Amphiphilic Core−Shell Nanocarriers Based On Hyperbranched Poly

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Amphiphilic Core-Shell Nanocarriers Based On Hyperbranched Poly(ester amide)-star-PCL: Synthesis, Characterization, and Potential as Efficient Phase Transfer Agent Ying Lin,†,‡ Xiaohui Liu,† Zhongmin Dong,†,‡ Baixiang Li,† Xuesi Chen,† and Yue-Sheng Li*,† State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China, and Graduate School of the Chinese Academy of Sciences, Changchun Branch, Changchun 130022, China Received June 2, 2008; Revised Manuscript Received July 7, 2008

Amphiphilic biodegradable star-shaped polymer was conveniently prepared by the Sn(Oct)2-catalyzed ring opening polymerization of
-caprolactone (CL) with hyperbranched poly(ester amide) (PEA) as a macroinitiator. Various monomer/initiator ratios were employed to vary the length of the PCL arms. 1H NMR and FTIR characterizations showed the successful synthesis of star polymer with high initiation efficiency. SEC analysis using triple detectors, RI, light scattering, and viscosity confirmed the controlled manner of polymerization and the star architecture. Because of the hydrophilic PEA core and hydrophobic PCL shell, the obtained star polymers displayed inverted unimolecular micellar structure confirmed by dynamic light scattering. Three water soluble dyes, congo red, methyl orange, and bromophenol blue, were used to investigate the host-guest behavior of the micelles. It proved that the core-shell unimolecular reverse micelles were able to transport polar dyes from water to the organic phase with a high efficiency of up to 22.6 dyes per polymer, indicating a great potential of the micelles as drug carriers. The influence of arm length and core size on the load efficiency of the nanocarrier was also evaluated.

Introduction Amphiphilic core-shell polymers have recently attracted significant attention due to their ability to self-assemble into micelles possessing a nanoscale, well-defined structure.1 As one example, dendrimers having amphiphilic core-shell structures were shown to exhibit interesting unimolecular micelle properties.2 In contrast to conventional micelles, which are generally weak physical aggregates of low molecular weight surfactants or amphiphilic block copolymers, the unimolecular micelles are stable to various environmental effects, such as dilution, shear force, temperature, and pH value, due to their unique dendritic characteristics and the covalent linkage between the hydrophobic and hydrophilic segments. In addition, the unimolecular micelles will not burst release the guest molecules out of control. These features make them attractive candidates as molecular nanocarriers for potential applications including controlled drug delivery and release,3 dye phase transfer,4 and building blocks for nanomaterials.5 Whereas dendrimers have well-defined molecular architecture on the nanometer length scale, numerous internal cavities, and a large modifiable surface functionality,6 the main drawback to dendrimers is their multistep tedious synthesis, which limits their general applicability. Therefore, it is desirable to develop more efficient ways to obtain dendritic molecular nanocarriers. So, without sacrificing the desirable architecture and properties of the dendrimer, hyperbranched polymer prepared by a costeffective methodology of one-step polymerization of ABx (x > 2) monomers was considered as an alternative.7 In contrast to the perfectly branched dendrimers, hyperbranched polymers * To whom correspondence should be addressed. Tel.: +86-43185262124. Fax: +86-431-85262039. E-mail: [email protected]. † State Key Laboratory of Polymer Physics and Chemistry. ‡ Graduate School of the Chinese Academy of Sciences.

have randomly branched structures. This imperfection in the structure of hyperbranched polymers, however, actually is an advantage for the nanocarrier applications. Because rigid and compact dendrimers have defined interior cavities, they only accept guest molecules with certain sizes. In contrast, the relatively less compact hyperbranched polymers have flexible interiors and can encapsulate a variety of guest molecules, which improved the load capacity. As a consequence, amphiphilic hyperbranched polymer with core-shell structure is of potential interest for the development of ideal nanocarriers. Mostly, the core-shell architectures can be achieved by the conjugation of different segments to their end functional groups. Currently, there are mainly two kinds of amphiphilic hyperbranched polymers. One is a hydrophobic core surrounded by a hydrophilic shell, which is applied in delivering hydrophobic compounds,8 and the other consists of a hydrophilic core and a hydrophobic shell with their application in transferring watersoluble compounds.9 The general approach for preparing the second materials was hydrophobization of hydrophilic hyperbranched cores, such as alkylation of hyperbranched polyglycerols,9a,9b,9c hyperbranched poly(amide-amine),9d,e and hyperbranched polyester.9f However, the introduction of a long alkyl chain leads to unbiodegradability, limiting their potential biomedical applications. Linear poly(
-caprolactone) (PCL), polylactide (PLA), and other aliphatic polyesters are of great interest due to their biodegradability, biocompatibility, and nontoxicity. They have been applied in a wide range of biological systems ranging from drug delivery to tissue engineering.10 Therefore, the incorporation of these biodegradable aliphatic polyesters into hyperbranched polymers represents a new way of creating welldefined core-shell architectures. Typically, the attachment of the polymeric shell in these core-shell systems occurs by either a “grafting-onto” (also named “arm-first”)11 or “grafting-from”

10.1021/bm800607a CCC: $40.75  2008 American Chemical Society Published on Web 08/13/2008

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(also named “core-first”) method.12 Dendritic polymers bearing abundant end functional groups are attractive macroinitiators for the preparation of core-shell star polymers by the “corefirst” strategy. Moreover, by tailoring the ratio of active sites to the amount of monomer, the materials obtained via “corefirst” strategy were more controllable than “arm-first” strategy. To date, a number of amphiphilic star polymers based on aliphatic polyesters have already been prepared by “core-first” strategy utilizing hyperbranched polymers as macroinitiators.13 However, most of the work was focused on a detailed synthesis and structural characterization, and little attention has been paid to their host-guest properties. In this context, recently, Chen14 and Adeli15 independently reported the use of commercial hyperbranched poly(ethylene imine) as initiator-core to prepare multiarm star PCL and PLA, respectively. Both of the resultant amphiphilic polymers displayed properties of encapsulation of water-soluble guests. Nevertheless, up to now, research in this area is still limited and deserves further exploration. Systematically investigation of factors influencing encapsulate efficiency is desired. In this contribution, we report the synthesis of a novel class of amphiphilic core-shell star polymers using core-first strategy. The star polymers consist of hydrophilic hyperbranched poly(ester amide) as core and hydrophobic PCL as shell. Both of them are fully biodegradable and biocompatible. To explore potential drug delivery application of this system, a series of dye phase transfer experiments were carried out. Polymers with different arm lengths and varying core sizes were used to investigate factors influencing the encapsulate efficiency.

Experimental Section Materials. Tris(hydroxymethyl)aminomethane was purchased from Acros and used as received. Succinic anhydride was purchased from the domestic market and purified by vacuum sublimation before use. Stannous 2-ethylhexanoate (Sn(Oct)2) was purchased from Aldrich and used as received. The
-caprolactone was purified by vacuum distillation over CaH2. The polar dyes, congo red (CR), methyl orange (MO), bromophenol blue (BB), were purchased from domestic market and used as received. Ultra-Pure deionized water was prepared from Millpore Filtration System (Millpore, U.S.). Other solvents such as chloroform and toluene were purified by M. Braun solvent purification system. Characterizations and Measurements. 1H NMR spectra were recorded on a Bruker AV 300 MHz spectrometer with CDCl3 or DMSO-d6 as the solvent. FTIR spectra were recorded on a Bio-Rad FTS-135 spectrophotometer. UV-vis absorption spectra were carried out on a Shimadzu UV-3600 spectrophotometer. And the measurements were made from solutions, using optical grade solvents and quartz glass cuvettes with a 10 mm path length. Size exclusion chromatography (SEC) was performed on a Waters 1525 separation module (Waters Corp.) connected with M302 triple detector array (Viscotek Corp., Houston, Texas), a combination of refractive index, light scattering (LS angle, 7° and 90°; laser wavelength, λ ) 670 nm), and viscosity detector. Two mixed bed SEC columns (GMHHR-M, GMHHR-H, Viscotek Corp.) were used. CHCl3 was used as mobile phase at a flow rate of 1.0 mL/min and an operating temperature of 25 °C. Data were collected and analyzed using OminSEC software version 4.1 (Viscotek Corp.). Weight-average molecular weights were calculated based on absolute measurements using light scattering detector. Size distribution of micelles was measured by dynamic light scattering (DLS) with a vertically polarized He-Ne laser (DAWN EOS, Wyatt technology). The scattering angle was fixed at 90° and the measurement was carried out with constant temperature at 25 °C. Before the measurement, the samples were filtered using 0.45 µm PTFE membrane filters to eliminate any dust particles.

Lin et al. Scheme 1. Chemical Structure Presentation of Hyperbranched Poly(ester amide) (PEA) Used in this Study

Synthesis of Hyperbranched Poly(ester amide). The hyperbranched poly(ester amide) was prepared according to the procedure described previously,16 and its chemical structure is presented in Scheme 1. In a cylindrical glass-reactor equipped with a mechanical stirrer, 10.00 g of succinic anhydride (0.1 mol), and 12.11 g of tris(hydroxymethyl)aminomethane (0.1 mol) were introduced under a nitrogen atmosphere. The reaction vessel was placed into an oil-bath preheated to 120 °C, with vigorous stirring for 32 h. The evolving water was removed by a slow stream of nitrogen and ultimately was distilled off under reduced pressure for 2 h. A total of 20.89 g (94% yield) of white block solid polymer was given. General Procedure for Synthesis of Multiarm Star Polymers. The multiarm polymer was prepared by ring-opening polymerization of CL using OH-terminated PEA as macroinitiator and Sn(Oct)2 as a catalyst. The typical polymerization was carried out as follows. The PEA was dried by an azeotropic distillation with toluene for 8 h, and then the toluene was evaporated completely under reduced pressure. Under the protection of N2, prescribed amounts of
-caprolactone and Sn(Oct)2 were added into the Schlenk tube containing PEA. An exhausting-refilling N2 process was operated three times. The bulk polymerization was carried out at 120 °C in an oil bath for 24 h with stirring. The polymerization was terminated by cooling to room temperature. The crude product was dissolved in a small amount of chloroform, filtered through a 0.45 µm-pore membrane filter, and precipitated into excess diethyl ether. The resultant polymer was dried at 50 °C in vacuo for 24 h to give wax or viscous solid depending on the molecular weight. The monomer conversion was determined gravimetrically. 1H NMR (CDCl3): δ 4.18-4.35 (C(C H2O)n(CH2OH)3-n in PEA), 4.07 (t, -CH2-C H2-OC(dO)-), 3.66 (t, -C H2OH in terminal PCL), 3.50-3.60 (C(CH2O)n(C H2OH)3-n in PEA), 2.52-2.68 (group, OOCC H2C H2CONH in PEA), 2.32 (t, -OC(dO)-C H2-CH2-), 1.65 (m, -CH2-C H2-CH2-C H2-CH2-), 1.39 (m, -CH2CH2-C H2-CH2CH2-). FTIR (cm-1): 3365, 2950, 2870, 1726, 1565, 1548. General Procedure for Determination of Transfer Capacities of PEA-star-PCL. The water-soluble dye CR, MO, and BB were used as anionic guest molecule to determine the transport capacities of star polymers, and chloroform was used as organic phase for the insolubility of CR, MO, and BB. A representative experimental transport procedure is given as the following: Different CR aqueous solution (10 mL) was mixed with PEA-star-PCL chloroform solution (10 mL, 1.0 mg/mL) and manually agitated for some minutes. Only the concentration of CR in water was changed, the concentration of PEA-star-PCL in chloroform remained unchanged. After standing for 3 h, phase

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Scheme 2. Synthesis and Encapsulate Polar Dye of PEA-Star-PCL Star Polymer

separation was completed and then an aliquot of the chloroform phase (3 mL) was transferred into a UV-vis cuvette and its absorption spectrum was measured. When the concentration of the dye was below saturation concentration, CR was totally extracted from the aqueous layer into the chloroform phase, and the water phase became colorless. Once the saturation concentration of the dye in the organic phase was reached, the absorption maximum in organic phase remained but the water phase became red. The average maximum load of dye molecules per polymer can be calculated from these UV-vis experiments (concentration should be converted into mol/L).

Results and Discussion Synthesis and Structural Characterization of CoreShell Star Polymer. Water-soluble PEA was prepared in onepot process from commercially available monomers and is readily available on a large scale.16 Importantly, due to the existence of aliphatic ester groups, the hyperbranched polymers are biodegradable. Benefiting from abundant hydroxyl terminal groups, the PEA was used as macroinitiator to polymerize
-CL in the presence of catalytic amounts of Sn(Oct)2 to provide amphiphilic multiarm star polymers having a hydrophilic core and a hydrophobic shell, as described in Scheme 2. Ringopening polymerization of cyclic esters catalyzed by Sn(Oct)2 is usually described as a coordinated insertion mechanism,17 wherein hydroxyl groups act as active propagation sites. The quasi-living controlled type of polymerization allows the control of molecular weights by the monomer/OH ratio. In the current study, all polymerization experiments have been performed in bulk at 120 °C for 24 h under rigorously anhydrous conditions. Three hyperbranched PEA samples with different molecular weight PEA26 (Mn ) 2600 Da, PDI ) 1.53), PEA41 (Mn ) 4100 Da, PDI ) 1.80), and PEA58 (Mn ) 5800 Da, PDI ) 2.62) were employed as the initiator-core. The crucial step for the controlled synthesis of the multiarm star polymers is to dry the PEA macroinitiators carefully to avoid initiation by traces of water that leads to cocurrent homopolymerization and, thus, an undesired blend of linear and star PCL. For PEA58, different CL monomer/hydroxyl group ([CL]/[OH]) ratios were used to prepare hyperbranched core-shell architectures with different arm lengths. They were denominated as PEA58CL2.5, PEA58CL5, PEA58CL7.5, and so on, according to the molecular weight of PEA and the [CL]/[OH] ratio. The number of hydroxyl groups per polymer (NOH) was calculated from the SEC molecular weight, assuming an average of two hydroxyl groups per repeat unit,18 because the AB3 type of monomer was applied in the preparation of PEA. The NOH for PEA58, PEA41, and PEA26 were determined to be 52, 37, and 23, respectively. It was inefficient to precipitate the obtained polymers completely when using a good precipitator for linear PCL such as cold methanol. And only very little amounts of the precipitants were collected, due to the higher polarity of the resulting multiarm

star polymers versus the linear PCL. Thus, the resultant star polymer is dissolved in CHCl3 since the uninitiated PEA is insoluble but the star polymer is soluble. After filtration and precipitation of the filtrate in diethyl ether, the polymers were obtained as sticky solid. The purified PEA-star-PCL star polymers were characterized by 1H NMR spectroscopy, as shown in Figure 1. It could be seen that peaks a, b, c, d, and d′ were attributed to PCL, where a, b, c, and d were assigned as methylene protons in the repeat units, and d′ was characteristic of methylene protons in the terminal units. According to the assignment, the average degree of polymerization of PCL arm (DParm) was calculated from the 1 H NMR spectra by dividing the average integration values of peak d by peak d′:

DParm )

I(d) +1 I(d′)

It should be noted that this calculation is based on the absence of PCL homopolymer, which was confirmed by SEC data, as will be discussed below. The data listed in Table 1 showed that the experimental values of DParm are comparable to the theoretical values. Remarkably, several new signals could be detected in Figure 1b-f, including peaks 1-3, which indicated the introduction of PEA. The ratio of the peak area of the PEA core to the PCL arms was deceased upon an increase in the [CL]/[OH], which is related to the increased PCL arm length. Compared with the 1H NMR spectrum of hyperbranched PEA (Figure 1a), the integration of peak 3 attributable to the methylene protons adjacent to ester groups is relatively bigger

Figure 1. 1H NMR spectra of hyperbranched PEA in DMSO (a) and star polymers with various [CL]/[OH] feed ratios in CHCl3: (b) 2.5/1; (c) 5/1; (d) 7.5/1; (e) 10/1; (f) 15/1 (one attached PCL arm was shown for simplicity).

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Table 1. Characterization Data of the Multiarm Star Polymers from NMR, SEC, and DLS sample

[CL]/[OH]

conv (%)

Mn,th (kDa)b

DParmc

Mn,SEC (kDa)d

PDId

[η] (dL/g)e

Re

Rv (nm)f

Rh (nm)g

PEA58CL2.5a PEA58CL5 PEA58CL7.5 PEA58CL10 PEA58CL15 PEA41CL10 PEA26CL10

2.5/1 5/1 7.5/1 10/1 15/1 10/1 10/1

81 84 88 90 94 91 93

17.8 26.9 39.2 53.5 83.7 42.5 24.3

3.1 5.5 7.3 9.2 12.7 9.3 9.5

17.9 25.0 35.8 47.1 68.5 32.2 20.6

2.34 1.87 1.66 1.41 1.37 1.39 1.25

0.106 0.145 0.180 0.264 0.422 0.157 0.135

0.32 0.37 0.35 0.40 0.41 0.35 0.36

3.22 3.83 5.48 7.61 9.06 4.80 3.45

3.19 3.86 5.55 7.53 9.30 4.87 3.40

a PEA58CL2.5: PEA58, a hyperbranched poly(ester amide) with Mn ) 5800 is the core. b Theoretical number average molecular weight calculated according to following equation, Mn,th ) Mn,PEA+ NOH × [CL]/[OH] × conv × 114.14, where 114.14 is the molecular weight of CL unit. c Calculated from 1 H NMR measurement. d Measured by SEC using light scattering and RI detector. e Mark-Houwink equation measured by SEC using viscosity detector. f Hydrodynamic radius determined by SEC using viscosity detector. g Hydrodynamic radius determined by DLS.

Figure 3. SEC elution profiles of the star polymers with different arm lengths. Figure 2. FTIR spectra of hyperbranched PEA (a) and star polymers with different arm lengths: (b) PEA58CL2.5; (c) PEA58CL5; (d) PEA58CL7.5; (e) PEA58CL10; (f) PEA58CL15.

than peak 2 attributable to the methylene protons adjacent to OH groups, confirming a successful grafting of the PCL arms onto the PEA core. The disappearance of peak 2 in Figure 1d-f showed that the polymerization of CL was initiated by the most of the hydroxyl groups. However, incomplete initiation occurred from the chain ends of the PEA at low [CL]/[OH] ratios (Figure 1b and c for PEA58CL2.5 and PEA58CL5, respectively). The same phenomenon was also observed by other researchers when using multifunctional initiator to graft PCL or PLA at low monomer/ initiator ratio.13a,b,19 The initiation efficiency, that is, the percent of hydroxyl groups of PEA initiated CL, could be estimated according to the 1H NMR spectrum by comparing the proton integration of 2 and 3. Unfortunately, due to the slight overlapping of peak d′ with peak 2, it is difficult to precisely determine the initiation efficiency of OH groups. For further confirmation of the grafting of PCL to PEA, FTIR spectra were investigated. Figure 2 displays the FTIR spectra of the star polymers with different arm lengths. The peak centered at 1726 cm-1 was characteristic of the CdO stretching mode of ester in PCL. Besides, two absorption bands centered at 1656 and 1548 cm-1, which were assigned to the CdO stretching and N-H bending mode for amide group in PEA core, were appeared. The intensity decreased with increasing the PCL content, indicating that PCL was grafted successively onto PEA. Compared with Figure 2a, the low intensity of the peak of the hydroxyl groups (in the region 3500-3100 cm-1) in Figure 2d-f indicates that the majority of the O-H groups of PEA has been initiated. From all the above discussions, the polymers with hydrophilic PEA as core and hydrophobic PCL as shell have been successfully synthesized.

Molecular Size and Inverted Unimolecular Micelle Characterization. It is already established that conventional SEC analysis has only limited suitability to determine the molecular weight of star-shaped polymers due to their more compact structure and smaller hydrodynamic volume in comparison with their linear analogues with identical molecular weights.13b,d,e,20 Because of the use of linear standards, the molecular weights of star polymers are usually underestimated by SEC. To resolve this problem, SEC equipped with triple detector array: refractive index, viscosity and light scattering detectors was employed to yield absolute molecular weight, regardless of the architecture of the macromolecule.12d,21 Figure 3 shows the typical SEC traces of multiarm star polymers based on the PEA58 core. The polymers all showed symmetrical and unimodal peaks as well as the absence of signals at lower molecular weight in the SEC elution curves, meaning no detectable PCL homopolymers were formed during the ringopening polymerization, which strongly supports the formation of graft polymers. As illustrated in Table 1, the PDI of the polymers are almost under 2.0 (except PEA58CL2.5 with 2.34), indicating that the polymers were synthesized in a controlled way. Additionally, it is interesting to note that the molecular weight distribution (MWD) of the stars becomes progressively narrower as the arm length increases. Narrowing of the MWD is quantified by the relative decrease in PDI from 2.34 to 1.37 due to the coupling of the distributions of the PCL chains at the star cores.13e Furthermore, the increase of the molecular weight with an increasing ratio of [CL]/[OH] is evident according to the SEC results. On the other hand, when PEA with different molecular weights was used as initiators, while keeping molar ratios of [CL]/[OH] constant, the molecular weights of star-shaped polymers increased with an increase in core size. The above

Amphiphilic Core-Shell Nanocarriers

Figure 4. Mark-Houwink plot for PEA58CL7.5 and its linear analogue. The slope of the line indicates the value of R.

results demonstrate that the molecular weights of the star-shaped polymers can be controlled by changing the core size and molar ratios of monomer to initiator. In contrast to the significant underestimation of molecular weights resulted from conventional SEC in similar system,13d,e,14 our molecular weights estimated by light scattering measurements (Mn,SEC in Table 1) are slightly lower than the theoretical molecular weights (Mn,th). The results verified the good control and pseudoliving character of the polymerization. Star polymers are well-known to exhibit unusual solution behavior, such as intrinsic viscosity [η] and the value of the Mark-Houwink exponent R are different from their linear analogues, also due to the compact character of the branched structure.22 To further support the formation of the star-shaped structure in this synthetic procedure, viscosity behavior of the resulting polymers was studied. The relationship between [η] and the molecular weight allows to judge the topology architecture of the polymers in solution, using the empirical Mark-Houwink equation [η] ) KMR.23 Figure 4 showed the corresponding double logarithm plots of intrinsic viscosity against molecular weight. For the purpose of comparison, linear PCL was synthesized via ring opening polymerization initiated with methanol. Obviously, the intrinsic viscosity of the star products is lower than that of the linear analogues, suggesting a denser and compacter structure for the former. The compact structure is confirmed by the Mark-Houwink exponent R value which is significantly depressed (0.32) relative to that of linear analogues (0.63). The R values of the resultant star polymers listed in Table 1 are in the range of 0.32 and 0.41, which is within the typical range for star polymers.24 Though containing ester linkage, PCL still have five methylene groups in its repeating unit, which renders PCL the hydrophobic nature. Therefore, it is soluble in a large variety of apolar organic solvents, for example, toluene, chloroform, dichloromethane, and tetrahydrofuran. In contrast to PCL, hyperbranched PEA is more hydrophilic due to abundant OH and amide groups. Therefore, it is only soluble in extremely polar solvents, such as water, methanol, DMF, and DMSO. As a consequence, attachment of the hydrophobic PCL chains to the hydrophilic PEA can lead to core-shell like inverted micelle in selective solvents, just as reported for PLA stars with poly(ethylene imine) core.15 To confirm the micellar property of the multiarm star polymers, the hydrodynamic radii (Rh) of the synthesized core-shell materials were measured by DLS

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in CHCl3, a good solvent for PCL. DLS measurement was performed with all the samples at the concentration of 1 × 10-4 g/mL at an angle of 90°. The measured average values of Rh are given in Table 1. The PEA58 macroinitiator showed the Rh value of 1.40 nm, while the Rh values of PEA41 and PEA26 macroinitiators were smaller than 1.0 nm, which is out of the range of the instrument. As expected, the Rh values of the star polymers (3.19-9.30 nm) were larger than those of their respective macroinitiators (