Novel amphiphilic triblock copolymer based on PPDO, PCL, and PEG: Synthesis, characterization, and aqueous dispersion

Colloids and Surfaces A: Physicochem. Eng. Aspects 292 (2007) 69–78

Novel amphiphilic triblock copolymer based on PPDO, PCL, and PEG: Synthesis, characterization, and aqueous dispersion Remant Bahadur K.C. a , Shanta Raj Bhattarai a , Santosh Aryal a , Myung Seob Khil b , N. Dharmaraj c,d , Hak Yong Kim d,∗ b

a Department of Bionanosystem Engineering, Chonbuk National University, Jeonju 561-756, Republic of Korea Center for Healthcare Technology Development, Chonbuk National University, Jeonju 561-756, Republic of Korea c Department of Chemistry, Government Art College, Udumalpet 64216, India d Department of Textile Engineering, Chonbuk National University, Jeonju 561-756, Republic of Korea

Received 27 March 2006; received in revised form 9 June 2006; accepted 13 June 2006 Available online 20 June 2006

Abstract Amphiphilic triblock copolymer, poly(p-dioxanone-co-caprolactone)-block-poly(ethylene oxide)-block-poly(p-dioxanone-co-caprolactone) (PPDO-co-PCL-b-PEO-b-PPDO-co-PCL) was synthesized by ring opening polymerization (ROP) of p-dioxanone and ␧-caprolactone initiated through the hydroxyl end of poly(ethylene glycol) (PEG) in the presence of stannous 2-ethyl hexanoate [Sn(oct)2 ] as a catalyst. Polymerization and structural features of the polymers were analyzed by different physicochemical techniques (GPC, 1 H NMR, 13 C NMR, FT-IR, DSC and TGA). The splitting of 1 H NMR resonance at δ 2.3 and δ 4.1 ppm reveals the random copolymerization. Polymeric nanoparticles were prepared in phosphate buffer (pH 7.4) by co-solvent evaporation technique at room temperature (25 ◦ C). Existence of hydrophobic domains as cores of the micelles were characterized by 1 H NMR spectroscopy and further confirmed with fluorescence technique using pyrene as a probe. Critical micelle concentration (CMC) of the polymer in phosphate buffer (pH. 7.4) was decreased from 2.3 × 10−3 to 7.6 × 10−4 g/L with the fraction of PCL. Polymeric nanoparticles observed by atomic force microscopy (AFM) were uniform and spherical, with smooth textured of around 50–30 nm diameter. Dynamic light scattering (DLS) and electrophoretic light scattering (ELS) measurements showed a monodisperse size distribution of around 113–90 nm hydrodynamic diameters and negative zeta (ζ) potential (−4 to −14 mV), respectively. The investigations for the polymeric nanoparticles in aqueous medium showed that the composition of the hydrophobic segment of amphiphilic block copolymer makes a significant influence on its physicochemical characteristics. © 2006 Elsevier B.V. All rights reserved. Keywords: Biomaterials; Block copolymers; Micelles; Self-assembled; Nanoparticles

1. Introduction Colloidal system, in the form of micro- and nano-sized particle, has been frequently used in biomedical field to encapsulate hydrophobic drugs and other bioactive molecules [1,2]. The fascinating aspect of this system in biomedical field is for increasing drug stability, drug solubility, and transport properties of pharmaceutical materials [3]. The frequently used colloidal nanoparticles are amphiphilic nanoparticles (formed by hydrophobic interactions), polyion complex nanoparticles (PICN;resulting from electrostatic interactions), and nanoparti-

∗

Corresponding author. Tel.: +82 63 270 2351; fax: +82 63 270 2348. E-mail address: [email protected] (H.Y. Kim).

0927-7757/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2006.06.009

cles stemming from metal complexation [4]. Polymers containing at least two distinct blocks (hydrophobic and hydrophilic), covalently bound at one point, lead to form an polymeric nanoparticles via intra- or intermolecular associations between hydrophobic moieties in aqueous medium since this medium is thermodynamically unfavorable to the hydrophobic segment [5]. Polymeric nanoparticles made up of synthetic biodegradable block copolymers have much attention due to their coreshell geometry in which hydrophobic domain (core) serves as a reservoir for the incorporated drugs and hydrophilic domain (shell) serves as a stabilizing interface of particles [6–8]. An important realization in drug delivery system (DDS) via polymeric nanoparticle is their surface functionalization with non-ionic hydrophilic polymers, which improves the

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Scheme 1. Structure of polymeric nanoparticles formed by amphiphilic triblock copolymers in aqueous solution; PPDO-b-PEO-b-PPDO polymers form nanoparticles with nearly crystalline cores (A), PPDO-co-PCL-b-PEO-b-PPDO-co-PCL polymers forms nanoparticles with an inhomogeneous semicrystalline core (B) and PCLb-PEO-b-PCL polymers form nanoparticles with homogenious semicrystalline cores (C).

physicochemical and biological properties. This can be accomplished in a regulated fashion by constructing nanoparticles from varieties of amphiphilic block copolymers [9–12]. Among the surface modifiers (hydrophilic segment), PEG has received a great attention due to its well-known physico-chemical and biological properties, including hydrophilicity, solubility in both water and organic solvents, lack of toxicity and antigenicity and immunogenicity [13–15]. PEG has also received Food and Drug Administration (FDA) approval for its possible internal consumption [16]. Opsonization probability is high enough in diblock copolymeric nanoparticles due to their exposed surface conformation [7]. Therefore, triblock copolymeric nanoparticles have received an increasing attention in DDS since the hydrophilic segment (PEG) is anchored into the core through its both terminals (Scheme 1) that results in the formation of a closed surface conformation consequently limits the possible opsonization [17]. Many biodegradable amphiphilic block copolymers based on aliphatic polyesters, e.g. PLA-b-PEG, PLGA-b-PEG, and PCLb-PEG-b-PCL, have been synthesized and frequently applied in DDS due to their safety profiles [17,18,19]. So far, research works have mainly been focused on the preparation of drugloaded polymeric nanoparticles by varying polymer/drug ratio, hydrophobic/hydrophilic constituent of the polymer; thereby study the micelle yield, drug loading content, entrapment efficiency and drug release profiles [20–23]. However, less attention has been paid for the chemical modification of core forming segment that could be an excellent engineering to incorporate varieties of drugs with diverse characteristics, e.g. genes, proteins, etc. There is no report on the use of PPDO based polymeric nanoparticles in DDS. Central focus on tackling this

issue, our research group has been involved for the synthesis of random triblock copolymers based on PLLA and PPDO using PEG as a central block and their application in DDS [24]. The increased attention for the synthesis of PPDO based copolymers is due to their well-known biodegradable, biocompatible and mechanical properties [25]. Kricheldorf and co-workers [26,27] suggested that the PDO undergoes polymerization through equilibrium, i.e. PDO and PPDO are easily polymerizable and depolymerizable materials, respectively. Therefore, the effective conversion of PDO into copolymer is still a challenging task [24(a)]. Chemical composition of the hydrophobic segment is the most important part of amphiphilic block copolymers that control various physicochemical properties of polymeric nanoparticles. Similarly, particle size and their surface characteristics are the major determinants of the clearance kinetics and biodistribution for polymeric nanoparticles. The objective of this research was the synthesis of a novel amphiphilic triblock copolymer, PPDO-co-PCL-b-PEG-b-PPDO-co-PCL, and preparation of nanoparticles in aqueous medium. The random increase in PCL fraction could improve the flexibility and hydrophobicity of the resulting polymer consequently a novel poly (ester-alt-ether) based polymeric nanoparticles of reasonable physicochemical characteristics can be prepared [23(b, c)]. We have selected PEG of high molecular weight (10,000 g/mol) to get polymeric nanoparticles with reasonable size and stability [2,24(b)]. Polymerization and molecular composition of the polymer were analyzed by different physicochemical techniques (1 H NMR, 13 C NMR, FT-IR, GPC, TGA and DSC). Selfassembly of polymers was formulated in phosphate buffer (pH 7.4) using co-solvent evaporation technique [23(b)]. Character-

K.C. Remant Bahadur et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 292 (2007) 69–78

ization of the polymeric nanoparticles was performed through NMR, fluorescence probe technique, AFM, DLS, and ELS methods. 1H

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tated in cold hexane and repeatedly washed with cold diethyl ether. 2.3. Preparation of PPDO-co-PCL-b-PEOb-PPDO-co-PCL nanoparticles

2. Experimental 2.1. Materials ␧-Caprolatone (␧-CL) (Aldrich Chemical Co.) was distilled over freshly powdered CaH2 prior to use. 1,4-dioxan-2-one (PDO), provided by Meta Biomedical Co. Ltd. Korea, was used as received. Poly(ethylene glycol) (PEG) (Mn = 10,000 g/mol) (Aldrich Chemical Co.) was used after re-precipitation using dichloromethane and diethyl ether as solvent and non-solvent, respectively, followed by azeotropic distillation [24(a)]. Stannous 2-ethyl hexanoate [Sn(oct)2 ] (Aldrich Chemical Co.) was used after being dissolved in dry toluene (0.039 M). All the other chemicals used in this research were purchased from Showa Chemical Ltd., Japan. 2.2. Polymer synthesis Triblock copolymers (PPDO-co-PCL-b-PEO-b-PPDO-coPCL) were synthesized by using ROP of PDO and ␧-CL (Scheme 2) in the presence of PEG and stannous octoate as macroinitiator and catalyst, respectively [24(a)]. In a typical experiment, stannous octoate (0.45 ml) was poured into three necked round bottom flask containing PEG (6.0 g) and was heated for 1 h at 100 ◦ C to ensure the complete drying and then cooled down to room temperature (25 ◦ C) using argon flow. PDO (5.0 g) and ␧-CL (13.0 g) were injected into the flask. The flask was vacuumed for 3 h and degassed with argon for 1 h. The resulting reaction mixture was stirred for 10 h at 160 ◦ C and kept at 100 ◦ C for another 24 h and the solid mass obtained was dissolved in CH2 Cl2, precipi-

PPDO-co-PCL-b-PEO-b-PPDO-co-PCL nanoparticles were prepared in phosphate buffer (pH 7.4) using co-solvent evaporation technique [24(b)]. The polymer (5.0 mg) was dissolved in acetonitrile (1.0 ml) and dropped into 5.0 ml of phosphate buffer (pH 7.4) under moderate stirring (50.0 rpm) at room temperature. The organic phase was allowed to evaporate under reduced pressure until the final volume of the aqueous suspension was reduced to 5 ml. Finally the suspension was filtered by a micro filter with 0.2 ␮m pore size to remove polymer aggregates. 2.4. Polymer characterization Nuclear magnetic resonance (NMR) spectra of the samples were recorded by using a JNM-Ex 400 FT-NMR spectrometer (Japan), operating at 400 MHz (1 H) and 100 MHz (13 C); 6% (1 H) and 13% (13 C) (w/v) solution in CDCl3 using tetramethyl silane (TMS) as the internal reference. Fourier-transform infrared (FT-IR) spectra were recorded as KBr pellets using an ABB Bomen MB 100 spectrometer (Canada). Gel permeation chromatographic (GPC) measurements were made via a Waters 15 ◦ C apparatus equipped with a refractive index detector and Waters Styragel® columns (HR1 , HR2 , and HR4 ) with a size of 7.8 nm × 300 nm using chloroform as the mobile phase at a flow rate of 1.0 ml min−1 . Thermogravimetric analysis (TGA) was performed by a TA 2010 type thermogravimetric analyzer at a heating rate of 15 ◦ C min−1 in the range of 50–600 ◦ C under the steady flow of nitrogen. Differential scanning calorimetry (DSC) analysis of polymer was performed by a DSC Q100 V7.3 Build 249 (DSC standard cell) at a heating rate of 20 ◦ C min−1 in the range of −90 to 150 ◦ C under the steady flow of nitrogen.

Scheme 2. Copolymerization of PDO and ␧-CL with PEG. The subscripts x, y and z represent the variable units.

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2.5. Nanoparticles characterization 1H

NMR spectrum of the polymeric nanoparticles was recorded by a FT-NMR spectrometer (JNM-Ex 400 Japan) at room temperature operating at 400 MHz in deuterium oxide containing (TMS) as the internal reference. CMC of the polymer was measured by the steady-state pyrene fluorescence method [28]. Steady-state fluorescent excitation spectra (λem = 390 nm) of pyrene were measured at various polymer concentrations using a F-200 fluorescence Spectrophotometer 2510221-07 (Hitachi, Ltd., Japan). Concentrations of polymer and pyrene were in the range of 1 × 10−4 to 1 g/L and 6 × 10−7 M in phosphate buffer (pH 7.4), respectively. CMC of the polymer was calculated at the intersection of two straight lines, one of which was the fitted line of the curve at low polymer concentrations and the other was the fitted line on the rapidly rising part of the curve. Size and surface morphology of polymeric nanoparticles were observed by AFM Nanoscope IV multimode (Digital Instrument, Mikro masch, USA) in tapping mode using a Si cantilever with a spring constant of 3.5 N/m and a resonance frequency of 75 kHz. Scanning was performed at a scan speed of 1.85 Hz with a resolution of 512 × 512 pixels. Samples for AFM observation were prepared as a drop-coated film on the argon dried Si (1 1 1) wafers. Average particles size and size distribution were determined by DLS (Malvern System 4700 instrument) equipped with vertically polarized light supplied by an argon-ion laser (Cyonics) operated at 20 mW. All experiments were performed at room temperature (25 ◦ C) of measuring angle 90◦ to the incident beam. ζ-potential of the nanoparticles was determined by ELS measurement (ELS 8000/6000 Otsuka electronics Co., Japan) at 25 ◦ C with a measuring angle of 20◦ when compared to the incident beam. Before each analysis, samples were sonicated in an ultra-sonicator bath for one minute. All the measurements were repeated three times and the values reported are the mean ± S.D. 3. Results and discussion 3.1. Polymer synthesis Molecular weight distribution and composition of the polymers were determined using GPC and 1 H NMR spectroscopy, respectively. Feed ratio [PEG/␧-CL/PDO] and the results of polymerization are summarized in Table 1. All the polymers showed relatively a wide molecular weight distribution and poly-

Fig. 1. GPC chromatogram of PPDO-b-PEO-b-PPDO (T1 ) (A), PPDO-coPCL-b-PEO-b-PPDO-co-PCL (T2 ) (B), PPDO-co-PCL-b-PEO-b-PPDO-coPCL (T3 ) (C), PPDO-co-PCL-b-PEO-b-PPDO-co-PCL (T4 ) (D) and PCL-bPEO-b-PCL (T5 ) (E), 1% (w/v) solution in CHCl3 .

dispersity [Mw /Mn ] of around 2 (Fig. 1). This fact might be caused by the intermolecular ester interchange at high temperature, until the establishment of the most probable molecular weight distribution [29]. Typical 1 H NMR spectrum (Fig. 2) exhibited the following characteristic resonance corresponding to different repeating units of the polymer. A sharp singlet (h) centered at δ 3.6 ppm was attributed to the –CH2 −CH2 -O– proton of PEO segment. A pair of triplets with equal intensities at δ 4.03 (–CH2 -O–) and δ 2.28 ppm (–CO-CH2 –), two multiplets at δ 1.61 (–CH2 -CH2 CH2 –) and δ 1.36 ppm (–CH2 -CH2 -CH2 –) denoted as d, g, e, and f respectively, were ascribed to the repeating units of PCL segments. Similarly, two intense triplets at δ 4.25 (–CH2 -CH2 O–) and δ 3.75 ppm (–CH2 -CH2 -O–), and a singlet at δ 4.08 ppm (–O-CH2 -O–) denoted as a, b and c, respectively, were assigned to the repeating unit of PPDO segments. A distinct triplet at δ 2.3 ppm (g* ) due to the –CO-CH2 – protons of PCL segment linked to PEO and PPDO was well resolved. Similarly a multiplet at δ 4.1 ppm (d*) was characterized to –CH2 −CH2 -O– protons of PEO coupled with polyesters and –CH2 -O– protons of PCL connected with PPDO. A weak signal a* at δ 4.3 ppm was assigned to the –CH2 -CH2 -O– protons of PPDO. Interestingly, resonance peaks corresponding to the terminal proton of PCL fragment were not observed, which implies the absence of PCL as the terminal unit. This feature indicates that ␧-CL was active during the initial stage of the polymerization (high temperature),

Table 1 Characterization of PPDO-co-PCL-b-PEO-b-PPDO-co-PCL triblock copolymer Sample code

T1 T2 T3 T4 T5 a b

Mn of PEG

10000 10000 10000 10000 10000

Composition (wt%)b

Polydispersity (Mw /Mn )a

Feed ratio (wt%) PEG

␧-CL

PDO

PEG

␧-CL

PDO

1.20 1.20 1.20 1.20 1.20

20 20 20 25 20

0 30 40 55 80

80 50 40 20 0

24.1 28.3 36.0 30.6 25.3

0.0 26.3 34.0 58.1 74.7

75.9 45.4 30.0 11.3 0.0

Measured by GPC analysis. Determined from 1 H NMR spectroscopy (CDCl3 ).

Mn a

Mw a

Polydispersity (Mw /Mn )a

39784 40000 43690 28600 45031

49730 62900 92622 48000 63043

1.25 1.57 2.12 1.67 1.40

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Fig. 2. 1 H NMR spectra of PPDO-co-PCL-b-PEO-b-PPDO-co-PCL (T4 ), 6% (w/v) solution in CDCl3 using tetramethyl silane (TMS) as the internal reference.

where as PDO was active at low temperature, since the reactivity of PDO at high temperature is insignificant [24(a),27]. Splitting of the resonance signals of terminal protons of each segment implies the quantitative random copolymerization of PDO and ␧CL. Mole fraction of repeating units and molecular weight of all

Fig. 3.

13 C

the polymers were determined from the well-resolved 1 H NMR resonance at δ 2.28 ppm (PCL), 3.6 ppm (PEO), and 3.75 ppm (PPDO) proton. 13 C NMR spectrum of polymer is shown in Fig. 3. The spectrum showed a well-resolved peak k at δ 70.4 ppm attributed

NMR spectra of PPDO-co-PCL-b-PEO-b-PPDO-co-PCL (T4 ), 13% (w/v) solution in CDCl3 using tetramethyl silane (TMS) as the internal reference.

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Fig. 4. FT-IR spectra recorded from KBr plate of PEG (10,000) (A), PCLb-PEO-b-PCL (B); PPDO-b-PEO-b-PPDO (C) and PPDO-co-PCL-b-PEO-bPPDO-co-PCL (T4 ) (D).

to the –CH2 -CH2 – carbons of PEO [30]. Two distinct peaks d and j at δ 170.4 and 173 ppm were assigned to the carbonyl carbon of PPDO and PCL, respectively [31]. Signals at δ 63, 68.2 and 69.2 ppm, and δ 25, 25.9, 28.1, 34, 64 ppm were assigned to the methylene carbon of PPDO and PCL, respectively. FT-IR spectra of three different copolymers are presented in Fig. 4. The vibration characteristics of C H and C O C of PEO were observed at 2873 and 1104 cm−1 (curve A). After copolymerization with ␧-CL and PDO, the above vibration showed a quantitative shift (curve D) and appeared at 2864 and 1141 cm−1 , respectively [30]. Strong absorption bands at 1734 and 2944 cm−1 attributed to the C O and C H stretching of PCL and PPDO units, respectively are also observed (curve D). Similarly, absorption bands at 3555 and 1100–1271 cm−1 attributed to the terminal OH and C O and O C O group were well resolved in the polymer’s spectra (curve D). These spectral features corresponding to the PPDO, PCL, and PEG units indicate their presence in the copolymer. Thermal characteristics of polymer investigated through DSC analysis are summarized in Table 2. Glass transition temperature (Tg ) and melting temperature (Tm ) of the polymer were resolved in both first and second runs where as the crystallization temperature (Tc ) only in second run DSC thermograms (Fig. 5).

Fig. 5. DSC thermograms of PPDO-b-PEO-b-PPDO (T1 ) (A), PPDOco-PCL-b-PEO-b-PPDO-co-PCL (T2 ) (B), PPDO-co-PCL-b-PEO-b-PPDOco-PCL (T3 ) (C), PPDO-co-PCL-b-PEO-b-PPDO-co-PCL (T4 ) (D) and PCL-b-PEO-b-PCL (T5 ) (E) obtained by heating the samples from −90 to 150 ◦ C at 20 ◦ C/min under the steady flow of nitrogen. Solid line (–) represents the first run DSC thermograms of the polymers and dot lines (- - -) represent the second run thermograms after quenching the polymers in liquid nitrogen.

Results showed that Tg , Tm and Tc of the polymer were linearly decreased with the fraction of PCl. In every polymer we found a single Tg , Tm and Tc . It implies the complete mixing of all the segments of constituent polymers within the copolymers [24(a)]. TGA is the best method for characterizing the copolymers [32]. Polymeric composition can be obtained by the qualitative characterization of degradation process illustrated by the inflection point temperature (Td ) and the weight loss percentage (W). TG curve of the copolymer (sample T4 ) presented in Fig. 6, showed two weight loss steps. The calculated Td and W values suggested that the first degradation step was due to the PPDO/PCL components and the second one due to the PEG segments. Td of the PPDO/PCL was observed at 336 ◦ C, whereas that of PEO was at 436 ◦ C.

Table 2 Thermal properties of PPDO-co-PCL-b-PEO-b-PPDO-co-PCL triblock copolymer derived from DSC analysis Sample codes

T1 T2 T3 T4 T5

First run

Second run

Tm

Tc

Tg

Tm

Tc

Tg

107.67 43.84 47.25 47.63 60.06

– – – – –

– −42.72 −52.30 −56.20 –

100.84 43.32 47.03 40.07 56.11

22.08 – −21.38 – –

−23.33 −42.86 −53.79 −57.48 –

Tm , Tc , and Tg , are the melting, crystallization and glass transition temperature of the polymers.

Fig. 6. TG curve of PPDO-co-PCL-b-PEO-b-PPDO-co-PCL (T4 ) obtained by heating the samples from 50 to 600 ◦ C at 15 ◦ C/min under the steady flow of nitrogen. Solid line (–) represents the TGA curve and dot lines (- - -) represent the DTA curve.

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Fig. 7. 1 H NMR spectrum of PPDO-co-PCL-b-PEO-b-PPDO-co-PCL (T4 ), 6% (w/v) solution in CDCl3 (A) PPDO-co-PCL-b-PEO-b-PPDO-co-PCL (T4 ), nanoparticles dispersed in D2 O (1 mg/ml) (B) using tetramethyl silane (TMS) as the internal reference.

3.2. Preparation of PPDO-co-PCL-b-PEO-b-PPDO-coPCL nanoparticles It is a well-known fact that a self-assembly of block copolymers can be accomplished by different techniques, e.g.; direct dissolution, solvent evaporation/film formation, dialysis, and cosolvent evaporation [7,8]. In the present study, we have selected co-solvent evaporation technique (acetonitrile/water system) for the purpose. Stability of the nanoparticles was increased with the fraction of PCL due to increase in surface charge [33]. In the case of the nanoparticles formed by the samples T1 and T2, where PPDO fraction was greater than 45%, aggregation was observed after a month. As shown in Scheme 1 the core of the PPDO-b-PEO-b-PPDO polymer forms nanoparticles with nearly crystalline core, which enables the penetration of water molecules into the system, consequently leads destabilization of the particles. On the other hand, PPDO-co-PCLb-PEO-b-PPDO-co-PCL and PCL-b-PEO-b-PCL polymeric forms nanoparticles with inhomogeneous and homogeneous semicrystalline core, respectively, which resist the possible penetration of water molecules thereby enhance the stability of particles. 1 H NMR spectroscopic analysis of the polymer in CDCl 3 and D2 O revealed its geometric variation in nonselective and selective solvent (Fig. 7) [34]. All the characteristic peaks in CDCl3 (spectrum “A”) were in the agreement with the basic structure of polymer where as the existence of only a signal (h*) at δ 3.6 ppm in D2 O (spectrum “B”) was related to the basic structure of polymeric nanoparticles. Such a significant variation may be only due to the complete masking of hydrophobic

segment in aqueous medium, which implies the self-assembly of the polymer into a core-shell system. Fluorescence spectra of pyrene provide significance information about its local environments. Fig. 8 shows the typical fluorescence excitation spectra of pyrene in PBS (pH 7.4) in presence of polymer (sample T4 ) in various concentrations. The characteristic feature of pyrene excitation spectra was the linear red shift of band from 334 to 337 nm with the concentration of polymers. The linear red shift was due to the fact that pyrene

Fig. 8. Fluorescence excitation spectra of pyrene as a function of PPDO-coPCL-b-PEO-b-PPDO-co-PCL (T4 ) nanoparticles concentration; 0.0001 (A), 0.0015 (B), 0.01 (C), 0.065 (D), 0.4 (E), 1.0 (F) mg/mL in PBS (pH 7.4) (λem : 390.0 nm).

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Table 3 Characterization of PPDO-co-PCL-b-PEO-b-PPDO-co-PCL nanoparticles Sample code

Particle size (nm) mean ± S.D. AFM

T1 T2 T3 T4 T5

50 46 47 43 30

± ± ± ± ±

ζ-Potential (mV) mean ± S.D.

Polydispersity mean ± S.D.

−4.00 −9.92 −9.47 −12.21 −14.00

0.13 0.20 0.18 0.13 0.09

CMC (g/L)

DLS 0.5 0.3 0.2 0.6 0.4

113 100 93 92 90

± ± ± ± ±

2.3 2.6 1.9 2.8 1.5

± ± ± ± ±

0.14 0.12 0.14 0.18 0.12

± ± ± ± ±

0.02 0.05 0.01 0.04 0.03

2.3 × 10−3 4.7 × 10−4 4.8 × 10−4 6.1 × 10−4 7.6 × 10−4

± ± ± ± ±

0.05 0.03 0.05 0.01 0.02

S.D., standard deviation; n = 3.

was preferentially partitioned into hydrophobic domains and moved from polar environment to hydrophobic micelle cores as the micelles were formed [35]. Unlike the previous report, pyrene excitation spectra of our polymer showed slightly broad band [28]. The possible cause behind this phenomenon could be due to the lesser hydrophobicity of the nanoparticles core compared to poly(styrene), as found in polyester based block copolymeric nanoparticles [36]. CMC value of the polymer was determined using the intensity ratio of band (0, 2) 337 nm to the band (0, 0) 334 nm of pyrene in excitation spectra. The intensity ratio of I337 /I334 against polymer concentration (log c) in pyrene excitation spectra was plotted [28]. At low polymer concentrations, these ratios give the value of pyrene in PBS where as at high concentration it gives the value of pyrene in the hydrophobic environment. We found that the value of CMC was linearly increased with the Tg of the polymer (Table 3). The standard deviation of CMC was relatively large probably due to the variation in hydrophobicity of the particles resulted by the wide polydispersity of the polymers. Fig. 9 shows the comparative plot of polymer samples T1 , T4 , and T5 . These results indicate that the formation of nanoparticles is facilitated by hydrophobicity of the polymer [37]. Morphology and size of the polymeric nanoparticles analyzed by AFM observation are summarized in Fig. 10 and Table 3. AFM observation revealed that most of the particles from all the samples were discrete, smooth, and regular with 50–30 nm diameter. DLS measurement showed a uni-modal size distribution (Fig. 11) with a mean hydrodynamic diameter in the range of 113–90 nm (Table 3). The linear decrease in particle size with the fraction of PCL (i.e. with Tg ), was in good agreement with

Fig. 9. Intensity ratio (I337 /I334 ) from pyrene (6 × 10−7 m) excitation spectra (λem : 390.0 nm) vs. nanoparticles concentration (log C) in PBS (pH 7.4): PPDOb-PEO-b-PPDO (T1 ), PPDO-co-PCL-b-PEO-b-PPDO-co-PCL (T4 ) and PCLb-PEO-b-PCL (T5 ).

the AFM observation. This may be probably due to the remarkable solubilization and hydrophobic effect of PPDO and PCL, respectively. The wide variation in particle size measured by AFM and DLS was due to the fact that DLS measurement gives only the hydrodynamic diameter rather than the actual diameter of nanoparticles. ζ-potential is one of the most important physicochemical characteristics of nanoparticles [4]. In the present study, we found that ζ-potential was negative correlated with PCL (Table 3). The value of ζ-potential was in the range of −4.00 to −14.00 mV with the fraction of PCL (i.e. with Tg ). It was due to the increase in the fraction of ionizable carboxyl

Fig. 10. AFM image recorded from the drop-cast film of PPDO-b-PEO-b-PPDO (T1 ) (A), PPDO-co-PCL-b-PEO-b-PPDO-co-PCL (T4 ) (B) and PCL-b-PEO-b-PCL (T5 ) (C) polymeric nanoparticles dispersed in PBS (pH 7.4) (1 mg/ml).

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Fig. 11. Size distribution measured from PPDO-b-PEO-b-PPDO (T1 ) (A), PPDO-co-PCL-b-PEO-b-PPDO-co-PCL (T4 ) (B) and PCL-b-PEO-b-PCL (T5 ) (C) nanoparticles dispersed in PBS (pH 7.4) (1 mg/ml).

groups in the polymer since the PCL units were increased as well [33]. Thus, we found a distinct variation in the physicochemical properties of polymeric nanoparticles (crystallinity and hydrophobicity) with the content of PCL, i.e., Tg , and Tc of the polymer. It implies that the micelle yield, drug loading content and entrapment efficiency of PPDO-co-PCL-b-PEO-bPCL-co-PPDO polymeric nanoparticles could be significantly enough. 4. Conclusion The silent feature of the present research was the identification of effect of PCL content on ABA type random triblock copolymers, poly(p-dioxanone-co-␧-caprolactone)-block-poly (ethylene oxide)-poly(p-dioxanone-co-␧-caprolactone). PPDOb-PEG-b-PPDO was modified into a semicrystalline and high flexible state by the random insertion of PCL segment. CMC was linearly decreased with increasing the fraction of PCL thereby decreasing particle size. DLS measurement showed that the particle size was less than 120 nm and the hydrophobic segment makes a significant influence on the mean diameter. The investigation for the polymeric nanoparticles into an aqueous medium showed that the composition of the hydrophobic segment makes a significant influence on its physicochemical characteristics. Hence, we believe that these nanoparticles could give a reasonable drug release profile and solubilization effect on the hydrophobic drugs associated with intravenous administration. Acknowledgements This work was supported by the Korean Research Foundation (KRF) Grant Funded by Korean Government (MOEHRD)

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