural properties of both PEG–PLA block copolymers and PLGA on the properties of PEG-modified PLGA nanoparticles via a block copolymerassisted emulsification/evaporation method.
cation was applied to nanoparticles preparations. • The nanoparticles with a diameter of 200 nm were obtained under optimized conditions. • The block copolymers greatly affected the release properties of nanoparticles.
a r t i c l e
i n f o
Article history: Received 16 November 2014 Received in revised form 1 January 2015 Accepted 4 January 2015 Available online 15 January 2015 Keywords: Nanoparticle Surface modification Poly(ethylene glycol) (PEG) Poly(lactide-co-glycolide) (PLGA) Drug delivery system
a b s t r a c t We show here a one-pot facile preparation of poly(ethylene glycol) (PEG)-modified poly(lactideco-glycolide) (PLGA) nanoparticles by means of a novel “block copolymer-assisted” emulsification/evaporation method. In the present article, we clarified the effects of preparation conditions and the structural properties of both polymeric modifiers and particle-forming hydrophobic polymers on the properties of the PEG-modified PLGA nanoparticles. We first clarified that the concentration of PLGA, the volume ratio of an organic solvent to a PVA solution, and a stirring rate were major factors affecting the diameter of the nanoparticles. We successfully prepared the nanoparticles, with a diameter of approximately 200 nm under the optimized conditions. The additive amount and the composition of PEG–PLA block copolymers affected the surface charge of the nanoparticles. Finally, we clarified that the compositions and molecular weights of the block copolymers greatly affected the release properties of PEG-modified nanoparticles. © 2015 Elsevier B.V. All rights reserved.
1. Introduction A drug delivery system (DDS) is a time- and site-specific system whereby drugs are delivered to a target site, consequently
∗ Corresponding author at: Department of Organic and Polymer Materials Chemistry, Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Tel.: +81 42 388 7387; fax: +81 42 388 7387. E-mail address: [email protected] (Y. Murakami). http://dx.doi.org/10.1016/j.colsurfa.2015.01.011 0927-7757/© 2015 Elsevier B.V. All rights reserved.
a b s t r a c t
resulting in both a reduction of side effects and an enhancement of the drugs’ pharmaceutical effects. Important factors for regulating DDS efficiency are an enhancement of absorption, a controlled release, and a targeting of drugs. Drug targeting is an important technique for achieving effective chemotherapy for cancers. Successful targeting requires the selection of suitable drug carriers including liposomes [1–3], micelles [4–9], and nano/microparticles [10,11]. Liposomes are suitable for encapsulating hydrophobic drugs, whereas polymeric micelles are suitable for encapsulating hydrophilic drugs. Although both of the carriers have been
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actively developed, the preparation of liposomes is sometimes difficult, whereas polymeric micelles exhibit a lack of drug preservation. By contrast, nano/microparticles can stably encapsulate both hydrophilic and hydrophobic drugs by both being prepared from stable emulsions where drugs dissolve in the emulsion droplet and blocking the invasion of water molecules into the particles. For effective administration into blood, it is important to control the diameter, surface properties, and drug preservation of drug carriers. Drug carriers are easily entrapped by a reticuloendothelial system (RES), because the carriers are recognized as foreign substances in blood vessels. Because the increased hydrophobicity of the carriers’ surface makes the carriers prone to easy entrapment by an RES, much attention has been paid to the development of effective surface-modification methods. Polyethylene glycol (PEG) has been a well known biocompatible modifier of drug carriers [12,13] and protein particles [14–18], because the presence of PEG both renders the carriers unrecognizable to phagocytes and reduces the aggregation of the carriers. However, a conjugation of PEG to carrier surface is difficult, because conjugation chemistry needs to be optimized for each polymer–particle combination [19]. Furthermore, poly(lactide-co-glycolide) (PLGA) and polylactide (PLA) have been used commonly in the preparation of particles [20–22], because the polymers are biocompatible and exhibit easily controllable degradation behaviors. However, PLGA and PLA particles have a reputation as being materials for which surface modification is difficult, because PLGA and PLA have almost no reactive groups in their molecules. In addition, although some investigations concerning surface modifications by electrostatic coupling reactions have been reported [23], the resulting interactions between a given particle and modifiers remain so weak as to be inapplicable to drug carriers. In this regard, we have proposed a novel one-pot facile technique for preparing surface-modified nano/microparticles to address the above problems [24-26]. Fig. 1 shows the proposed scheme for preparing the surface-modified particles via a “block copolymer-assisted” emulsification/evaporation method. First, o/w emulsion is prepared by the addition of an organic solvent dissolving both hydrophobic polymers and amphiphilic block copolymers into an aqueous solution containing emulsion stabilizers. In the first step, amphiphilic block copolymers present on the emulsion surface help stabilize the emulsion. In the next step, the hydrophobic polymers and the hydrophobic part of the amphiphilic block polymers gradually get tangled during the organic solvent’s evaporation from o/w emulsion. Consequently, the hydrophilic polymer chains are introduced on the surface of the finally resulting particles. The surface properties and drug-release behaviors of particles are controllable by changes to the molecular compositions, molecular weights, concentrations and mixed ratios of both hydrophobic polymers and amphiphilic block copolymers. We have succeeded in preparing nanoparticles for intravenous drug delivery [24] and microparticles for pulmonary drug delivery by means of the newly developed method [25,26]. However, in the former case, the diameter of the obtained particles was large (approximately 600–900 nm), because a membrane emulsification technique was used for the preparation of the particles. Furthermore, the drugrelease properties of the surface-modified particles still require clarification. The present paper describes two important topics: an effort to prepare PEG-modified nanoparticles (of which diameter is approximately 200 nm) by means of a high-speed homogenization (instead of membrane emulsification) based on our novel emulsification/evaporation techniques; and the compound-release behaviors of the resulting PEG-modified nanoparticles. This is the first report concerning the effects of “physically” combined surfacemodifiers on the release properties of the resulting polymeric nanoparticles.
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Fig. 1. The scheme of the novel block polymer-assisted emulsification/evaporation technique for preparing surface-modified nanoparticles. The hydrophobic polymers and the hydrophobic parts of amphiphilic block polymers gradually got tangled as the organic solvent evaporated from the o/w emulsion.
2. Materials and methods 2.1. Materials Ethylene oxide (Sumitomo Seika Chemicals Co., Ltd., Osaka, Japan) was purified by distilling it with CaH2 . dl-Lactide (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was recrystallized twice from ethyl acetate. 2-Methoxyethanol was distilled with sodium under reduced pressure. Potassium naphthalene was obtained by mixing potassium and naphthalene in anhydrous tetrahydrofuran (THF) for 18 h. PLGA7520, PLGA7510, PLGA7505, PLGA5020 and PLGA5005 (PLGA hijk represents PLGA whose composition ratio of lactic acid and molecular weight is hi and jk kg/mol, respectively) were purchased from Wako Pure Chemical Industries (Osaka, Japan) and stored in a freezer prior to use. Poly(vinyl alcohol) (PVA, the degree of polymerization: 500, saponification degree: 86–90 mol%) as an emulsion stabilizer was purchased from Wako Pure Chemical Industries and used without further purification. Rhodamine B was purchased from Wako Pure Chemical Industries. All the other reagents were of analytical grade and were used without further purification.
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tape and coating a thin platinum film (approximately 5 nm in thickness) on the sample under a reduced pressure with an MSP-1S ion coater (Vacuum Device Inc., Ibaraki, Japan). The volume-averaged diameter and the zeta potential of the particles were determined by dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, UK). The zeta potentials were determined under the condition that the concentration of NaCl was 1 × 10−4 M. 2.4. Release of hydrophobic compounds from surface-modified and surface-unmodified PLGA nanoparticles
Fig. 2. The scheme of the synthesis of a methoxy-terminated poly(ethylene glycol)–poly(lactic acid) (PEG–PLA) block copolymers by means of employing ringopening polymerization from ethylene oxide and dl-lactide.
2.2. Synthesis of methoxy-terminated poly(ethylene glycol)–poly(lactic acid) (PEG–PLA) block copolymers A methoxy-terminated poly(ethylene glycol)–poly(lactic acid) (PEG–PLA) block copolymer was synthesized by ring-opening polymerization of both ethylene oxide and dl-lactide in THF according to the previously reported method with slight modifications [24–30], as shown in Fig. 2. First, 2-methoxyethanol (0.3–1.0 mmol) and potassium naphthalene (1 molar equivalent of 2-methoxyethanol) were mixed in THF for 1 h. The purified ethylene oxide (80–131 mmol) was added to the obtained potassium 2-methoxyethoxide solution (total volume: 50 ml). After the solution was stirred for 48 h, THF solution of purified dl-lactide (17–70 mmol) was added to the solution. After the reaction, the resulting block copolymer was precipitated into cold 2-propanol, stored in a freezer for 12 h, centrifuged at 10,500 rpm (SS-1500, Sakuma Seisakusho Co., Ltd., Tokyo, Japan), and lyophilized in benzene. The average molecular weight of the obtained block copolymer was determined by the use of gel permeation chromatography (GPC) (system: LC2000plus, JEOL Ltd., Tokyo, Japan; column: TSKgel G3000HHR , TOSOH, Tokyo, Japan; eluent: DMF in the presence of 10 mM LiBr, flow: 1 ml/min; column temperature: 40 ◦ C) and 1 H NMR (AL-300, 300 MHz, JEOL Ltd., Tokyo, Japan). 2.3. Preparation and characterization of surface-modified and surface-unmodified PLGA particles The surface-modified PLGA particles were prepared according to an emulsification/evaporation technique by means of homogenization of the emulsion solution, as shown in Fig. 1. PLGA7510 (0.06–1.25 w/v%), and PEG–PLA block copolymers were dissolved in a dichloromethane–toluene mixed solvent where the molar ratio of PEG–PLA/PLGA was 1–5. PVA as an emulsion stabilizer was dissolved in pure Milli-Q water (0.2–2 w/v%). The organic solvent was added to the PVA solution where the volume ratio of the organic solvent/the PVA solution was 0.25–2, and subsequently, an o/w emulsion was formed by homogenization (5000–20,000 rpm, 2–10 min). The emulsion was then stirred at 150 rpm overnight at room temperature with a propeller type impeller so that the organic solvent from the o/w emulsion droplets would evaporate. Finally, we obtained purified surface-modified particles by centrifuging the solution at 7500 rpm and washing the particles with pure Milli-Q water three times. Surface-unmodified PLGA particles were prepared by means of the same method except that PEG was absent. The surface morphology of the surface-modified and surfaceunmodified PLGA particles was observed by means of a scanning electron microscope (SEM, VE-9800, KEYENCE Co., Ltd., Osaka, Japan). We prepared the specimens for SEM observation by mounting the sample on an aluminum plate with double-sided adhesive
We prepared rhodamine B-loaded nanoparticles by dissolving rhodamine B (0.01 w/v%) in the organic solvent in the preparation stage of the o/w emulsion. The obtained nanoparticles were dispersed in water at room temperature. At specific time intervals, the particles were collected by centrifugation at 7500 rpm, the amount of released compounds in the supernatant solution was determined by spectrofluorophotometer (ex : 500 nm, em : 576 nm, FP-6500, Jasco Inc., Tokyo, Japan), and the particles were redispersed. 3. Results and discussion 3.1. Synthesis and characterization of the PEG–PLA block copolymer The obtained PEG–PLA block polymers were characterized by 1 H NMR and GPC. The 1 H NMR spectra of the PEG–PLA block copolymer shown in Fig. 3 indicate that the block copolymers were synthesized successfully (the peak at around 1.8 ppm corresponds presumably to some impurities). Table 1 summarizes the results of the synthesis of the PEG–PLA block polymers A–E with different compositions. The number–average molecular weights, Mn , of the PEG and PLA units were determined on the basis of GPC and 1 H NMR, respectively. It was confirmed that the obtained Mn of PEG and PLA were 2650–9380 and 3260–7150, and the distribution of Mw /Mn was narrow. 3.2. Effects of preparation conditions on the diameter of nanoparticles The passive targeting of drug carriers is commonly based on the enhanced permeability and retention (EPR) effect wherein molecules of a specific size (approximately 20–200 nm) accumulate selectively in solid tumors so that the molecules exhibit therapeutic
Fig. 3. Typical 1 H NMR spectra of a PEG–PLA block copolymer.
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Table 1 Characterization of synthesized methoxy-terminated poly(ethylene glycol)-block-poly(lactic acid) (PEG–PLA) block copolymer. Code
A B C D E
PEG (including methoxy terminus)
PLA
PEG–PLA (including methoxy terminus)
Mn
Mw /Mn
Mn
Mn
4690 2650 4160 7840 9380
1.10 1.05 1.05 1.07 1.05
4490 4440 7150 3730 3260
effects for a long period [31]. In this study, we evaluated the effects of preparation conditions (concentration of an emulsion stabilizer (PVA) and a particle-forming hydrophobic polymer (PLGA), a molar ratio of PEG–PLA to PLGA, a volume ratio of the organic solvent to an aqueous PVA solution, a stirring time, and a stirring rate) on the diameter of the PEG-modified nanoparticles. Fig. 4 shows the effects of preparation conditions on the diameter of the PEG-modified particles, which was determined by means of DLS. PVA acts as an emulsion stabilizer by adsorbing the surface of the emulsion and by consequently making its droplets stably disperse in a continuous phase. However, it was confirmed that the emulsion solution foamed as the concentration of PVA increased. The foam prevented the successful formation of uniformly dispersed droplets of o/w emulsions. As a result, the diameter of the obtained nanoparticles increased as the concentration of PVA increased, as shown in Fig. 4a. The phenomenon was observed when PVA concentration was over 0.3 w/v%. In general, the presence of foam reduces emulsion stability and consequently makes resulting nanoparticles heterogeneous. By contrast, the emulsion was unstable when the concentration of PVA was zero, suggesting that PVA had an important role in enhancing the stability of o/w emulsion. The concentration of PVA was thus optimized to be 0.3 w/v%. Next, the concentration of polymers (particle-forming hydrophobic polymers and surface modifiers) was evaluated. In general, the diameter of the obtained nanoparticles becomes small when the concentration of particle-forming polymers decreases [32]. In our case, we also confirmed the same phenomena, as shown in Fig. 4b. However, the decrease in the concentration of PLGA will reduce the productivity of the resulting nanoparticles. Furthermore, in some cases, we observed large particles or aggregate (with a diameter over 300 nm) when PLGA concentration was over 0.2 w/v%. The concentration of PLGA was thus optimized to be
9180 7090 11,310 11,600 12,600
Mw /Mn 1.19 1.16 1.13 1.07 1.06
0.125 w/v%. It was noteworthy that the concentration of PEG–PLA block copolymers had almost no influence on the diameter of the resulting nanoparticles, as shown in Fig. 4c, presumably because the concentration of PEG–PLA block copolymer affected mainly the density of PEG that was introduced on the particle surface. In addition to chemical compounds present in emulsion systems, the volume ratio of a dispersed phase to a continuous phase is an important factor governing the diameter of emulsion droplets. In our case, the diameter of the nanoparticles decreased as the volume ratio of organic solvent to PVA solution decreased, as shown in Fig. 4d. The diameter of the particles became constant below the ratio of 0.5. Furthermore, the decrease in the amount of an organic solvent where the polymeric compounds have been dissolved will generally reduce the productivity of the resulting nanoparticles. Thus, the volume ratio of organic solvent to PVA solution was optimized to be 0.5. In general, when an emulsion is prepared, stirring conditions are important factors affecting the diameter of the nanoparticles. In our study, the diameter of nanoparticle decreased while the stirring rate increased or the stirring time increased, as shown in Fig. 4e and f, because the increased stirring rate and stirring time made emulsion droplets small. As the stirring time increased, the temperature of the resulting emulsion increased and consequently decreased the stability of the emulsion. The stirring rate and stirring time were thus optimized to be 20,000 rpm and 5 min, respectively. Furthermore, in our recent study, we found that emulsions exhibited the highest stability when the density of a dispersed phase (an organic solvent) was adjusted to be 1.00 g/cm3 , which is the same as the density of a continuous phase (an aqueous solution), because the differences in the densities of the two phases play a critical role in regulating the stability of emulsions [24]. Finally, the nanoparticles having a diameter of approximately 200 nm and a narrow size distribution were successfully prepared
Fig. 4. The effects of preparation conditions on the diameter of the PEG-modified particles that was determined by means of DLS (arrows refer to optimized conditions).
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Fig. 5. SEM image of nanoparticles obtained under optimized conditions.
with high reproducibility, as shown in Fig. 5 (DLS-based diameter of the particles were 214 ± 11 nm), under the preceding optimal conditions (the concentration of PVA: 0.3 w/v%; the concentration of PLGA: 0.125 w/v%; the molar ratio of PEG–PLA to PLGA: 1; the volume ratio of an organic solvent to a PVA solution: 0.5; the stirring rate: 20,000 rpm; and the stirring time: 5 min) in the presence of a dichloromethane–toluene mixture having a density of 1.00 g/cm3 as a dispersed phase. 3.3. The surface properties of surface-modified PLGA nanoparticles Controlling the surface properties of nanoparticles was important for their successful performance as drug carriers. We observed the surface morphology of surface-modified PLGA nanoparticles by means of SEM. The nanoparticles exhibited a spherical shape and a diameter of about 200 nm. The results of our SEM-image observation support the preceding DLS-based results, wherein the nanoparticles having a diameter of 200 nm were successfully prepared with high reproducibility. Furthermore, we evaluated the surface property of the nanoparticles by using zeta potential, an important factor for the regulation of the stability of the nanoparticles. In the preparation stage of the nanoparticles, PLGA and the hydrophobic part of the amphiphilic PEG–PLA block copolymer gradually got tangled as the organic solvent evaporated from the o/w emulsion. Consequently, the PEG chain, which was introduced on the surface of the finally obtained nanoparticles, would improve the low intravital stability of the particles, because the PEG chain both prevents nanoparticles from contacting RES and reduces their nonspecific interactions with blood vessels. Thus, it is important to control the surface charge of the nanoparticles by suitably selecting the composition and the additive amount of the PEG–PLA block copolymers. Fig. 6 shows the zeta potential of the PEG-modified PLGA nanoparticles, which were prepared from PLGA and PEG–PLA with different compositions. The surface of the surface-unmodified PLGA nanoparticles had a negative charge, approximately −24 mV, which was attributable to the carboxyl groups of PLGA present on the particle surface. We found that a PLA chain length of PEG–PLA block copolymers hardly affected the surface charge of the nanoparticles (by the comparison of the effects that block copolymers A and C had), whereas an increased PEG chain length of PEG–PLA block copolymers increased surface charge (by the comparison of the effect that block copolymers A, D and E had). The differences between effects of PLA and PEG were attributed to the fact that PLA and PEG chains of PEG–PLA block copolymers were present inside and on the surface of PLGA particles. Fig. 6 shows that, as the molar
Fig. 6. The zeta potentials of the PEG-modified PLGA nanoparticles prepared from PLGA and PEG–PLA with different compositions.
ratio of PEG–PLA to PLGA increased, the surface electric charge of the particle increased or decreased, and then reached saturated values when the molar ratio of PEG–PLA to PLGA was over 2. These phenomena are presumably due to two possible factors: (1) in general, it is difficult to introduce all added PEG–PLA block copolymers at the surface of emulsion droplets when the emulsions form, and (2) in general, the PEG density on particles’ surfaces hardly changes when PEG–PLA block copolymers are added because the resulting nanoparticles grow significantly in size. Moreover, we found that the zeta potential of the PEG-modified nanoparticles decreased as the molar ratio of PEG–PLA to PLGA increased in cases where the block copolymer B was used, because the PEG chain length was too short to be present at the interface of emulsions. 3.4. Release of hydrophobic compounds from surface-modified and surface-unmodified PLGA nanoparticles It is important to prepare nanoparticles exhibiting desired drugrelease properties for the design of drug carriers that circulate in blood vessels suitably and result in successful drug effects at target sites. In this study, thus, we have evaluated the effect of nanoparticles’ preparation conditions on the nanoparticles’ drug-release properties. Fig. 7 shows an effect of the preparation conditions on the drugrelease properties of PEG-unmodified and PEG-modified PLGA particles. The preparation conditions include factors such as the hydrophobicity of inclusions and both the compositions and molecular weights of both hydrophobic polymers and block copolymers. In all the evaluations, the diameter and dispersion of the nanoparticles did not change during the experiment of the release. The average diameters of the particles used were almost the same (218 ± 6 nm). Therefore, at least, it was no effect of the particles’ diameter on the drug-release properties shown in Fig. 7. For the PEG-unmodified nanoparticles, the molecular weight of PLGA affected the release profile of rhodamine B. As shown in Fig. 7a, the release of rhodamine B was repressed as the length of the PLGA chain increased because of an enhanced entanglement of particle-forming polymers. The effect of particle-forming polymers on the release properties of the particles was consistent with previously reported results concerning self-assembling nanoparticles of PLGA-grafted pullulan [33], PEG-grafted PLA nanoparticles [34], and PLGA nanoparticles [35]. For the PEG-modified nanoparticles, we found that compositions of amphiphilic block copolymers were attributable to the
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Fig. 7. The effect of the preparation conditions on the drug-release properties of (a) PEG-unmodified and (b–d) PEG-modified PLGA nanoparticles. The block copolymers used were block copolymers E, A, and C for (b), (c), and (d), respectively.
release profiles of rhodamine B from PEG-modified nanoparticles, as shown in Fig. 7b–d. The release results shown in Fig. 7b–d were obtained by the use of PEG-modified PLGA nanoparticles that were prepared in the presence of PEG–PLA block copolymer E, A and C, respectively. In all the evaluations, the molar ratio of PEG–PLA block copolymers to PLGA was fixed at 2 during preparation of the nanoparticles. By comparing the results concerning PEG-unmodified and PEGmodified PLGA nanoparticles where a PEG chain length was long (Fig. 7a and b), the release of rhodamine B from the PEG-modified PLGA nanoparticles was reduced when PLGA5005 and PLGA7505 with molecular weights of 5000 were used as particle-forming polymers. Basically, the release from the nanoparticles prepared from PLGA with a low molecular weight is fast, because the particle-forming PLGA chains entangle partially and sparsely. However, because the presence of PEG–PLA block polymers made both emulsion droplets more stable and polymeric compounds more highly packed, the resulting PEG-modified nanoparticles exhibited reduced drug-release properties. In addition, the presence of PEG chains and simultaneously formed hydration layers possibly reduced the drug-release properties of the particles. When PLGA5020 and PLGA7520 (PLGAs with high molecular weights) were used, the release of rhodamine B from the resulting particles was slow intrinsically, because the PLGA chains entangled densely. Thus, we observed almost no effect of the presence of block copolymers on the change in the particles’ release properties. By contrast, we conducted a comparison between the results concerning PEG-unmodified PLGA nanoparticles and PEG-modified PLGA nanoparticles where a PEG chain length was short (Fig. 7a and c): the PEG–PLA block polymer had almost no effect on the change in the particles’ release properties. These results shown in Fig. 7a–d exemplify an important aspect of PEG present on the surface of the particles for the regulation of their release properties. We compared the results for PEG-unmodified PLGA nanoparticles with the results for PEG-modified PLGA nanoparticles where a PLA chain length was long (Fig. 7a and d). We found that the release from the PEG-modified PLGA nanoparticles was reduced when
PLGA5005 and PLGA7505 (with molecular weights of 5000) served as particle-forming polymers, presumably because of the formation of densely entangled PLGA–PLA layers. We observed almost no effect of the presence of block copolymers on the change in the particles’ release properties when PLGA5020 and PLGA7520 were used as particle-forming polymers, because the polymers with a high molecular weight were sufficiently entangled to incorporate compounds effectively even though no block copolymers coexisted in the preparation stage of emulsions. 4. Conclusions In the present article, we have used “block copolymer-assisted” emulsification/evaporation method to investigate the effects that preparation conditions and structural properties of both PEG–PLA block copolymers and PLGA might have on the properties of PEG-modified nanoparticles. We investigated several preparation conditions: concentrations of an emulsion stabilizer (PVA), concentration of a particle-forming hydrophobic polymer (PLGA), a molar ratio of PEG–PLA to PLGA, a volume ratio of an organic solvent to aqueous PVA solution, a stirring time, and a stirring rate. The results of our DLS measurements and SEM observations show that the concentration of PLGA, the volume ratio of an organic solvent to a PVA solution, and the stirring rate are major factors affecting the diameter of the nanoparticles. We successfully prepared the nanoparticles, with a diameter of approximately 200 nm under optimal conditions (the concentration of PVA: 0.3 w/v%; the concentration of PLGA: 0.125 w/v%; the molar ratio of PEG–PLA to PLGA: 1; the volume ratio of an organic solvent to a PVA solution: 0.5; the stirring rate: 20,000 rpm; and the stirring time: 5 min) in the presence of a dichloromethane–toluene mixture having a density of 1.00 g/cm3 as a dispersed phase. In addition, the additive amount and the composition of PEG–PLA block copolymers affected the surface charge of the nanoparticles. Finally, we clarified that the compositions and molecular weights of the block copolymers greatly affected the release properties of PEG-modified nanoparticles. For fully characterizing PEG-modified nanoparticles, in vivo
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