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“Bitter-Sweet” Polymeric Micelles Formed by Block Copolymers from Glucosamine and Cholic Acid Kun Zhang, Yongguang Jia, I-Huang Tsai, Satu Strandman, Li Ren, Liangzhi Hong, Guangzhao Zhang, Ying Guan, Yongjun Zhang, and Julian X. X. Zhu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01640 • Publication Date (Web): 17 Jan 2017 Downloaded from http://pubs.acs.org on January 18, 2017

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“Bitter-Sweet” Polymeric Micelles Formed by Block Copolymers from Glucosamine and Cholic Acid Kun Zhang,a Yong-Guang Jia,a I-Huang Tsai,a Satu Strandman,a Li Ren,b Liangzhi Hong,b Guangzhao Zhang,b Ying Guan,c Yongjun Zhang,c and X. X. Zhu*a a

Département de Chimie, Université de Montréal, C.P. 6128, Succ. Centre-ville,

Montreal, QC, H3C 3J7, Canada; b

School of Materials Science and Engineering, South China University of Technology,

Guangzhou, China; c

Institute of Polymer Chemistry, Nankai University, Tianjin, China.

Abstract Natural compounds glucosamine and cholic acid have been used to make acrylic monomers which are subsequently used to prepare amphiphilic block copolymers by reversible addition-fragmentation chain transfer (RAFT) polymerization. Despite the striking difference in polarity and solubility, three diblock copolymers consisting of glucosamine and cholic acid pendants with different hydrophilic and hydrophobic chain lengths have been synthesized without the use of protecting groups. They are shown to self-assemble into polymeric micelles with a “bitter” bile acid core and “sweet” sugar shell in aqueous solutions as evidenced by dynamic light scattering and transmission electron microscopy. The critical micelle concentration varies with the hydrophobic/hydrophilic ratio, ranging from 0.62 to 1.31 mg/L. Longer chains of polymers induced the formation of larger micelles in range of 50–70 nm. These micelles can solubilize hydrophobic compounds such as Nile Red in aqueous solutions. Their load capacity mainly depends upon the hydrophobic/hydrophilic ratio of the polymers, and may be also related to the length of the hydrophilic block. These polymeric micelles helped to have a tenfold increase in the aqueous solubility of

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paclitaxel and showed no cytotoxicity below the concentration of 500 mg/L. Such properties make these polymeric micelles interesting reservoirs for hydrophobic molecules and drugs for biomedical applications. Keywords: Polymeric micelles, block copolymers, glucosamine, cholic acid.

Introduction Polymeric micelles self-assembled by amphiphilic block copolymers have been studied for various applications, particularly for drug delivery systems (DDS), because of their nanoscale size and core-shell structure. The hydrophobic blocks form a core that can encapsulate molecules insoluble in water, while the hydrophilic blocks generate an outer shell to surround and protect the drug-loaded core. The small size of the micelles (usually 10 - 100 nm)1 helps to reduce the risk of embolism in capillaries, in contrast of larger carriers.2 Also, their size and the hydrophilic corona can help them to avoid body clearance by renal filtration or the reticuloendothelial system, thereby resulting in prolonged blood circulation time.3-5 First, polymeric micelles can be preferentially accumulated in tumor or inflammation sites by the enhanced permeability and retention effect, which is called passive targeting.4, 6 Additionally, active targeting can also be implemented through conjugation with site-specific ligands such as a carbohydrate7-9 or an antibody10, 11 on the surface of these aggregates. Polymeric micelles are regarded as a versatile and desirable drug delivery system in many cases. Another essential requirement for DDS is biocompatibility, which is defined as “the quality of not having toxic or injurious effects on biological systems”.12 Poly(ethylene glycol) (PEG) is by far the most popular polymer used as the biocompatible hydrophilic block.13, 14 However, PEG also has some shortcomings such as the lack of targeting ability without functionalization4 and the reduced targeting ability by shielding the functional end groups in the shell15 as well as allergic reactions of certain population.16 Glycopolymers may be the useful alternatives to PEG, and contain carbohydrate moieties as pendant groups. Numerous glycopolymers have been made


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and investigated as biomaterials.17-24 Apart from their high hydrophilicity and biocompatibility, a more important reason for the interest is that carbohydrate could recognize a variety of receptor proteins.25-28 However, most monosaccharides or disaccharides have weak interactions with proteins. This specific recognition ability of glycopolymer gives them an advantage over PEG as the shell of polymeric micelles through improving targeting effects. However, use of some non-biocompatible blocks to form the core of the micelles may limit the applications of these polymeric micelles as DDS. To improve the biocompatibility of materials, a common approach is to design and make polymers from the natural compounds or biocompatible constituents. Cholic acid, a natural bile acid existing in large quantities in the gallbladder of humans and animals, helps in the digestion of fats in the gastrointestinal tract.29 It is an ideal building block for biomimetic systems due to the rigidity of its steroid ring, amphiphilic property and the possibility of functionalization.30-35 Previously, cholic acid was incorporated into the hydrophilic polymers and self-assembled into the core of the polymeric micelles,36-39 serving as DDS. Meanwhile, glucosamine is a natural amino monosaccharide present in shellfish shells, animal bones, bone marrow and cell walls of fungi. In the human body, glucosamine plays an important role in building joint cartilage.40 It is thus attractive to prepare the polymeric micelles as DDS via the combination of hydrophilic glucosamine forming the “sweet” shell and hydrophobic cholic acid forming the “bitter” core. Herein, we have used the reversible additionfragmentation chain transfer (RAFT) polymerization method to prepare diblock copolymers of the acrylic derivatives of glucosamine and cholic acid without protecting groups. The self-assembly of the block copolymers and their potential applications as a reservoir for a hydrophobic molecule such as Nile Red or paclitaxel have been investigated. Experimental Section Materials. All chemicals, except those noted separately, are purchased from Aldrich (Oakville, ON, Canada) and used without further purification. These include cholic acid (98%), D-glucoamine hydrochloride (99%), 4-(dimethylamino)pyridine (DMAP) (99%). The 3 ACS Paragon Plus Environment

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solvents were purchased from Fisher Scientific (Whitby, ON, Canada). Tetrahydrofuran (THF), N,N-dimethylformamide (DMF) were dried by using a solvent purification system from Glass Contour. Water was purified with a Millipore Milli-Q system. Acrylic anhydride was synthesized from acrylic acid and acryloyl chloride. 2’-Hydroxyethyl 3α,7α,12αtrihydroxy-5β-cholan-24-ate (CAEG) was prepared from cholic acid and ethylene glycol with concentrated hydrochloric acid as the catalyst.41 The free radical initiator 2,2’-azobis(2methylpropionitrile) (AIBN, from Aldrich) was purified by recrystallization in methanol. 3Benzylsulfanylthiocarbonyl sulfanylpropionic acid (BSPA) was used as a chain transfer reagent (CTA) for RAFT polymerization and prepared according to a literature procedure from 3-mercaptopropionic acid, carbon disulfide and benzyl bromide.42 Characterization. H and

13

C NMR spectra were recorded on a Bruker AV400 NMR

spectrometer (1H, 400 MHz;

13

C, 100 MHz). The chemical compositions and the self-

assembly properties of the polymers were investigated by 1H NMR spectra with CDCl3, D2O and DMSO-d6 as solvents. Mass spectra were obtained on an Agilent LCMSD TOF spectrometer. Size exclusion chromatography (SEC) was performed on a Waters Millennium system equipped with a Waters 717 plus autosampler, a Waters 600 controller, and a Waters 2414 refractive index detector using two consecutive columns (Phenogel, 5 µm, 1000 Å, 300 mm × 7.8 mm, Styragel HR4, 5 µm, 300 mm × 7.8 mm). DMF containing 0.01 M LiBr was filtered by using 0.2 µm nylon Millipore filters for eluent. The flow rate of the eluent was 1 mL/min. Poly(methyl methacrylate) standards (2500 - 296000 g/mol) were used for calibration of molecular weight. Dynamic light scattering (DLS) measurements were performed on a Malvern Zetasizer NanoZS instrument equipped with a 633 nm 4 mW He-Ne Laser. The polymer concentration was 1.0 g/L in PBS (0.1 M, pH 7.4) and 0.1 g/L in water, and the samples were filtered through 0.2 µm Millipore filters to remove dust. Disposable sizing cuvettes were used and each sample was measured in triplicates. The samples for transmission electron microscopy (TEM) tests were prepared by placing a drop of PACAEG-b-PAGA solution (0.1 g/L, in water) on 300 mesh carbon-coated copper grids (Beijing Xinxing Braim, China). The solution was frozen in liquid nitrogen, followed by the removal of water through freeze-drying. Microscopy was done on a


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FEI Tecnai G2 F20 transmission electron microscope operating at a 200 kV accelerating voltage. The pyrene fluorescence excitation spectra were recorded with a fluorescence spectrophotometer from Photon Technology International (PTI) at room temperature. A given amount of pyrene (Fluka, 99%) stock solution in acetone was placed in empty vials. The solvent was evaporated, and 5mL aliquots of PACAEG-bPAGA aqueous solutions of various concentrations were then added to these vials. The final pyrene concentration was 5 × 10-7 M. The mixtures were equilibrated by shaking overnight at room temperature, and then let to stand for 4 h. The fluorescence excitation spectra of pyrene were recorded at the excitation wavelength range from 300 to 360 nm, and the detection wavelength was fixed at 390 nm. The slit widths of excitation and emission were both 2 nm Preparation of monomers. Acryloyl glucosamine (AGA) was synthesized according to a procedure previously reported43 with minor changes. Typically, to a solution of Dglucosamine hydrochloride (8.6 g, 40 mmol) in 2 M aqueous solution of K2CO3 (40 mL) with 0.08 g of NaNO2, acryloyl chloride (7.24 g, 80 mmol) was added dropwise at 0 °C under vigorous stirring. The mixture was reacted at 0 °C for 2 h, and then let warm to room temperature. After stirring for 24 h, the solution was poured into 200 mL of ethanol, and the precipitate was then filtered out. The product was recrystallized in a water/ethanol/ethyl ether mixture at 4 °C. Yield: 4.0 g, 43%; 1H NMR (400 MHz, D2O): δ (ppm) = 6.14 – 6.29 (2H, m, CH2=CHCO and CH2=CHCO, cis), 5.72 (1H, m, CH2=CHCO, trans), 5.15 (0.85H, d, β-H1), 4.70 (0.15H, m, α-H1), 3.42 – 3.91 (6H, m, H2 – H6). 13C NMR (100 MHz, D2O): δ (ppm) = 169.1, 168.8, 129.9, 129.6, 128.0, 127.9, 94.9, 90.8, 76.0, 73.9, 71.6, 70.7, 70.0, 69.8, 60.7, 60.6, 56.8, 54.2. Mass (ESI Pos): m/z calcd for C9H15NO6 [M + H]+ 234.097, [M + Na]+ 256.079, found [M + H]+ 234.098, [M + Na]+ 256.080. The

monomer

(ACAEG)

was

2’-acryloyloxyethyl

synthesized

methacryloyloxyethyl

according

3α,7α,12α-trihydroxy-5β-cholan-24-ate to

a

method

used

3α,7α,12α-trihydroxy-5β-cholan-24-ate

41

to

make

with

2’-

minor

modification. Typically, 2.0 g of CAEG (4.4 mmol) was dissolved in 40 mL of THF with 2.2 g of triethylamine (22.1 mmol) and 0.08 g of DMAP (0.65 mmol). The solution was cooled to 0 °C, and 0.84 g of acrylic anhydride (6.6 mmol, in 10 mL of

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THF) was then added dropwise with vigorous stirring in an ice bath. After reacting at room temperature for 4 h, the solvent was removed under reduced pressure. The solid was dissolved in DCM, and washed successively with water, 5% aqueous solution of Na2CO3, 1 M HCl, and brine, and then dried over anhydrous Na2SO4. The crude product was purified by column chromatography (silica gel, ethyl acetate as the eluent). Yield: 1.75 g, 78%; 1H NMR (400 MHz, CDCl3): δ (ppm) = 0.70 (s, 3H, 18CH3), 0.91 (s, 3H, 19-CH3), 1.00 (d, 3H, 21-CH3, J = 6.11), 3.48 (m, 1H, 3β-H), 3.87 (s, 1H, 7β-H), 4.00 (s, 1H, 12β-H), 4.36 (m, 4H, COOCH2CH2OCO), 5.88 – 5.91 (m, 1H, CH2=CHCO, trans), 6.13 – 6.20 (m, 1H, CH2=CHCO), 6.44 – 6.48 (m, 1H, CH2=CHCO, cis). 13C NMR (100 MHz, CDCl3): δ (ppm) = 174.0, 165.9, 131.4, 128.0, 73.0, 72.0, 68.4, 62.4, 62.0, 47.1, 46.5, 41.9, 41.5, 39.7, 39.6, 35.2, 35.1, 34.7, 34.6, 31.1, 30.8, 30.6, 28.3, 27.5, 26.6, 23.2, 22.6, 17.3, 12.6. Mass (ESI Pos): m/z calcd for C29H46O7 [M + NH4]+ 524.358, [M + Na]+ 529.314, found [M + NH4]+ 524.359, [M + Na]+ 529.315. Polymerization. Homopolymerization of ACAEG was performed using AIBN as the initiator and BSPA as the CTA. Typically, 1.0 g ACAEG (1.97 mmol), 32.3 mg of BSPA (0.12 mmol) and 1.9 mg of AIBN (0.012 mmol) ([ACAEG]:[BSPA]:[AIBN] = 50:3:0.3) were dissolved into 6 mL of DMF. The solution was transferred to a Schlenk tube and then thoroughly deoxygenated by three freeze-pump-thaw cycles. The tube was then sealed and placed in an oil bath at 70 °C. After 3.5 h, the solution was poured into cold ethyl ether. The precipitate was collected and dried in vacuum at room temperature to yield PACAEG12 as a yellowish solid. The molecular weights of PACAEG were determined by SEC and estimated by 1H NMR in DMSO-d6 from the ratio of NMR peak integrations (Figure 1A) of the methyl protons of cholic acid pendant (18-CH3, 3H, δ = 0.58 ppm) and of the phenyl protons of the RAFT agent (5H, δ = 7.13 – 7.34 ppm). PACAEG18 was prepared under the similar condition.


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Scheme 1. Synthesis of monomers and polymers.

The diblock copolymer was synthesized by a similar procedure using PACAEG as the macro-CTA. A certain amount of PACAEG and AIBN were dissolved in 6 mL of 7 ACS Paragon Plus Environment

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DMF and then mixed with 1.5 mL of aqueous solution of the AGA monomers. The solution was transferred to a Schlenk tube and purged with argon for 30 min, and then reacted at 70 °C for 7 h for PACAEG12-b-PAGA84 ([AGA]:[PACAEG]:[AIBN] = 100:1:0.3),

or

6

h

for

PACAEG12-b-PAGA110

and

PACAEG18-b-PAGA129

([AGA]:[PACAEG]:[AIBN] = 200:1:0.3). The solution was subsequently plunged into cold methanol to obtain the block copolymer as a white precipitate. After filtration, the residuals were dissolved in water and then yield the block copolymers through freezedrying. The molecular weights of the block copolymers were calculated from the ratio of NMR peak integrations of the methyl protons of cholic acid pendant (18-CH3, 3H, δ = 0.54 ppm) and that of the methine protons of saccharide pendant (β-H1, 0.85H, δ = 4.85 – 5.43 ppm in Figure 1B). Acetylation of the block copolymers PACAEG-b-PAGA. SEC initially failed to provide the molecular weights of the amphiphilic glycopolymers, probably caused by the binding of the glycopolymers onto the chromatographic column. Therefore the sugar residues were acetylated to reduce the polarity of the glycopolymers to make the sample suitable for SEC measurements.44 The copolymers PACAEG-b-PAGA cannot be directly dissolved in an organic solvent. To solve this problem, we first dissolved ca. 20 mg of PACAEG-b-PAGA in 1 mL of water, followed by the addition of 3 mL of DMF. The water was removed from the system by freeze-drying, and the DMF solution was purged with argon. Under argon, an excess of acetic anhydride (0.4 mL) and anhydrous pyridine (0.8 mL) were added. After reacting at room temperature for 24 h, the solution was poured into cold ethyl ether to obtain the acetylated product (19 mg, 60%). Solubilization of hydrophobic compounds. To prepare the aqueous solutions of PACAEGb-PAGA, appropriate amounts of the copolymers were directly dissolved in phosphate buffer solution (0.1 M, pH 7.4) or water, and then the mixture was equilibrated by shaking overnight at room temperature. A fixed amount of excess Nile Red (from Aldrich, technical grade) or a measured volume of the THF solution of excess paclitaxel (from Aladdin, China, 98%) was added in 3 mL of PACAEG-b-PAGA solution (1.0 g/L, THF/water = 2/1) and


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dispersed evenly by sonication for 2 min. After the evaporation of the solvents, a measured volume of PBS (0.1 M, pH 7.4) was added dropwise with vigorous stirring. After equilibration, the solution was filtered through 0.45 µm Millipore filters to remove the residual dye. The solubilization of Nile Red by PACAEG-b-PAGA was studied

by

UV-vis

spectrophotometry

(Agilent

Cary

5000

UV-vis-NIR

spectrophotometer), while the concentration of paclitaxel in the solution was determined by HPLC (Agilent 1260) equipped with a UV detector at 227 nm using an Agilent Poroshell 120 EC-C18 column (2.7 µm, 4.6 mm × 50 mm). The mixture of methanol, acetonitrile and water (25/45/30) was filtered through 0.2 µm nylon Millipore filters and used as the eluent with a flow rate of 0.6 mL/min. Cytotoxicity study. L929 mouse fibroblast cells were cultured in RPMI 1640 medium (from Gibco) supplemented with 5% (v/v) fetal bovine serum (FBS, from Hyclone) in a humidified atmosphere at 37 °C and 5% CO2. Cells were seeded in 96-well plates at a density of 1500 cells/well and incubated for 24 h. The culture medium was replaced by fresh medium with different concentrations of PACAEG18-b-PAGA129, and the cells were incubated further for 48 h. Fresh culture medium without polymers was used as control, and each sample was replicated in five wells. Ten microliters of Cell Counting Kit-8 (CCK-8, from Dojindo) solution were added to each well, and the cells were incubated further for 3.5 h in the same condition. The absorbance was measured at 450 nm with a reference wavelength of 600 nm by a BioTek synergy HT microplate reader. Cell viability (%) = (ODsample-ODblank)/(ODcontrol-ODblank) × 100%, where ODcontrol and ODsample were obtained in the absence or presence of polymer samples, respectively, and ODblank was obtained with RPMI 1640 medium and CCK-8 solution alone. Results and Discussion Synthesis and characterization of monomers and polymers. The hydrophilic glycomonomer AGA was synthesized by reacting glucosamine hydrochloride with acryloyl chloride in alkaline aqueous solution, while the hydrophobic monomer ACAEG was made from acrylic anhydride and ethylene glycol monocholate. RAFT polymerization was used to


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synthesize the amphiphilic block copolymers with cholic acid and glucosamine as pendants (Scheme 1). In order to improve the water solubility of the copolymers, the hydrophobic block was designed to be shorter than the hydrophilic one. Two PACAEG macro-CTA of various lengths (PACAEG12, Mn, NMR = 6300, PDI = 1.17; PACAEG18, Mn, NMR = 9400, PDI = 1.25) were obtained via the homopolymerization of ACAEG. The growth of the second block (PAGA) was controlled by using the PACAEG macro-CTA. To investigate the relationship between the properties and the structure of the block copolymers, we prepared three diblock copolymers PACAEG12-b-PAGA84, PACAEG12-b-PAGA110 and PACAEG18-bPAGA129. The former two have the same chain length of the hydrophobic block, and the latter two have close chain lengths of the hydrophilic block. The detailed information of these polymers is shown in Table 1. Table 1 Polymerization Conditions, Molecular Weights of the Polymers and Size of the Aggregates. Dh of aggregates (nm) Polymer

Ratio in feeda

MnNMR

PACAEG12

50:3:0.3

6 300

PACAEG18

100:3:0.3

9 400

CMC (g/L)

1.0 g/L in 0.1 M PBS (pH 7.4)

0.1 g/L in water

6 700 (1.17)

-

-

-

9 000 (1.25)

MnGPC (Đ)

-

-

-

b

PACAEG12-b-PAGA84

100:1:0.3

25 900

23 700 (1.28)

51.3 ± 0.7

51.8 ± 0.2

0.62

PACAEG12-b-PAGA110

200:1:0.3

32 000

28 800b (1.36)

60.8 ± 0.8

61.4 ± 0.6

1.31

39 500

b

70.8 ± 1.5

68.7 ± 0.8

1.31

PACAEG18-b-PAGA129 a

200:1:0.3

32 900 (1.46)

Ratio of [Monomer]:[CTA]:[Initiator] in the feed. b These values are calculated from the Mn

of the acetylated copolymers measured by SEC. It is to be noted that the copolymers in this study have been successfully made without the use of any protecting groups, even with a highly hydrophobic cholic acid block. There have been only rare examples of hydropobic polymers such as poly(5’-Omethacryloyl uridine),45 poly(N-isopropylacrylamide)46 and poly(butyl acrylate)47 directly connected to the unprotected glycopolymers. In most cases, the carbohydrate moieties need to be protected with nonpolar groups in order to improve their solubility in organic solvents. For example, Stenzel and co-workers48 prepared a series of


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amphiphilic diblock copolymers with acetylated galactosyl groups via nitroxidemediated polymerization. In another instance, the trimethylsilyl group (TMS) was used to protect the glycomonomer 2-methacrylamido glucopyranose.15 Obviously, the protection and deprotection of glycomonomer add more steps to the synthesis and reduce the overall yield of the final products. In the present work, no protecting groups were used even though cholic acid and glucosamine have highly disparate solubility properties. The polymerization of AGA was conducted in a DMF/water mixture to resolve the problem of dissolving the highly hydrophobic PACAEG macro-CTA and the hydrophilic monomer AGA in a common solvent. The solubility problem renders the determination of the molecular weights of PACAEG-b-PAGA by SEC difficult, if not impossible. Here we chose to acetylate the glucosamine moieties in the block copolymers to reduce their polarities so that an organic solvent may be used as the eluent. Comparing the 1H NMR spectra of PACAEG-b-PAGA and their acetylated products, the signals of glycosamine methylene and methine groups are shifted to low field region (from 3.0 – 5.4 to 3.5 – 6.3 ppm) after acetylation, which indicate a complete acetylation of all the hydroxyl groups of glycosamine residues. In contrast, the proton signal on position 3 (3.18 ppm) of cholate almost disappeared (Fig. 1A), whereas no chemical shift change was observed for the protons on positions 7 and 12 (3.61 and 3.78 ppm, respectively), indicating that only hydroxyl group on position 3 has been acetylated (Fig. 1D). Therefore, the Mn of PACAEG-b-PAGA may be deduced from those of their acetylated products measured by SEC and is listed in Table 1. The Mn values of the PACAEG-b-PAGA copolymers measured by SEC are also approximations since the calculation of the molecular weights was based on a different polymer, i.e., poly(methyl methacrylate), as calibration standards. The self-assembly properties of the block copolymers. These copolymers can selfassemble in aqueous solutions as shown by the fluorescence spectroscopy with pyrene as a hydrophobic probe. When pyrene enters a hydrophobic environment from water, the ratio of the intensities of the vibrational bands one to three (I1/I3) in the emission spectrum decreases,49 and the (0,0) band in the excitation spectra shifts from 333 to 336 nm.50 The plot of fluorescence intensity ratio (I3/I1) of pyrene versus the 11 ACS Paragon Plus Environment

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concentration of the block copolymers (Fig. 2) shows the abrupt increase of the I3/I1 value at a certain concentration for the PACAEG-b-PAGA copolymer, reflecting the transfer of pyrene into the hydrophobic environment upon the aggregation of the polymer. This concentration corresponds to the critical micelle concentration (CMC), defined as the intersection point of the two linear segments of the curve. The CMC values of these block copolymers in PBS (0.1 M, pH 7.4) were estimated to be ca. 0.62, 1.31 and 1.31 mg/L for PACAEG12-b-PAGA84, PACAEG12-b-PAGA110 and PACAEG18-b-PAGA129, respectively. These results indicate that all the block copolymers

form

aggregates

in

aqueous

solutions

and

the

high

hydrophobic/hydrophilic ratio of the block copolymers leads to a low CMC value. Other factors may also affect their CMC values. For example, for PACAEG12-bPAGA110 and PACAEG18-b-PAGA129, the proportional lengths of their hydrophilic blocks are closer than those of their hydrophobic blocks, they both have a similar CMC value within the experimental error even though their hydrophobic/hydrophilic ratios are different.

Fig. 1 The 1H NMR spectra of (A) PACAEG12 in DMSO-d6, (B) PACAEG12-b-PAGA84 in DMSO-d6 and (C) in D2O, (D) acetylated PACAEG12-b-PAGA84 in DMSO-d6. The numbers shown in the spectrum are in accordance to the molecular structure presented in Scheme 1.


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The formation of aggregates in water was further studied by the 1H NMR spectra of PACAEG12-b-PAGA84. The signals of the methyl groups (0.5 – 1.0 ppm in Fig. 1B) of the cholic acid moiety disappeared when the solvent was changed in from DMSO-d6 to D2O (Fig. 1C),51,

52

indicating that the core formed by the PACAEG block was

surrounded by the PAGA block in water.

Fig. 2 The intensity ratio (I3/I1) in the fluorescence emission spectra of pyrene as a function of concentration of the block copolymers (A) PACAEG12-b-PAGA84, (B) PACAEG12-bPAGA110, (C) PACAEG18-b-PAGA129. The concentration of pyrene was 5 × 10-7 M in PBS (0.1 M, pH 7.4). DLS was used to study the sizes of the aggregates of the PACAEG-b-PAGA block copolymers. The results showed that the block copolymers formed aggregates in aqueous solutions, and the sizes of the aggregates in PBS (0.1 M, pH 7.4) at the polymer concentration of 1.0 g/L were 51.3 ± 0.7, 60.8 ± 0.8, and 70.8 ± 1.5 nm for PACAEG12-b-PAGA84,

PACAEG12-b-PAGA110,

and

PACAEG18-b-PAGA129,

respectively (Fig. 3A and Table 1.). The size of the aggregates appears to increase with


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the length of the hydrophilic block of the copolymers, but not related to the hydrophobic/hydrophilic ratio of the block copolymers.

Fig. 3 The intensity average size distribution of the aggregates for the block copolymers (A) 1.0 g/L, in 0.1 M PBS (pH 7.4) and (B) 0.1 g/L, in water. (I) PACAEG12-b-PAGA84, (II) PACAEG12-b-PAGA110, and (III) PACAEG18-b-PAGA129. The morphology of the aggregates formed by PACAEG-b-PAGA was observed by TEM. In order to avoid the agglomeration of the aggregates during drying and minimize the interference with background from salts, the samples were prepared in pure water at a lower concentration (0.1 g/L). The particle sizes of these block copolymers in water obtained on DLS were similar as that in PBS (0.1 M, pH 7.4) at the concentration of 1.0 g/L (Fig. 3B and Table 1). The TEM images show that these block copolymers self-assembled into spherical micelles with the sizes of 10 ~ 20, 25 ~ 35, and 40 ~ 70 nm for PACAEG12-b-PAGA84, PACAEG12-b-PAGA110 and PACAEG18-b-PAGA129, respectively (Fig. 4). Here, the smaller particle sizes observed by TEM than the hydrodynamic diameter measured by DLS (Table 1.) may be due to the collapse of aggregates during drying and the greater contribution to light-weighted size distributions from larger scatterers.53


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Fig. 4 TEM images of the aggregates formed by block copolymers (0.1 g/L, in water): (A) PACAEG12-b-PAGA84, (B) PACAEG12-b-PAGA110 and (C) PACAEG18-b-PAGA129. Load capacity. The micelles formed by these copolymers in aqueous solutions are less than 100 nm in diameters, and may be the excellent carriers to entrap some hydrophobic molecules as well as drugs in their core. Their load capacities were measured by the solubilization of a model hydrophobic Nile Red, which is known to be practically insoluble in water (solubility in the order of 80 µg/L. Excess Nile Red was mixed with the polymers in aqueous solution to yield purple solutions (Fig. 5). The purple color normally indicates that Nile Red is close to a polar environment, as it shows a yellowish color in non-polar organic media. It is possible that the hydrophobic core formed by the cholic acid residues has a certain polarity. The UVvis absorption results of the filtrates show that PACAEG-b-PAGA can incorporate Nile Red into the micelles. Nile Red load capacity seems to depend on the hydrophobic/hydrophilic ratio of the block copolymers, and PACAEG12-b-PAGA84 shows the highest load capacity. It may be also related to the length of the hydrophilic PAGA block. For example, PACAEG12b-PAGA110 and PACAEG18-b-PAGA129 with a different hydrophobic/hydrophilic ratio but similar hydrophilic block lengths showed similar loading capacities for Nile Red.

Fig. 5 The solubility and the UV-vis spectra of Nile Red in PBS (0.1 M, pH 7.4) with 1.0 g/L (A) PACAEG12-b-PAGA84, (B) PACAEG12-b-PAGA110, and (C) PACAEG18-b-PAGA129 and (D) without the polymer. PACAEG12-b-PAGA84 was chosen as an example and used to investigate the load capacity for real drugs due to its highest Nile Red load capacity. Paclitaxel, one of the most effective antitumor drugs used in the treatment of a variety of cancers in the


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market today, is known to have a poor water solubility (~ 0.25 mg/L) and was tested as a model drug. The HPLC results indicated that the concentration of paclitaxel in aqueous solution was increased tenfold after loading into the micelles self-assembled by PACAEG12-b-PAGA84 at a concentration of 1.0 g/L, and the drug content of the micelles is 0.25 wt% (Figure S2). Cytotoxicity studies. A CCK-18 assay was performed to evaluate the cytotoxicity of these block copolymers against L929 cells. Here, PACAEG18-b-PAGA129 was selected as an example. The copolymer showed no obvious cytotoxicity (> 80% cell viability) below a concentration of 0.5 g/L for L929 cells (Fig. 6). At higher concentrations, the copolymer could not be completely dissolved in the culture medium. The insoluble part of the copolymer may cause a negative effect on cell growth. In addition, the thiocarbonylthio moiety of the copolymers (the RAFT end group) may be a source of potential toxicity,54 but may be easily removed in case of need.55

Fig. 6 Cytotoxicity assay against L929 cells (1000 cells/well) after incubation with PACAEG18-b-PAGA129 at 37 °C and 5% CO2 for 48 h.

Conclusions RAFT polymerization was successfully used in the preparation of the block copolymers PACAEG-b-PAGA, in which the hydrophilic and hydrophobic blocks are both made of derivatives of natural compounds, glucosamine and cholic acid, respectively. The striking difference in solubility of cholic acid and glucosamine


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derivatives in both water and organic solvents poses a serious challenge in the synthesis and the subsequent characterization of such copolymers. The synthetic strategy used in this work eliminated the need of protecting groups for the glycosamine units, thereby enlarging the choice of monomers for various applications. The block copolymers self-assemble into “bitter-sweet” polymeric micelles with diameters of 50-70 nm in aqueous solutions. These aggregates are stable in aqueous solutions with low CMC and high load capacity for Nile Red and paclitaxel, which may endow their potential use as a drug delivery system. The self-assembly of the copolymers depends not only on the hydrophobic/hydrophilic ratio of the copolymers, but also the lengths of both blocks. These polymeric micelles may be further optimized as a drug delivery vehicle by changing the hydrophobic or hydrophilic chain length to improve the drug content of the micelles. More importantly, these copolymers also exhibited a lower cytotoxicity, but issues regarding their biocompatibility, targeting effect and the potential improvement the drug efficiency are subjects of further studies.

Acknowledgments The financial support from NSERC of Canada, FQRNT of Quebec, and the National Natural Science Foundation of China (grant # 21228401, 50803018 and 51273072) is gratefully acknowledged. The authors of Université de Montréal are members of CSACS funded by FQRNT and GRSTB funded by FRSQ.

Supporting Information Mass spectra of the monomers. 1H NMR spectra and SEC traces of the polymers. HPLC chromatograms of paclitaxel before and after being loaded into a polymer.

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Table of Contents (TOC) Amphiphilic diblock copolymers containing natural compounds cholic acid and glucosamine as pendant groups are prepared by RAFT polymerization. These copolymers could self-assemble into “bitter-sweet” polymeric micelles in aqueous solutions.


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