Amphiphilic cylindrical copolypeptide brushes as potential nanocarriers for the simultaneous encapsulation of hydrophobic and cationic drugs

Colloids and Surfaces B: Biointerfaces 94 (2012) 324–332

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Amphiphilic cylindrical copolypeptide brushes as potential nanocarriers for the simultaneous encapsulation of hydrophobic and cationic drugs Xiang Zeng, Jinhu Li, Jinhong Zheng, Ying Pan, Jinzhi Wang, Lumian Zhang, Xiaoying He, Daojun Liu ∗ Medical College, Shantou University, 22 Xinling Road, Shantou 515041, China

a r t i c l e

i n f o

Article history: Received 11 November 2011 Received in revised form 7 February 2012 Accepted 7 February 2012 Available online 16 February 2012 Keywords: Drug delivery system Peptide Polymer brush Hydrophobic drug Cationic drug

a b s t r a c t Cylindrical copolypeptide brushes PLLF-g-(PLF-b-PLG) with poly(l-lysine-co-l-phenylalanine) (PLLF) as the backbone and poly(l-phenylalanine)-b-poly(l-glutamic acid) (PLF-b-PLG) as the side chains have been synthesized and evaluated as drug delivery carriers. The synthesized copolypeptide brushes were characterized by 1 H NMR, gel permeation chromatography (GPC), and transmission electron microscopy (TEM). In aqueous solution, the copolypeptide brushes adopt cylindrical morphologies and resemble unimolecular polymeric micelles with a hydrophobic poly(l-phenylalanine) core and a hydrophilic poly(l-glutamate) shell. An encapsulation study demonstrated that these water soluble, biodegradable copolypeptide brushes encapsulate hydrophobic compounds and cationic hydrophilic guest molecules simultaneously. Furthermore, the encapsulated cationic model compounds exhibit a pH-responsive releasing property. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Micelles formed from amphiphilic block copolymers have recently attracted much attention in biomedical fields such as those involved in drug delivery [1,2]. The hydrophobic blocks of the copolymer should segregate into the core of the micelles and serve as a microenvironment for the solubilization of various therapeutic compounds with low solubility and/or low stability. However, polymeric micelles are thermodynamically unstable in infinitely dilute environments such as the bloodstream. Once the concentration drops below the critical micelle concentration (CMC), the disruption of micellar structures leads to a burst release of entrapped drugs. To improve the micellar stability, unimolecular polymeric micelles have been developed from dendritic polymers such as dendrimers and hyperbranched polymers [3–6]. The shell functionalization of these branched macromolecules has been used to produce a wide variety of core–shell star polymers that can be used as nanocarriers in drug delivery [7–13]. The common structure of these potential drug-delivery scaffolds consists of a hydrophobic interior and a hydrophilic shell, which is mostly intended to entrap and deliver nonpolar guest molecules via hydrophobic interactions. Cylindrical polymer brushes are a type of graft copolymer in which multiple polymer chains (side chains) are grafted to a linear polymer (backbone) [14,15]. Various synthetic techniques,

∗ Corresponding author. Tel.: +86 754 88900499; fax: +86 754 88557562. E-mail address: [email protected] (D. Liu). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2012.02.012

including anionic polymerization, cationic polymerization, ringopening polymerization (ROP) and controlled/living radical polymerization, have been used to obtain well-defined brush polymers through the synthesis strategies of “grafting onto”, “grafting from”, and “grafting through” [14,15]. Their nonspherical macromolecular geometries and lengths of up to a few hundred nanometers have found numerous potential applications in nanoscience, such as templates for inorganic particles [16], precursors for nanocapsules [17], nanotubes [18,19], and carbon nanostructures [20]. Brush polymers can be designed to possess a core–shell architecture that resembles unimolecular polymeric micelles, which make them attractive for drug delivery applications. Drug molecules can be chemically linked to the interior of polymer brushes or physically entrapped into the polymer brush micelles with a high loading capacity and high temporal stability [21–28]. The physical entrapment of drug molecules into polymer brushes could be realized through hydrophobic interactions, ionic interactions or hydrogen bonding and can therefore be applied to a large number of drug molecules. Comparatively, chemical incorporation of drug molecules onto the backbone of polymer brushes via labile linkages may lead to well-shielded environments for drug moieties and thus well-controlled drug loadings and continuous release. However, the synthesis of polymer brush-drug conjugates remains challenging. The unique architecture of cylindrical brush polymers may have advantages in drug loading and release over star, block, and dendritic copolymers. Nevertheless, the use of brush polymers as drug delivery carriers has been less explored relative to the vast number of reports on their synthesis, stimuli–responsive properties [29–33] and self-assembled nanostructures [34–39].

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Herein, we report on the design and synthesis of a type of amphiphilic cylindrical copolypeptide brush, PLLF-g-(PLF-bPLG) with a poly(l-lysine-co-l-phenylalanine) (PLLF) backbone and poly(l-phenylalanine)-b-poly(l-glutamic acid) (PLF-b-PLG) side chains, as a potential drug delivery nanocarrier. The designed cylindrical copolypeptide brushes resemble unimolecular micelles with a poly(l-phenylalanine) hydrophobic core and a negatively charged poly(l-glutamate) hydrophilic shell at physiological pH and can therefore serve as potential nanocarriers to encapsulate hydrophobic and cationic drugs and entrap them simultaneously. 2. Materials and methods 2.1. Materials l-Phenylalanine, pyrene, Oil-Red O (OR), and crystal violet (CV) were obtained from Sigma–Aldrich (St. Louis, MO, USA). ␧-Benzyoxycarbonyl-l-lysine (ZLL) and ␥-benzyl-l-glutamic acid (BLG) were from Sichuan Tongsheng Amino Acid Co., China. Doxorubicin hydrochloride (DOX) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products, China. Trifluoroacetic acid (TFA), dichloroacetic acid and HBr/CH3 COOH (33 wt%) were obtained from Shanghai Darui Co., China. Dimethylsulfoxide (DMSO), ethyl acetate and dichloromethane (DCM) were obtained from Aladdin Co., China, and distilled over CaH2 . Petroleum ether (Aladdin, China) was refluxed with sodium and distilled immediately before use. Regenerated cellulose dialysis tubing (molecular weight cut-off, MWCO, 14 kDa) was obtained from Viskase, USA.

Ultrahydrogel Linear column and a 2414 RI detector. An aqueous solution of 0.5 wt% NaHCO3 was used as the eluent at a flow rate of 0.5 mL min−1 at 40 ◦ C. Molecular weights were calibrated on poly (ethylene glycol) standards (Supplementary data Fig. S1). The morphology of the synthesized polymer was visualized by the use of a JEOL JEM-1400 transmission electron microscope (TEM) at an operating voltage of 100 kV. A quantity of 0.5 mg mL−1 of copolypeptide brush in a 0.5 wt% NaHCO3 aqueous solution was filtered through ultrafiltration membranes and deposited onto a carbon-coated copper grid. The excess copolymer solution was wiped off with filter paper, and the grid was dried in the ambient atmosphere for 1 h. UV–vis absorption spectra were recorded on a UVPC 2501 spectrophotometer (Shimadzu). Fluorescence spectra were recorded on a RF-5301PC spectrofluorophotometer (Shimadzu). 2.3. Synthesis of ˛-amino acid N-carboxyanhydrides ␥-Benzyl-l-glutamate N-carboxyanhydride (BLG-NCA), ␧benzyoxycarbonyl-l-lysine N-carboxyanhydride (ZLL-NCA), and l-phenylalanine N-carboxyanhydride (Phe-NCA) were prepared by the same method as described previously [40]. 1 H NMR (400 MHz, CDCl3 ) ı (ppm): BLG-NCA: 7.34–7.41 (m, 5H, ArH), 6.39 (br, 1H, NH), 5.14 (s, 2H, CH2 ), 4.37 (t, 1H, CH), 2.60 (t, 2H, CH2 ), 2.07–2.33 (m, 2H, CH2 ); ZLL-NCA: 7.26–7.37 (m, 5H, ArH), 6.68 (br, 1H, NH), 5.11 (s, 2H, CH2 ), 4.89 (br, 1H, NH), 4.27 (m, 1H, CH), 3.21 (m, 2H, CH2 ), 1.39–1.99 (m, 6H, CH2 CH2 CH2 ); Phe-NCA: 7.19–7.37 (m, 5H, ArH), 6.13 (br, 1H, NH), 4.53 (q, 1H, CH), 2.98–3.33 (m, 2H, CH2 ). 2.4. Synthesis of poly(l-lysine-co-l-phenylalanine) (PLLF) (Scheme 1)

2.2. Measurements 1H

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NMR spectra were recorded on a Bruker DMX 400 MHz spectrometer with D2 O, CDCl3 , or DMSO-d6 as the solvent. The molecular weights and molecular weight distributions of the backbone were determined on a Waters 515 gel permeation chromatograph (GPC) equipped with Shodex KD804 and KD802.5 columns and a 2414 RI detector. DMF in the presence of 50 mmol L−1 LiCl was used as the eluent at a flow rate of 0.5 mL min−1 at 40 ◦ C. The molecular weights and molecular weight distributions of the final products were determined on a Waters 515 gel permeation chromatograph equipped with an

n-Hexylamine (0.036 mmol) was added into a mixed solution of ZLL-NCA (0.55 g, 1.80 mmol) and Phe-NCA (1.03 g, 5.39 mmol) in anhydrous DCM (120 mL). The reaction mixture was stirred at room temperature for 2 days. Monomer conversion, which was higher than 99%, was determined by absorbing the released CO2 using a standard NaOH solution and then back titrating with a standard HCl solution. The obtained random copolypeptides poly(␧-benzyoxycarbonyl-l-lysine-co-lphenylalanine) (PZLLF) were isolated by precipitation in diethyl ether and dried under vacuum. Yield: 1.25 g (98%).

Scheme 1. Synthesis of PLLF by ring-opening polymerization of ZLL-NCA and Phe-NCA mixtures.

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Scheme 2. Synthesis of PLLF-g-(PLF-b-PLG) by PLLF-initiated sequential ring-opening polymerization of Phe-NCAs and BLG-NCAs.

Subsequently, PZLLF (0.20 g) was dissolved in TFA (6 mL), HBr/CH3 COOH (5 mL) was added, and the mixtures were stirred for 2 h at room temperature. The deprotected random copolypeptides PLLF were precipitated in diethyl ether, filtrated, and washed with excess diethyl ether until it was an off-white color. The obtained products were dried under vacuum and characterized by GPC and 1 H NMR. Yield: 0.15 g (93%). 2.5. Synthesis of poly(l-lysine-co-l-phenylalanine)-g-[poly(lphenylalanine)-b-poly(l-glutamic acid)] (PLLF-g-(PLF-b-PLG)) (Scheme 2) Phe-NCAs and BLG-NCAs were polymerized sequentially in DCM using PLLF as the macroinitiator to produce poly(l-lysine-co-l-phenylalanine)-g-[poly(l-phenylalanine)-bpoly(␥-benzyl-l-glutamate)] (PLLF-g-(PLF-b-PBLG)). An aliquot of

PLLF (0.20 g) solution in DMSO (5 mL) was added into a solution of Phe-NCA (0, 0.14, or 0.28 g) in DCM (50 mL). The reaction mixture was stirred at room temperature for 6 h. Subsequently, a given amount of BLG-NCA (1.88 g) in DCM was added into the reaction system and stirred for an additional 12 h. The obtained PLLF-g-(PLF-b-PBLG) was isolated by precipitation in diethyl ether, dried under vacuum and characterized by 1 H NMR. Yield: 93–97%. The ␥-benzyl protection groups of PBLG were removed in the presence of HBr to generate the final product PLLF-g-(PLF-b-PLG) [41]. HBr/CH3 COOH (6 mL) was added to a solution of PLLF-g-(PLFb-PBLG) (0.5 g) in dichloroacetic acid (5 mL). After the reaction mixture was stirred at 30 ◦ C for 2 h, the product was isolated by precipitation in diethyl ether and filtration. The obtained solid was dissolved in an aqueous solution of 0.5 wt% NaHCO3 , dialyzed thoroughly against water, and lyophilized to obtain the final product

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PLLF-g-(PLF-b-PLG). Yield: 60–70%. The final products were characterized by 1 H NMR, GPC, and TEM.

2.9. Simultaneous encapsulation of hydrophobic and cationic hydrophilic molecules by PLLF-g-(PLF-b-PLG)

2.6. Aggregation behavior of PLLF-g-(PLF-b-PLG) determined by fluorescence measurements

Hydrophobic pyrene and cationic hydrophilic CV or DOX were chosen for the simultaneous encapsulation study because of the slight overlap in their respective absorption spectra. For the simultaneous encapsulation study, the experiments were conducted for pyrene and then CV or DOX following the same procedures as described above. Finally, the absorption spectra were recorded using a UV–vis spectrophotometer.

The aggregation behavior of PLLF-g-(PLF-b-PLG) in aqueous solution was investigated by fluorescence measurement using pyrene as a probe [42]. Briefly, an aqueous solution of pyrene with a concentration of 6.0 × 10−7 mol L−1 was prepared. A given amount of PLLF-g-(PLF-b-PLG) was then added to prepare a series of solutions with fixed pyrene concentration and a varying concentration of PLLF-g-(PLF-b-PLG) that ranged from 0.0001 to 0.5 g L−1 . The solutions were equilibrated by shaking overnight at room temperature. Steady-state fluorescence excitation spectra were recorded on the spectrofluorophotometer at a detection wavelength of 390 nm. 2.7. Encapsulation of hydrophobic dyes by PLLF-g-(PLF-b-PLG) The copolypeptide brushes PLLF-g-(PLF-b-PLG) were dissolved in water at concentrations varying from 0.1 to 2.0 g L−1 , and an excess of pyrene or OR solid (structure shown in Fig. 1) was added. The mixture was vigorously stirred at room temperature for 2 days. The insoluble dye residues were then removed by centrifugation at 14,000 rpm for 20 min, and the obtained clear supernatant was analyzed by UV–vis spectrophotometer for its absorbance. The amounts of dye guest molecules solubilized in the polymer solution were estimated using a calibration curve (Supplementary data Fig. S2) constructed from standard solutions of dyes in THF.

2.10. In vitro release of hydrophilic model compounds from PLLF-g-(PLF-b-PLG) The release of hydrophilic model compounds from PLLF-g-(PLFb-PLG) was investigated using a dialysis method (MWCO, 14 kDa) at 37 ◦ C with a model compound-loaded polypeptide brush solution (5 mL) against water (150 mL) at varying pH (3.0–7.4). The model compound-loaded brush polypeptide solution was prepared as described in Section 2.8. The solution pH was adjusted using NaOH or HCl because there is no buffer system that can be used to cover such a wide pH range. At selected time intervals, a given volume of release media was withdrawn and replenished with an equal volume of fresh release media. The amounts of released model compounds were determined with a UV–vis spectrophotometer. The release of free model compounds with the same concentration in the absence of polymers was also conducted under the same conditions to serve as the control. 3. Results and discussion

2.8. Encapsulation of cationic hydrophilic molecules by PLLF-g-(PLF-b-PLG) A given amount of CV or DOX (structure shown in Fig. 1) was added into a PLLF-g-(PLF-b-PLG) solution in water (5 mL), 0.01 mol L−1 Tris buffer (pH 7.4, 5 mL), or 0.16 mol L−1 Tris buffer (pH 7.4, 5 mL). After being sonicated for 2 min at room temperature, the clear solution was dialyzed thoroughly against water or Tris buffer under sink conditions to remove free guest molecules. The absorbance of encapsulated guest molecules was measured with a UV–vis spectrophotometer and then, compared with the calibration curve (Supplementary data Fig. S3) of the guest molecules to estimate the loading capacity.

Fig. 1. Chemical structures of pyrene, OR, CV, and DOX.

3.1. Synthesis and characterization of copolypeptide brushes PLLF-g-(PLF-b-PLG) The designed copolypeptide brushes PLLF-g-(PLF-b-PLG) consisted of a random copolypeptide PLLF backbone and block copolypeptide PLF-b-PLG side chains, and their syntheses were achieved by two consecutive steps as depicted in Schemes 1 and 2. First, a random copolypeptide PLLF backbone was prepared by the ring-opening polymerization of Phe-NCA and ZLL-NCA mixtures initiated by the terminal primary amino groups of nhexylamine (Scheme 1). The ring-opening polymerization of amino acid-NCAs is a well-known, efficient method for producing oligoor polypeptides, and this method had been used to prepare polypeptide brushes [43] or brush-like polymers having either polypeptide backbones [44] or polypeptide sidechains [36,45–47]. In the present study, poly(l-lysine) was employed as part of the backbone because its pendant amino side chains were used to initiate the subsequent polymerization of amino acid-NCAs to graft the side chains of the copolypeptide brushes. However, there were two challenges for the use of the poly(l-lysine) homopolymer as the polypeptide backbone. First, the homopolymer was slightly soluble in the subsequent polymerization solvent (DMSO). Additionally, it was found that the ROP of amino acid-NCAs initiated by the poly(llysine) homopolymer cannot proceed efficiently due to the densely packed amino side chains. Therefore, poly(l-phenylalanine) was incorporated as part of the backbone because it could increase the solubility of the backbone in DMSO and also dilute the amino side chains of poly(l-lysine) so that the subsequent ROP proceeded in a controlled manner. The addition of different molar ratios of Phe-NCA monomer to ZLL-NCA monomer (1:2, 1:1, 2:1, 3:1) were examined, and a ratio of 3:1 was determined to be optimal with respect to the two criteria discussed above. Because the final copolypeptide brushes were to be utilized as potential drug delivery vehicles, a total degree of polymerization of 200 for the

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Fig. 2.

1

H NMR spectra of (a) PZLLF and (b) PLLF in DMSO-d6 .

copolypeptide backbone was designed so that the length of the final polymer brushes was on the order of 100 nm. Because of the steric effect among the multiple polypeptide side chains, a fully extended conformation of the backbone is expected. Thus, the contour length (L) of the backbone can be approximated as follows: L = 200 × 0.37 nm = 74 nm. After the ROP of the mixed Phe-NCAs and ZLL-NCAs, the protective benzyl groups of PZLL were removed in the presence of HBr. The 1 H NMR spectra of the backbone before and after the deprotection procedure are shown in Fig. 2. The deprotected product PLLF in Fig. 2b showed no signals (ı = 4.97 ppm) caused by the benzyl groups, which confirmed that the deprotection was complete. The degrees of polymerization for poly(l-phenylalanine) and poly(llysine) in PLLF backbone can be estimated using the signal of the terminal methyl group as a reference. The obtained values (100 for poly(l-phenylalanine) and 36 for poly(l-lysine)) were much smaller than the expected values (150 for poly(l-phenylalanine) and 50 for poly(l-lysine)), which is most likely caused by the noise

signal in the terminal methyl group. The molecular weight of PLLF backbone and its distributions were determined with GPC using DMF in the presence of 50 mmol L−1 LiCl as the eluent; the obtained molecular weight was 24 kDa with a polydispersity index of 1.79. In the second step, the side chains of copolypeptide brushes were incorporated with a “grafting from” method, and this was realized by the sequential ROP of Phe-NCAs and BLG-NCAs initiated by the pendant amino groups of a PLLF macroinitiator (Scheme 2). The structure of copolypeptide brushes can be adjusted by tailoring the feeding ratios of respective amino acid-NCA monomer to the initiating amino groups in the PLLF backbone. In the current study, the feeding ratio of BLG-NCA monomer to the initiating amino groups was fixed at 20:1, whereas the feeding ratio of Phe-NCA monomer to the amino groups varied from 0:1 to 2:1 to 4:1. A relatively longer poly(l-glutamate) segment improved the aqueous solubility of the synthesized polypeptide brushes. After the sequential ROP of Phe-NCAs and BLG-NCAs, the protective benzyl groups of PBLG were removed in the presence of HBr to obtain the final products

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Fig. 3.

1

329

H NMR spectra of (a) PLLF-g-(PLF-b-PBLG) in DMSO-d6 and (b) PLLF-g-(PLF-b-PLG) (P2) in D2 O.

PLLF-g-PLG (P0), PLLF-g-(PLF2 -b-PLG) (P2), and PLLF-g-(PLF4 -bPLG) (P4), where the subscripts 2 and 4 represented the number of amino acid units in the poly(l-phenylalanine) blocks. Fig. 3 shows the 1 H NMR spectra of PLLF-g-(PLF2 -b-PBLG) and PLLF-g-(PLF2 -b-PLG) (P2). The disappearance of the signals (ı = 5.03 ppm) of the benzyl groups confirmed that the deprotection was complete. GPC was then used to measure the molecular weights and molecular weight distributions of the copolypeptide brushes. A typical GPC chromatogram of P2 is shown in Fig. 4, and the results are summarized in Table 1. P0, P2, and P4 had very similar molecular weights, regardless of the difference in the calculated values. GPC measurements only reflect the relative hydrodynamic size. Because the side chains of the polypeptide brush force the main chain to stretch out and the whole molecule to adopt a cylindrical shape (vide infra), the long axis determines the size of the particles. Therefore, the fact that their relative molecular weights do not differ significantly is not overly surprising. The molecular weights obtained from the GPC measurements were lower than the calculated values for all the polymer brushes. This result may be attributed to the smaller hydrodynamic volumes of polymer brushes when compared to linear polymers, which is because the side chains of the polymer brushes were forced to stay close to one another.

The structural characteristics of PLLF-g-(PLF-b-PLG) in aqueous solution were monitored by fluorescence spectroscopy using pyrene as a hydrophobic probe. The fluorescence excitation spectra of pyrene in P2 solutions of various concentrations are shown

Fig. 4. GPC chromatogram of P2.

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Table 1 Molecular weights of the copolypeptide brushes and their loading capacities. Polymer brushes

P0 P2 P4

Molecular weights [×103 g mol−1 ]

Loading capacity [nguest /nhost ]a

Calc.

GPC

PDI

In water

157 172 187

92 95 100

1.56 1.48 1.50

In Tris (10 mM)

In Tris (160 mM)

Pyrene

OR

CV

DOX

CV

DOX

CV

DOX

1.1 2.1 2.4

2.6 4.1 6.7

270 272 275

275 280 290

79 87 94

44 54 62

2.3 3.3 4.9

39 45 46

a All loading capacities were determined in triplicate and the corresponding mean values were reported. Typically, the relative standard deviation of the measurements is in the range of ±10%.

in Fig. 5. The free pyrene demonstrated a maximum wavelength excitation (ex,max ) of 335 nm. It is generally accepted that this wavelength will red shift to approximately 340 nm once pyrene is solubilized in the hydrophobic interior of a micellar structure that is formed when the concentration of polymer is above its critical aggregation concentration [42]. However, the ex,max of pyrene in the present study remained at 335 nm for the whole concentration range of P2 from 0.00001 to 0.5 g L−1 , which suggests that P2 does not form aggregates but rather exists in the form of individual molecules. The designed polypeptide brushes possess multiple long PLG chains at the periphery, and the side chains are expected to be predominantly deprotonated and negatively charged in a neutral aqueous solution. The electrostatic repulsion between the polypeptide brushes might prevent them from aggregation. It should be noted that pyrene can be encapsulated into the hydrophobic core of P2 which will give rise to a red-shift in the wavelength of maximum absorption. However, this would require vigorous stirring for a significant amount of time. The morphologies of the polypeptide brushes P0, P2, and P4 were visualized by TEM. P0, P2, and P4 had approximately the same morphologies, sizes and size distributions. A typical TEM image of P4 is shown in Fig. 6, in which the polymer brushes adopted a cylindrical morphology. Sizes with a length of approximately 100 ± 20 nm and a width of approximately 25 ± 5 nm were obtained by analyzing 200 polymer brushes. The size and morphology of the synthesized copolypeptide brushes that were determined using TEM were consistent with the designed structure of the individual polymer brushes and confirms that the polymer brushes had been prepared successfully and were well dispersed in an aqueous solution as unimolecular micelles. 3.2. Encapsulation properties of copolypeptide brushes PLLF-g-(PLF-b-PLG) The synthesized cylindrical copolypeptide brushes consist of an amphiphilic core–shell structure: a hydrophobic core composed of poly(l-phenylalanine), which creates a microenvironment for the

Fig. 5. Excitation spectra of pyrene as a function of the concentration of P2 in water.

encapsulation of nonpolar guest molecules, and a hydrophilic shell of poly(l-glutamate), which makes the polymer brushes watersoluble and could also entrap cationic molecules via electrostatic interactions. In contrast with polymeric micelles that are selfassembled from amphiphilic block copolymers, polymer brushes with amphiphilic block copolymer side chains can adopt a stable unimolecular micellar structure of cylindrical shape, which cannot be dissociated upon dilution because of the covalent attachment of the side chains to the backbone. To evaluate the encapsulation property of the synthesized copolypeptide brushes, pyrene and OR were used as hydrophobic model compounds, and CV and DOX were used as cationic polar compounds. The encapsulation study showed that the hydrophobic model compounds pyrene and OR could be solubilized in an aqueous solution of copolypeptide brushes. The UV–vis spectra of pyrene encapsulated by the copolypeptide brushes exhibited a bathochromic shift in the wavelength of maximum absorption (max = 340 nm) when compared to that of free pyrene (max = 335 nm) (Fig. 7) caused by ␲–␲ interactions between the side phenyl rings in the core of the copolypeptide brushes and the entrapped pyrene guests. The encapsulation of cationic model compounds by the polypeptide brushes were performed in water or in 0.01 or 0.16 mol L−1 Tris buffer (pH 7.4). CV and DOX could be efficiently entrapped by the copolypeptide brushes with a high loading capacity as a result of the electrostatic interactions between the cationic guest molecules and the negatively charged side chains of poly(l-glutamate). The UV–vis spectra of DOX entrapped in the copolypeptide brushes demonstrated a bathochromic shift in the wavelength of maximum absorption (max = 506 nm) when compared to that of free DOX molecules (max = 479 nm) (Fig. 7), which suggests that the cationic guest molecules were encapsulated and located around the shell of polypeptide brushes. The loaded values of different guest molecules increased proportionally with the concentrations of copolypeptide brush solutions that ranged from 0.1 to 2 g L−1 . The loading capacities (nguest /nhost )

Fig. 6. TEM image of P4.

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Fig. 8. Release of loaded CV from P2 at various pH levels. Curves a, b, c, and d correspond to pH levels of 7.4, 5.0, 4.0, and 3.0, respectively; curve e represents the release of free CV at a pH of 3.0. The errors of the measurements are typically in the range of ±10%.

Fig. 7. UV–vis absorption spectra of simultaneously encapsulated guest molecules in P2 solutions: (a) pyrene and CV; (b) pyrene and DOX.

of various guest molecules, determined in all cases by UV–vis spectroscopy according to the theoretical molecular weights of copolypeptide brushes, are listed in Table 1. As expected, an increase in the length of the hydrophobic core gave rise to a corresponding increase in the loading capacity of hydrophobic dyes. The loading capacities of polymer brushes towards CV or DOX increased slightly from P0 to P2 to P4 (from 270 to 272 to 275 for CV and from 275 to 280 to 290 for DOX, respectively), which may be explained by the synergistic effect of hydrophobic and electrostatic interactions [48]. The loading capacities of CV or DOX in Tris buffer were lower than those in pure water and decreased further when the concentration of Tris buffer was increased from 0.01 to 0.16 mol L−1 . A plausible explanation might be that the presence of an electrolyte weakens the electrostatic interactions between the copolypeptide brush hosts and the CV or DOX guest molecules [49]. Notably, the synthesized polypeptide brushes retained considerable loading capacity towards DOX even at 0.16 mol L−1 Tris buffer (isotonic solution) as a result of the synergistic effect of hydrophobic and electrostatic interactions because DOX is more hydrophobic than CV. Because the hydrophobic model compounds are solubilized by the amphiphilic polypeptide brushes via hydrophobic interactions, their loading efficiency (LE) is very high regardless of the rather low loading capacity. When the amount of dyes is lower than their loading capacity, the LE approach quantitative values. For hydrophilic model compounds, the LE tends to be lower because the model compound is in a dynamic equilibrium among the outshell of polypeptide brushes between the solution inside the dialysis bag and the solution outside the dialysis bag. The LE is only 27–29% when the number of guest molecules the same as that of the glutamic acid side chains is fed. The LE can be improved by lowering the amount of model compounds. For example, the value of LE

increases to approximately 50% when only half of the model compound is added. Because the synthesized copolypeptide brushes demonstrated encapsulation of both hydrophobic dyes and cationic hydrophilic guest molecules, which were based on the two discrete domains and the specific interactions, these unimolecular micelles may serve as hosts for the simultaneous encapsulation of versatile guest molecules. As shown by the absorption spectra in Fig. 7, hydrophobic pyrene and cationic CV or DOX can be encapsulated sequentially by the copolypeptide brushes, and the loading capacity of each dye was not fundamentally affected by the other. The synthesized copolypeptide brushes can potentially be used as nanocarriers for the versatile and simultaneous encapsulation of hydrophobic and cationic drugs. 3.3. In vitro release of encapsulated model compounds from PLLF-g-(PLF-b-PLG) The properties of the in vitro release of encapsulated model compounds were investigated. Hydrophobic model compounds cannot be released spontaneously because of their poor aqueous solubilities. Therefore, the in vivo release of hydrophobic drugs is expected to be primarily dependent on the degradation kinetics of the biodegradable polymer brushes. The pKa of the side chains of PLG is approximately 4.0–4.5. Therefore, a decrease in the pH of the solution will gradually convert the side chains of PLG from the deprotonated form to the protonated form. This change in charge may weaken the electrostatic interactions with the encapsulated guest molecules and accelerate their release rate. The cumulative release profiles of polymer-encapsulated CV under different pH conditions as well as the cumulative release profile of free model molecules are shown in Fig. 8. As seen in Fig. 8, the pH of the solution significantly affected the release kinetics of CV from P2. At a pH of 7.4, the entrapped CV demonstrated a sustained release, and the cumulative release was less than 13% after 3 days. When the pH was decreased to 5.0, at which point a small percentage of the PLG side chains are expected to be protonated, the cumulative release increased to 25% after 3 days. However, at a pH of 4.0, at which point most of the PLG side chains exist in the protonated form, an accelerated increase to approximately 60% after 3 days was observed in the cumulative release. When the pH was further decreased to 3.0, at which point the side chains of PLG are expected to exist predominantly in the protonated form, a quantitative release was observed after 15 h. Even at a pH as low as 3.0, the release rate of the encapsulated drug

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molecules was still slower than that of the free guest molecules. The release of CV and DOX from other polypeptide brushes has demonstrated similar behaviors in response to pH. 4. Conclusions We successfully prepared the water-soluble and amphiphilic core–shell cylindrical copolypeptide brushes PLLF-g-(PLF-b-PLG). The backbone of random polypeptides PLLF was generated by the ring-opening polymerization of Phe-NCA and ZLL-NCA mixtures, and the diblock side chains were attached using the “grafting from” technique via sequential ring-opening polymerization of Phe-NCAs and BLG-NCAs. The synthesized copolypeptide brushes resembled unimolecular micelles with a hydrophobic poly(lphenylalanine) core and a hydrophilic poly(l-glutamate) shell at physiological pH. The encapsulation study showed that these water soluble, biodegradable copolypeptide brushes could encapsulate hydrophobic compounds and hydrophilic cationic guest molecules with high loading capacity and could even entrap them simultaneously. Furthermore, a pH-responsive release of the encapsulated cationic model compounds was observed. Because of the diverse polarities and functionalities of amino acids, the structure of these core–shell cylindrical copolypeptide brushes can be easily extended and modified. The versatile and simultaneous encapsulation properties of cylindrical core–shell copolypeptide brushes could be used for the transport of medicinal compounds and for many other multifunctional applications. Acknowledgements This work was financially supported by the National Natural Science Foundation of China (50973058) and Team Project of Natural Science Foundation of Guangdong Province (9351503102000001). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.colsurfb.2012.02.012. References [1] K. Kataoka, A. Harada, Y. Nagasaki, Adv. Drug Deliv. Rev. 47 (2001) 113. [2] E. Soussan, S. Cassel, M. Blanzat, I. Rico-Lattes, Angew. Chem. Int. Ed. 48 (2009) 274. [3] C.C. Lee, J.A. MacKay, J.M.J. Fréchet, F.C. Szoka, Nat. Biotechnol. 23 (2005) 1517. [4] U. Gupta, H.B. Agashe, A. Asthana, N.K. Jain, Biomacromolecules 7 (2006) 649. [5] D. Astruc, E. Boisselier, C. Ornelas, Chem. Rev. 110 (2010) 1857. [6] M. Calderón, M.A. Quadir, S.K. Sharma, R. Haag, Adv. Mater. 22 (2010) 190. [7] H. Türk, A. Shukla, P.C.A. Rodrigues, H. Rehage, R. Haag, J. Chem. Eur. 13 (2007) 4187. [8] M.R. Radowski, A. Shukla, H. von Berlepsch, C. Böttcher, G. Pickaert, H. Rehage, R. Haag, Angew. Chem. Int. Ed. 46 (2007) 1265.

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