Article pubs.acs.org/Langmuir
Core−Shell Structure, Biodegradation, and Drug Release Behavior of Poly(lactic acid)/Poly(ethylene glycol) Block Copolymer Micelles Tuned by Macromolecular Stereostructure Chenlei Ma,† Pengju Pan,*,† Guorong Shan,† Yongzhong Bao,† Masahiro Fujita,‡ and Mizuo Maeda‡ †
State Key Laboratory of Chemical Engineering, College of Chemical and Biological Engineering, Zhejiang University, 38 Zheda Road, Hangzhou 310027, China ‡ Bioengineering Laboratory, RIKEN, Hirosawa 2-1, Wako, Saitama 351-0198, Japan S Supporting Information *
ABSTRACT: Poly(ethylene glycol)-b-poly(L-lactic acid)-bpoly(D-lactic acid) (PEG-b-PLLA-b-PDLA) stereoblock copolymers were synthesized by sequential ring-opening polymerization. Their micelle formation, precise micelle structure, biodegradation, and drug release behavior were systematically investigated and compared with the PEG-b-poly(lactic acid) (PEG-b-PLA) diblock copolymers with various PLA stereostructures and PEG-b-PLLA/PEG-b-PDLA enantiomeric mixture. Stereoblock copolymers having comparable PLLA and PDLA block lengths and enantiomerically-mixed copolymers assemble into the stereocomplexed core−shell micelles, while the isotactic and atactic PEG-b-PLA copolymers formed the homocrystalline and amorphous micelles, respectively. The PLA segments in stereoblock copolymer micelles show smaller crystallinity than those in the isotactic and enantiomerically-mixed ones, attributed to the short block length and presence of covalent junction between PLLA and PDLA blocks. As indicated by the synchrotron radiation small-angle X-ray scattering results, the stereoblock copolymer micelles have larger size, micellar aggregation number, core radius, smaller core density, and looser packing of core-forming segments than the isotactic and enantiomerically-mixed copolymer micelles. These unique structural characteristics cause the stereoblock copolymer micelles to possess higher drug loading content, slower degradation, and drug release rates.
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INTRODUCTION Amphiphilic block copolymers can self-assemble into the core− shell micelles comprised of hydrophobic cores stabilized by the hydrophilic shell in aqueous solution due to the phase separation of immiscible blocks. The core−shell structure of such assemblies is ideally suited to the biomedical applications such as controlled drug delivery.1 The hydrophobic environment in micelle core is favorable for encapsulation of hydrophobic drugs. The noncovalent interactions between drug and core-forming segments such as hydrophobic interactions and H-bonds can be the driving forces of drug entrapment.2 The primary mechanism for the release of encapsulated drugs from micelle cores is diffusion and core degradation.3 Because the chemical structure and chain-packing mode of hydrophobic block significantly influence the degradation of micelle core and diffusion of drugs, they would be the critical factors governing the drug entrapment and release kinetics. Many core−shell micelles have been designed for drug delivery applications through the self-assembly of amphiphilic block copolymers. One of the most popular groups is the copolymers with poly(ethylene glycol) (PEG) as the hydro© 2015 American Chemical Society
philic block and biocompatible, biodegradable polyesters such as poly(lactic acid) (PLA)4−8 and poly(ε-caprolactone) (PCL)2,9 as the hydrophobic block. PLA has three stereoisomers, that is, poly(L-lactide) (PLLA), poly(D-lactide) (PDLA), and poly(D, L-lactide) (PDLLA). PLLA and PDLA are isotactic and semicrystalline, while PDLLA is atactic and amorphous. Equimolar PLLA/PDLA mixture can form a stereocomplexed polymorph.10 The amorphous, homocrystalline, and stereocomplexed PLAs have different chain-packing mode, intermolecular interaction,11,12 and degradation rate13−15 in the solid state. In the stereocomplexed PLA, the chain packing is more compact due to the enhanced intermolecular interactions between two enantiomers. The hydrophobic block of amphiphilic copolymer plays an important role in micelle formation and characteristics. The micelle characteristics such as critical micelle concentration (CMC) and micelle size have been modified by changing the molecular weight of hydrophobic block5 and incorporating Received: September 29, 2014 Revised: December 24, 2014 Published: January 2, 2015 1527
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comonomer into the hydrophobic block.3 The tune of tacticity and stereostructure of hydrophobic block is also a feasible method to control the micelle characteristics, because they significantly influence the packing mode, crystallinity, and crystalline polymorph of core-forming block. Owning to the diversified stereostructures of PLA, the structure and physical properties of PLA copolymer micelles such as CMC, micelle size,16,17 colloid stability,17−19 degradation, drug/protein loading and release behavior,20,21 cellular uptake,6 and function22 have been tailored by altering the stereostructure and crystallization of PLA block. For example, Ning et al. have reported that the PEG-b-PLLA/PEG-b-PDLA stereocomplexed micelles show higher colloid stability than the amorphous and homocrystalline PLA/PEG copolymer micelles.17 Chen et al. have found that the rifampin loading capacity and encapsulation efficiency of PEG-b-PLLA/PEG-b-PDLA stereocomplex micelles were higher than those of PEG-b-PLA micelles.20 Because the core structures of copolymer micelles are key parameters governing their properties and functions such as degradation rate, loading content, and release kinetics of drugs, clarifying the relationship between copolymer structure, micelle structure, and micelle properties are of fundamental importance to design the micellar materials with desired properties and functions. The precise internal structures of PLLA/PEG block copolymer micelles comprised of homocrystalline core have been investigated by the light8,23 and neutron5,24 scattering techniques. However, a systematical investigation on the precise structure and structural difference of stereocomplexed, homocrystalline, and amorphous micelles of PLA/PEG block copolymers, as well as their effects on the micelle properties and functions, are unexplored. In this study, we synthesized a new kind of PLA/PEG block with stereoblock PLA as the hydrophobic segment, that is, PEG-b-PLLA-b-PDLA. The core crystalline structure, thermal property, biodegradation, and drug release behavior of PEG-bPLLA-b-PDLA were studied and compared with those of atactic PEG-b-PDLLA, isotactic PEG-b-PLLA, and PEG-b-PLLA/ PEG-b-PDLA enantiomeric mixture. Precise micelle structures of block copolymers with various stereostructures were assessed by synchrotron radiation small-angle X-ray scattering (SAXS), which has been a powerful tool for structural analysis of micelle particles.25 On the basis of these results, the relationships between copolymer stereostructure, inner micelle structure, and micelle properties were elucidated. This proves that the precise structure and properties of PLA/PEG copolymer micelles can be feasibly tuned through controlling the stereostructure of PLA block. To our knowledge, this is the first systematic investigation on the effects of stereostructure on precise micelle structure, biodegradation, and in vitro drug release behavior of PLA/PEG block copolymers.
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(10.0 g, 2.0 mmol) and L-lactide (5.0 g, 34.7 mmol) were added into a flame-dried Schlenk flask and dried under reduced pressure at 50 °C for 0.5 h. Seventy milliliters of dried toluene was injected into the flask. Thirty milliliters of toluene was then distilled out under argon atmosphere to remove the trace water. After the injection of 30 mg of Sn(Oct)2 catalyst, the mixture was reacted at 110 °C for 12 h. After polymerization, the reaction mixture was diluted by dichloromethane and then precipitated in excess cold ethyl ether. The purified product was finally dried under vacuum to a constant weight (14.3 g, yield: 95.3%). The second-step ROP was conducted in a similar procedure with PEG-b-PLLA as the macroinitiator and D-lactide as the monomer. For comparison, PEG-b-PLA diblock copolymers comprised of isotactic PLLA, PDLA, and atactic PDLLA were synthesized by a similar method as the first-step ROP. Racemic lactide was used as the monomer in the synthesis of PEG-b-PDLLA. The stereoblock, isotactic, and atactic PLA/PEG block copolymers were, respectively, referred as ExLyDz, ExLy (or ExDy), and ExDLy, where E, L, D, and DL are the abbreviations of PEG, PLLA, PDLA, and PDLLA, respectively. The subscripts represent the degree of polymerizations (corresponding to lactic acid unit) of corresponding blocks, as derived from the 1H NMR data. Preparation of Copolymer Micelles and Drug-Loaded Micelles. Typically, 0.1 g of copolymer was dissolved in 10 mL of THF at room temperature, followed by the addition of deionized water (30 mL) under continuous stirring. The micelle solution was attained after evaporating THF at room temperature. DOX-loaded micelles were prepared according to a published method.9 Briefly, DOX·HCl (10 mg) and 3.0 equiv of triethylamine (TEA) were dissolved in 2.0 mL of DMSO. A 0.1 g sample of copolymer dissolved in THF (10 mL) was added into this solution. The mixed solution was added dropwise to 20 mL of deionized water under continuous stirring. The solution was then dialyzed against deionized water for 24 h (MWCO = 3500) to remove the free DOX and byproduct, during which the water was changed every 6 h. The solution was finally freeze-dried to give the DOX-loaded micelles in dark-red power. To determine the drug loading content (DLC), dry DOX-loaded micelles were dissolved in DMSO and the UV absorbance at 485 nm was measured on a UV-Vis spectrophotometer (UV-1800, Shimazu, Kyoto, Japan). DLC was defined as the weight percentage of DOX in micelles. DOX solutions (in DMSO) with various concentrations were prepared and the absorbances at 485 nm were measured to generate a calibration curve for DLC calculation. In Vitro Release Behavior of Drug-Loaded Micelles. Lyophilized micelles (5 mg) were dispersed in 2 mL of NaCl solution (0.9%) and the solution was then transferred to a dialysis bag (MWCO = 3500). The bag was placed into 10 mL of PBS (10 mM, pH 7.4) solution. Release study was conducted at 37 °C in an incubator shaker. At selected time intervals, PBS solution outside the dialysis tube was removed for UV−vis measurement and replaced with the fresh PBS solution. DOX concentration was calculated based on the absorbance at 485 nm. The release experiments were conducted in triplicate and the averaged data was used. Enzymatic Degradation of Micelles. Two milligrams of proteinase K and 1.0 mg of NaN3 were added into 10 mL of micelle solution (3.0 g/L) in deionized water. The solution was incubated at 37 °C for 2 h and then lyophilized. Molar ratio of lactic acid (LA)/ ethylene glycol (EG) unit in the partially degraded micelles was determined by 1H NMR spectroscopy.26 The degradation experiments of micelles were conducted in triplicate and the averaged data was used. Characterization. 1H NMR spectrum was measured on a 400 MHz Bruker AVANCE II NMR spectrometer (Bruker BioSpin Co., Switzerland) at room temperature with CDCl3 or D2O as the solvents. Molecular weights were measured on a gel permeation chromatography (GPC) consisting of a Waters degasser, a Waters 1515 isocratic HPLC pump, a Waters 2414 RI detector (Waters Co., Milford, MA, U.S.A.), and two PL-gel mix C columns at 30 °C. THF was used as the eluent at a flow rate of 1.0 mL/min and polystyrene was used as the standard. Specific optical rotation, [α], of copolymers was measured in chloroform at a concentration of 1 g/dL at 25 °C using an automatic
EXPERIMENTAL SECTION
Materials. Monomethoxy PEG (mPEG, Mn = 5000 g/mol) was purchased from Sigma-Aldrich. L- and D-lactide (> 99.9%) were purchased from Purac Co. (Gorinchem, The Netherlands) and recrystallized from ethyl acetate before use. Tin(II) 2-ethylhexanoate [Sn(Oct)2, Sigma-Aldrich], proteinase K (Aladdin), and doxorubicin hydrochloride (DOX·HCl) were used as received. Toluene was dried by sodium and distilled after being refluxed for 48 h. Synthesis of PLA/PEG Block Copolymers. PEG-b-PLLA-bPDLA stereoblock copolymers were synthesized via sequential ringopening polymerization (ROP). A typical procedure to prepare PEGb-PLLA-b-PDLA having the expected Mns of PLLA and PDLA blocks of 2500 g/mol is described as below. In the first-step ROP, mPEG 1528
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Figure 1. (a) Synthesis of PLA/PEG diblock and stereoblock copolymers. GPC traces of PEG-b-PLLA and PEG-b-PLLA-b-PDLA block copolymers for (b) E113L32 and E113L32D34 and (c) E113L20 and E113L20D44. ⎛ − q 2R 2 ⎞ g ⎟ I(q) = I(0)exp⎜⎜ ⎟ 3 ⎝ ⎠
polarimeter (P810, Hanon Instruments, China) with a wavelength of 589 nm. Fourier transform infrared (FTIR) spectrum was measured on a Nicolet 5700 spectrophotometer (ThermoElectron, Madison, U.S.A.) with 64 scans and a resolution of 2 cm−1. Wide-angle X-ray diffraction (WAXD) measurements were performed on a Rigaku RU200 (Rigaku Co., Tokyo, Japan) instrument with a Ni-filtered Cu Kα radiation (λ = 0.154 nm), working at 40 kV and 200 mA. Thermal properties of lyophilized micelles and copolymers were measured on a NETZSCH 214 Polyma DSC (NETZSCH, Germany) equipped with an IC70 intracooler. The lyophilized micelles and melt-crystallized copolymer films were heated from −70 to 190 (or 230) °C at a heating rate of 10 °C/min. Dynamic light scattering (DLS) analysis was performed on a Zetasizer 3000 HSA instrument (Malvern Instruments, Malvern, UK) with a scattering angle of 90°. The micelle solution (0.2 wt %) was passed through a syringe-driven filter unit (0.45 μm) prior to the measurement. Three repeated measurements were performed for each sample. Micelle morphology was observed on a JEM-1230 transmission electron microscope (TEM, JEOL, Tokyo, Japan) operated at an acceleration voltage of 80 kV. To prepare the TEM sample, the micelle solution (0.2 wt %) was dropped on a carbon-coated copper grid and the solvent was removed by a filter paper. CMC was determined by the surface tension method.27,28 Surface tensions of copolymer solutions with various concentrations were measured on a DataPhysics OCA 20 instrument (DataPhysics Instruments, Filderstadt, Germany) at 25 °C. SAXS patterns of micelle solutions (0.5 wt %) were measured at BL45XU RIKEN Structural Biology Beamline I of SPring-8 (Harima, Japan) using an incident X-ray with a wavelength of λ = 1.0 Å. Twodimensional (2D) SAXS patterns were collected using a Pilatus 300 KW (Rigaku Co., Tokyo, Japan) detector at room temperature. All the data was corrected from the background and air scattering. SAXS data was converted to the absolute intensity according to a published method.29 The 2D scattering pattern was integrated into to the 1D scattered intensity I(q) as a function of scattering vector q (q = 4π sin θ/λ, where 2θ is the scattering angle) by circularly averaging. SAXS data at low q was analyzed by Guinier equation30
(1)
where I(0) (cm−1) is the forward scattering intensity at q → 0 and Rg is the gyration radius of micelle. Rg and I(0) can be obtained from the slope and intercept of ln[I(q)] versus q2 plot. The average micellar aggregate number, Nagg, is calculated as follows29
Nagg =
Magg M polymer
=
I(0)NA M polymerc ΔρM2
(2)
where Magg and Mpolymer are the averaged molecular weights of micelle and block copolymer, c (g/cm3) is the copolymer concentration, NA is the Avogadro constant, and ΔρM (cm/g) is the scattering length difference per mass (ΔρM = Δρv). ̅ The difference of scattering length density Δρ can be determined from the known chemical composition of polymer and solvent. Specific volume of polymer, v ̅ (cm3/g), was calculated from the copolymer composition and bulk densities of PLA (1.25 g/cm3)31 and PEG (1.13 g/cm3).5 The core−shell structural model with two graded smooth interfaces between core and shell characterized by σin and between shell and solvent characterized by σout was employed in analyzing the SAXS data (see Supporting Information).32−35 The dimensions of central parts of core (Wcore) and shell (Wshell), dimensions of core−shell (2σin) and shell−solvent interfaces (2σout) were attained through fitting the SAXS profiles. On the basis of these parameters, the radius of core (Rin), thickness of shell (Lshell), and radius of particle (Rout) can be calculated by Rin = Wcore + σin, Lshell = Wshell + σin + 2σout, and Rout = Wcore + 2σin + Wshell + σout, respectively. The packing density of PLA chains in micelle cores (ρcore) and the surface area available per PEG chain at the core−shell interface (SPEG) were calculated as ρcore = Nagg × Mn,PLA/ (4/3π × Rin3NA) and SPEG = 4πRcore2/Nagg, respectively.
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RESULTS AND DISCUSSION Synthesis of PLA/PEG Block Copolymer. PLA/PEG stereoblock, isotactic, and atactic diblock copolymers having the
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Table 1. Molecular Structural Characteristics of PLA/PEG Block Copolymers samples
Mn,th (kDa)
Mn,GPCa (kDa)
Mw/Mnb
Mn,NMRc (kDa)
mPLA (%)
L/(L + D)
[α] (deg)
E113L32D34 E113L20D44 E113L6D57 E113L64/E113D71 E113L64 E113D71 E113DL58 E113L14D14 E113L32/E113D30 E113L32 E113D30 E113DL31
9.4 9.3 9.3
13.4 13.0 12.4
1.06 1.06 1.14
9.7 9.6 9.5
0.48 0.31 0.10
9.5 9.7 9.2 7.1
13.7 15.2 11.3 10.9
1.09 1.13 1.10 1.05
9.6 10.1 9.2 7.0
7.2 7.1 7.2
10.9 10.5 10.5
1.05 1.05 1.03
7.3 7.2 7.2
48.7 48.0 47.4 49.2 48.0 50.5 45.7 28.7 30.9 31.5 30.2 30.9
1.0 ± 0.10 27.1 ± 0.05 53.3 ± 0.05 1.1 ± 0.30 −75.4 ± 0.10 79.1 ± 0.05 −1.3 ± 0.05 −3.3 ± 0.10 −1.6 ± 0.05 −46.2 ± 0.10 43.5 ± 0.05 −0.55 ± 0.05
1.0 0 0.50 0.50 1.0 0 0.50
a
Number-average molecular weight (Mn) derived from GPC. bMw is the weight-average molecular weight and Mw/Mn represents the polydispersity index. cMn derived from 1H NMR.
Figure 2. Representative 1H NMR spectra of (a) block copolymers in CDCl3 and (b) lyophilized micelles in D2O.
Figure 3. WAXD patterns of lyophilized micelles for PLA/PEG block copolymers with different stereostructures and compositions. In panels a and b, the block copolymers have the Mn,NMR of about 10 kDa and 7.5 kDa, respectively.
1.15). For the stereoblock copolymers, the elution time decreases after the first- and second-step ROPs, implying the successive growth of PLLA and PDLA blocks. Theoretical molecular weight (Mn,th) of block copolymers was calculated from the monomer/initiator ratio and conversion. Mn of PLA segment in block copolymers was determined by comparing the intensity of methine proton signal of PLA block and the intensity of CH2 proton signal of PEG block in the 1H NMR spectrum (Figure 2a). Mn,NMR, representing the Mn derived from 1H NMR, was attained by summing the Mns of PEG and PLA blocks. The Mn values measured by GPC and NMR were consistent with those
expected LA/EG mass ratios of 1/1 and 1/2 were prepared via one- or two-step ROP using mPEG as the macroinitiator (Figure 1a). In the stereoblock copolymers, the molecular weights of PLLA and PDLA blocks were controlled by changing the lactide/macroinitiator molar ratio. The molar fraction of L-isomer to total LA unit in stereoblock copolymers varies in a wide range of 0.10−0.48 (Table 1). Molecular structure and molecular weight of block copolymers were characterized by GPC and 1H NMR. GPC traces of stereoblock and diblock copolymers display a narrow and single peak (Figures 1b,c and Supporting Information Figure S1), indicative of the narrow molecular weight distribution (PDI < 1530
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Figure 4. DSC heating curves for lyophilized micelles of PLA/PEG block copolymers with different stereostructures and compositions. In panels a and b, the block copolymers have the Mn,NMR of about 10 and 7.5 kDa, respectively.
Table 2. Physical Properties of PLA/PEG Block Copolymers and Their Micellesa lyophilized micelle
copolymer
samples
Tm,PEG (°C)
ΔHm,PEG (J/g)
Tm,PLA (°C)
ΔHm,PLA (J/g)
Tm,PEG (°C)
ΔHm,PEG (J/g)
Tm,PLA (°C)
ΔHm,PLA (J/g)
CMC (mg/L)
Rh (nm)
E113L32D34 E113L20D44 E113L6D57 E113L64/E113D71 E113L64 E113D71 E113DL58 E113L14D14 E113L32/E113D30 E113L32 E113D30 E113DL31
50.9 54.2 57.5 48.3 55.0 53.2 54.6 57.8 59.2 59.7 60.4 57.8
64.2 76.3 75.1 61.3 69.9 63.4 90.2 113.1 105.5 108.0 112.3 129.2
193.9 173.5 141.5 214.6 153.7 155.9
23.0 22.4 21.1 47.2 29.1 33.0
10.2 18.6 17.5 46.5 28.3 31.8
6.4 21.8 11.1 11.6
72.9 68.5 71.5 57.1 69.2 66.7 97.7 108.7 101.2 98.6 101.7 119.1
158.9 175.7 140.5 209.4 151.3 153.8
161.1 190.4 128.4 128.7
57.1 55.5 57.7 51.7 56.5 51.7 56.6 57.8 59.0 60.2 58.5 58.5
128.9 187.4 124.3 131.0
3.8 20.0 8.2 8.1
9.4 10.1 11.3 8.9 13.0 12.0 15.0 12.9 13.0 13.8 14.1 15.5
21.6 ± 0.6 21.6 ± 0.6 21.1 ± 0.5 17.7 ± 0.7 17.1 ± 0.7 17.5 ± 0.8 17.8 ± 0.5 15.4 ± 0.6 15.1 ± 0.7 13.1 ± 0.4 12.6 ± 0.5 14.9 ± 0.3
Tm,PEG, melting temperature of PEG block. Tm,PLA, melting temperature of PLA block. ΔHm,PEG, melting enthalpy of PEG block. ΔHm,PLA, melting enthalpy of PLA block.
a
micelles comprised of hydrophobic PLA core and hydrophilic PEG corona in aqueous solution.37 WAXD, FTIR, and DSC were applied to examine the solidstate structure and thermal properties of copolymer micelles, which were also compared with those of the copolymer films crystallized at 120 °C. In the WAXD patterns, the micelles show similar diffraction angles as the copolymer films (Figure 3, Supporting Information Figure S2). The lyophilized micelles of stereoblock copolymers with comparable L- and D-isomer contents (i.e., E113L32D34, E113L14D14, and E113L20D44) and enantiomerically-mixed copolymers display typical diffraction peaks of stereocomplexed PLA (2θ = 12 and 21°),10 rather than the diffractions of PLA homocrystals. This coincides with the report of Li et al. that the stereoblock PLLA-b-PDLA with L/D ratio of 1/1−2/1 mainly crystallizes in the stereocomplex form.38 Formation of stereocomplexed micelles in the stereoblock copolymers and enantiomeric mixture were further confirmed by FTIR (Supporting Information Figure S3) and DSC (Figure 4). The micelles comprised of stereoblock (e.g., E113L32D34 and E113L20D44) and enantiomerically-mixed copolymers exhibit lower wavenumber (1753−1751 cm−1) for ν(CO) band and higher melting points (Tm = 170−220 °C) of PLA blocks than those of isotactic diblock copolymers. The lower frequency of PLA ν(CO) vibration is an indicator of stereocomplexation, which is resulted by the H-bond interactions between the enantiomers.11,12
calculated theoretically (Table 1). Mass ratio of LA to EG unit in block copolymer was estimated by comparing the NMR integration ratio of PEG at 3.63 ppm (−O−CH2−CH2−) and PLA at 5.15 ppm (−CH−) (Figure 2a). The copolymer compositions measured by NMR were in agreement with those calculated from [α], assuming that the enantiopure PLLA and PDLA have the [α] values of −156° and +156°, respectively.36 The [α] values of symmetrical stereoblock (E113L32D34 and E113L14D14) and atactic block copolymers are close to zero, confirming the similar contents of L- and D-isomer in them. All these results confirm that the stereoblock and diblock copolymers with various stereostructures are synthesized in a well-controlled manner. Micelle Formation and Crystalline State of Micelle Core. Micelles composed by the stereoblock, isotactic, atactic, and enantiomerically-mixed PLA/PEG block copolymers were prepared by a precipitation/solvent evaporation method.5 1H NMR spectrum of lyophilized micelles dispersed in D2O was used to confirm the micelle formation (Figure 2b). The lyophilized micelles can well disperse in water and D2O to a transparent solution, although the block copolymers are insoluble under the same conditions. Compared to the 1H NMR spectrum collected in CDCl3 (Figure 2a), the NMR peaks of PLA block cannot be observed, while those of PEG block (δ = 3.7 ppm) can be clearly seen in the spectra measured in D2O. This demonstrates that the stereoblock, diblock, and enantiomerically-mixed copolymers all assemble into the 1531
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Figure 5. (a) One-dimensional SAXS profiles and (b) Guinier plots in small q for micelle solutions of PLA/PEG copolymer micelle solutions (0.5 wt %). The data are shifted vertically for clarity. Real lines represent the fits by core−shell model and linear fits in panel a and b, respectively.
Table 3. Structural Characteristics of Micelles Formed from PEG-b-PLA at 25 °C in Aqueous Solution (0.5 wt %)a samples
Rg (nm)
Rg/Rh
Nagg
Wcore (nm)
σin (nm)
E113L32D34 E113L20D44 E113L6D57 E113L64/E113D71 E113L64 E113DL58
10.3 9.4 9.3 8.0 9.2 9.3
0.48 0.43 0.44 0.45 0.54 0.52
254 274 270 241 231 221
8.64 ± 0.02 8.63 ± 0.01 8.35 ± 0.03 7.49 ± 0.01 7.51 ± 0.03 7.60 ± 0.01
0.011 ± 0.006 0.010 ± 0.004 0.010 ± 0.006 0.009 ± 0.005 0.011 ± 0.008 0.010 ± 0.003
Wshell (nm)
σout (nm)
Rin (nm)
Rout (nm)
Lshell (nm)
ρcore (g/cm3)
SPLA (nm2/chain)
± ± ± ± ± ±
1.00 ± 0.02 1.00 ± 0.01 1.00 ± 0.03 0.99 ± 0.02 1.01 ± 0.04 0.98 ± 0.02
8.65 8.64 8.36 7.50 7.52 7.61
15.8 15.8 15.5 14.6 14.6 14.7
8.12 8.11 8.11 8.09 8.12 8.06
0.73 0.77 0.83 1.09 0.99 0.84
3.70 3.43 3.25 2.93 3.08 3.28
6.11 6.10 6.10 6.09 6.09 6.09
0.01 0.01 0.01 0.01 0.06 0.01
Rg, gyration radius. Rh, hydrodynamic radius. Nagg, average micellar aggregation number. Wcore, dimension of central part of core. σin, half of the core−shell layer thickness. Wshell, width of central part of shell. σout, half of the shell-solvent layer thickness. Rin, radius of core. Rout, radius of micelle. Lshell, thickness of shell. ρcore, chain packing density in core. SPLA, surface area available per PEG chain at the core−shell interface. a
Similar as the isotactic block copolymers, the micelles composed by the stereoblock copolymers with shorter PLLA block (i.e., E113L6D57) show the characteristic diffractions of PLA homocrystals (e.g., 2θ = 17°)39 in the WAXD pattern (Figure 3) and a lower melting point (140−160 °C) for PLA block in the DSC curves (Figure 4). Neither stereocomplex nor homocrystal diffraction is observed in PEG-b-PDLLA, because of the amorphous nature of PDLLA. In the micellation process, the crystallization/stereocomplexation of PLA blocks and the formation of micelles take place simultaneously, causing that the PLA segments have sufficient time and higher mobility to crystallize. Hence, the PLA blocks in copolymer micelles exhibit larger melting enthalpies and crystallinities than those in the copolymer films (Table 2). As shown in Table 2, increasing the PLA block length leads to the enhancement of melting enthalpy, because of the increased crystallizability. However, even though the E113L32D34 stereoblock copolymer has similar content and block length of PLA (including both PLLA and PDLA) as the E113L64/E113D71 enantiomerically mixture, the former shows the smaller melting enthalpy of PLA core (ΔHm,PLA). Similar result is observed by comparing E113L14D14 with E113L32/E113D30 mixture (Table 2). These results are also confirmed by the copolymer films (Supporting Information Figure S4) and indicate the lower degree of crystallinity or stereocomplexation in stereoblock architecture. The lower degree of crystallinity in stereoblock copolymers may be ascribed to the shorter block length and the presence of linking junction between PLLA and PDLA blocks. In polymer crystallization, the chain ends and the junctions inside polymer chains would be expelled from the crystalline lamellae. When the PLLA and PDLA blocks are too short, it will be difficult to form the thick and order crystalline lamellae in crystallization. de Jong et al. have found that the stereocomplexation of enantiomerically-mixed PLA/PEG tri-
block copolymers are retarded when the PLLA and PDLA blocks are too short.40 Furthermore, the linking junction between PLLA and PDLA blocks in stereoblock copolymers would restrict the mobility and regular folding of PLA chain in crystallization, resulting in the decreased crystallinity. CMC of PLA/PEG block copolymers was investigated by the surface tension method. When the concentration of amphiphilic copolymer is approaching CMC, the slope of surface tension versus concentration plot changes abruptly (Supporting Information Figure S5). CMC can be evaluated from the inflection of surface tension versus concentration plot (Table 2).27,28 At the similar composition, the stereoblock and enantiomerically-mixed copolymers show smaller CMCs than the diblock copolymers. The isotactic block copolymers (i.e., PEG-b-PLLA and PEG-b-PDLA) bearing the crystallizable core-forming blocks have smaller CMCs than their analogs with the atactic core-forming blocks (i.e., PEG-b-PDLLA); this is consistent with the case of PEG-b-poly(ε-caprolactone) and PEG-b-poly(ε-decalactone) micelles.3 Because the lower CMC generally means higher micellar stability,41,42 the CMC results suggest that the crystallization, especially stereocomplexation, of core-forming blocks enhances the stability of assembled micelles. Size and morphology of copolymer micelles were identified by DLS and TEM. The stereoblock, diblock, and enantiomerically-mixed micelles all exhibit a single peak in the distribution curves of hydrodynamic radius (Rh) (Supporting Information Figure S6). The micelles of block copolymers with Mn,NMR around 10 kDa and 7.5 kDa have Rh values ranging from 17−22 and 12−16 nm, respectively (Table 2). Increasing the PLA block length leads to the enhancement of Rh, because the micelles with larger cores would be formed to include more insoluble chains. Micelles comprised of diblock and enantiomerically-mixed copolymers have similar Rh values, which range 1532
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17−18 and 12−15 nm for the copolymers with Mn,NMR ≈ 10 and 7.5 kDa, respectively. Interestingly, the stereoblock copolymers assemble into larger micelles than their diblock counterparts due to the larger aggregation number and looser packing of PLA in the micelle core, as indicated in the following SAXS data. As observed from TEM images (Supporting Information Figure S7), most of the micelles have the spherical shapes and nanoscopic sizes with radius ranging from 15−25 nm, which are in accordance with the DLS results. Internal Structure of Micelle Analyzed by Synchrotron Radiation SAXS. The precise core−shell structures of micelles were analyzed by synchrotron radiation SAXS. Figure 5a shows the representative SAXS profiles of copolymer micelle solutions on an absolute scale. Rg and I(0) were obtained from the Guinier plot (Figure 5b). I(0) was also calculated from the fitted parameters of core−shell model, which was similar to that derived from Guinier plot (Supporting Information Table S1). Because Guinier law is universal for the scattering intensity at small q, I(0) derived from Guinier plot is used to calculate Nagg. Rgs of micelles for PLA/PEG copolymers with Mn,NMR ≈ 10 kDa are ranging 8.0−10.3 nm (Table 3). Notably, the enantiomerically-mixed copolymer micelles exhibit a smaller Rg than the other micelles, because it has a more compact core (as described below). Rg was compared with Rh measured by DLS. The Rg/Rh ratios are around 0.5 for all copolymer micelles and these values are lower than the theoretical value of hard spherical particles (Rg/Rh = (3/5)0.5 = 0.775). This suggests that the PLA/PEG micelle has larger electron density portion in inner part than that in the outer part, characteristic of the core−shell spherical particles.43,44 Naggs of copolymer micelles were calculated from eq 2. Naggs of block copolymers vary from 221 to 274 (Table 3), which are in a similar range as those measured by small-angle neutron scattering (SANS) for the PEG-b-PLA and PLA-b-PEG-b-PLA copolymers with similar compositions5,24 but larger than those derived from static light scattering (SLS).17 Among all the micelle samples, the stereoblock and enantiomerically-mixed copolymer micelles show larger Nagg, while the atactic copolymer micelles comprised of noncrystallizable cores have the smallest Nagg. Besides the hydrophobic interactions, crystallization, and stereocomplexation of core-forming segments are the additional driving forces for copolymer association and micelle formation. This stronger driving force may favor more copolymer chains self-assembling into one micelle, accounting for the larger Nagg. To attain the precise core−shell structures of PLA/PEG copolymer micelles, the SAXS data was further analyzed by a core−shell model. As seen from the real lines of Figure 5a, good fits were attained for the copolymer micelles. However, the fitted results show slight deviation from the experimental SAXS curves at high q (q > 0.4 nm−1). Even though many papers reported that PLA/PEG copolymer micelles are spherical,8,23 researchers also claimed that these micelles can be lamellar or ellipsoid due to the crystallization of PLA core.24 The deviation of micelle shape from spherical geometry may induce these differences between the fitted and experimental data. As displayed in Table 3, Rin of micelles changes from 7.50 to 8.65 nm. Rin and Rout of stereoblock copolymer micelles are slightly larger than those of the atactic, isotactic diblock, and enantiomerically-mixed copolymer micelles, in accordance with the results attained from DLS. Lshell of micelles is almost independent of the stereostructure of block copolymers, because the micelle shells are all comprised by the same length
of PEG. Because of the smaller Rin and relatively larger Nagg, the enantiomerically-mixed copolymer micelles have smaller SPEG than other micelles, indicating the denser grafting of PEG chains on the core surface. ρcore of micelle, which is considered to be a key factor for drug loading and release, is strongly dependent on the macromolecular stereostructure and architecture. The enantiomerically-mixed and isotactic copolymer micelles exhibit larger densities of 0.99−1.09 g/cm3, which are close to the density of bulk PLA (∼1.25 g/cm3). This indicates that the micelle cores with homocrystalline and stereocomplexed structure are highly dehydrated. Notably, the stereocomplexed micelles of enantiomerically-mixed copolymers have higher ρcore than that of homocrystalline micelles formed by PEG-b-PLLA, because of the stronger intermolecular H-bond interactions and tighter molecular packing in stereocomplexed PLA.11,12 Interestingly, despite of the stereocomplexed core structure, ρcore of stereoblock copolymer micelles (e.g., E 113 L 20 D 44 and E113L32D34) is much smaller, indicating the relatively looser packing of PLA chains in these micelles. This unique structure of stereoblock copolymer micelles may be ascribed to the lower degree of stereocomplexation and crystallinity induced by the shorter block length and the linking junction between PLLA and PDLA blocks, as confirmed by the aforementioned WAXD and DSC data. Enzymatic Degradation of Micelles. Enzymatic degradation of PLA/PEG micelles with various core structures was assessed in aqueous solution with the presence of proteinase K. After partial degradation, the micelle samples were analyzed by 1 H NMR spectroscopy (Supporting Information Figure S8) and the LA/EG molar ratio, which is an indicator of degradation rate, was calculated (Table 4). The LA/EG Table 4. Drug Loading Content of Copolymer Micelles and LA/EG Unit Ratios of Copolymer Micelles before and after Enzymatic Degradation for 2 h sample
LA/EG before degradation
LA/EG after degradation
degraded ratio (%)
drug loaded content (%)
E113L32D34 E113L20D44 E113L6D57 E113L64/E113D71 E113L64 E113D71 E113DL58
0.584 0.566 0.557 0.597 0.566 0.628 0.513
0.570 0.540 0.525 0.585 0.518 0.580 0.450
2.4 4.6 5.7 2.0 8.5 7.6 12.3
2.88 ± 0.18 2.65 ± 0.16 2.62 ± 0.20 1.22 ± 0.15 1.66 ± 0.15 1.61 ± 0.13 0.81 ± 0.11
molar ratios decrease after enzymatic degradation, indicating the partial decomposition of PLA segment. Degradation rate is strongly influenced by the solid-state structure of PLA in micelle core. The stereoblock and enantiomerically-mixed copolymer micelles composed by stereocomplexed cores degrade slower due to the strong interactions between two enantiomers. Because the amorphous phase with loose and disorder chain packing can be easily attacked by enzyme, the PEG-b-PDLLA micelles comprised of amorphous core degrade faster, which is in in agreement with the results of PLA homopolymer14 and block copolymer.15 Under the investigated conditions, the degradation rate of atactic copolymer micelles is 3−6 times of that of stereocomplexed micelles. In Vitro Release of DOX Drug from Micelles. A typical anticancer drug, DOX, was used to study the drug loading and 1533
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copolymers formed the micelles with stereocomplexed core, while isotactic and atactic copolymers assembled into the micelles having the homocrystalline and amorphous cores, respectively. The crystallinity of PLA in stereoblock copolymer micelles is smaller than those of the isotactic and enantiomerically-mixed ones. Compared to the isotactic and enantiomerically-mixed copolymers, the micelles of stereoblock copolymers show larger Rh, Nagg, Rcore, smaller ρcore, and looser packing of PLA chains in the micelle core. These structural characteristics of stereoblock copolymer micelles render them higher DLC, slower degradation, and drug release rate. This study demonstrates that the inner structure and physical properties of amphiphilic copolymer micelles can be precisely tailored from the stereostructure, crystallization, and stereocomplexation of core-forming segments. The results obtained here will serve as the basis for designing the micellar materials with tunable structure and optimized functions.
release behavior of PLA/PEG micelles. It is generally recognized that the physical encapsulation of hydrophobic drugs in polymeric micelles is mainly driven by the hydrophobic interactions between drug and hydrophobic segments of polymer. As shown in Table 4 and Figure 6, DLCs of various
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ASSOCIATED CONTENT
S Supporting Information *
Figure 6. In vitro DOX-release profiles of copolymer micelles in PBS solution.
Description of core−shell model, structural parameters of copolymer micelles, GPC, WAXD, FTIR, DSC, DLS, CMC, and TEM data of block copolymers, and 1H NMR spectra of partially degraded copolymer. This material is available free of charge via the Internet at http://pubs.acs.org.
micelles changes in the order of stereoblock > isotactic > enantiomerically-mixed > atactic copolymer micelle, while the DOX release rate changes in the order of atactic > isotactic > stereoblock > enantiomerically-mixed copolymer micelle. Interestingly, the stereoblock copolymer micelles exhibit larger DLC and slower DOX release rate. It is considered that chain packing and polymer/drug interactions inside the micellar cores are critical factors for the DLC and drug release rate. Larger DLC of stereoblock copolymer micelles can be attributed to the bigger size, looser chain packing, and stronger intermolecular interaction in micelle cores. The interactions between stereocomplex crystals and drugs and slow degradation of stereocomplexed PLA core may be responsible for the slow DOX release behavior of stereoblock and enantiomerically-mixed copolymer micelles. Because the too compact packing of PLA chains in enantiomerically-mixed copolymer micelles prevents the penetration and diffusion of drugs, these micelles show relatively lower DLC. PEG-b-PDLLA micelles comprised of amorphous core have the smallest DLC, even with the looser packing of PLA in micelle core. They also show much faster drug release than other micelles and the encapsulated DOX drugs are almost completely released after 5 h. In the first 10 h, the DOX release rate from PEG-b-PDLLA micelles is nearly twice of the other micelles. The small DLC and fast release rate may be explained by the weak drug/polymer interactions in micelle core. The easy release of drug during dialysis can be also responsible for the small DLC of PEG-b-PDLLA micelles measured in this study.
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AUTHOR INFORMATION
Corresponding Author
*author. Tel.: +86-571-87951334, email:
[email protected]. cn. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of China (51103127, 21274128, 21422406) and State Key Laboratory of Chemical Engineering (SKL-ChE12D06). Synchrotron radiation SAXS experiments were performed at BL45XU of SPring-8, Japan. We thank Dr. Takaaki Hikima, RIKEN SPring-8 Center, for his help with the SAXS experiment at SPring-8.
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REFERENCES
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CONCLUSIONS Micellation, micelle structure, biodegradation, and in vitro drug release behavior of PEG-b-PLLA-b-PDLA stereoblock copolymers, PEG-b-PLA diblock copolymers with different PLA tacticities, and PEG-b-PLLA/PEG-b-PDLA enantiomeric mixtures were investigated. The stereostructure, architecture, and crystallization of hydrophobic PLA blocks play an important role in the core structure, stability, thermal and physical properties of copolymer micelles. Stereoblock copolymers with similar L- and D-isomer contents and enantiomerically-mixed 1534
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