Thermo- and pH-Responsive Copolymers Bearing Cholic Acid and

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Thermo- and pH-responsive copolymers bearing cholic acid and oligo(ethylene glycol) pendants: self-assembly and pH-controlled release Yongguang Jia, and X. X. Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b06909 • Publication Date (Web): 19 Oct 2015 Downloaded from http://pubs.acs.org on October 20, 2015

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ACS Applied Materials & Interfaces

Thermo- and pH-responsive copolymers bearing cholic acid and oligo(ethylene glycol) pendants: self-assembly and pH-controlled release Yong-Guang Jia, X. X. Zhu* Département de Chimie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, QC, H3C 3J7, Canada

ABSTRACT: A family of block and random copolymers of norbornene derivatives bearing cholic acid and oligo(ethylene glycol) pendants were prepared in the presence of Grubbs’ catalyst. The phase transition temperature of the copolymers in aqueous solutions may be tuned by the variation of comonomer ratios and pH values. Both types of copolymers formed micellar nanostructures with a hydrophilic poly(ethylene glycol) shell and a hydrophobic core containing cholic acid residues. The micellar size increased gradually with increasing pH due to the deprotonation of the carboxylic acid groups. These micelles were capable of encapsulating hydrophobic compounds such as Nile Red (NR). A higher hydrophobicity/hydrophilicity ratio in both copolymers resulted in a higher loading capacity for NR. With similar molecular weights and monomer compositions, the block copolymers showed a higher loading capacity for NR than the random copolymers. The NR-loaded micelles exhibited a pH-triggered release behavior. At pH 7.4 within 96 h, the micelles formed by both kinds of copolymers released 56% and 97% NR, respectively. Therefore, these micelles may have promise for use as therapeutic nano-carriers in drug delivery systems.

KEYWORDS: Thermo- and pH-responsive; self-assembly; pH-controlled release; cholic acid and oligo(ethylene glycol).

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Introduction Self-assembly of amphiphilic copolymers yields aggregates with potential applications in biomedicine, biotechnology and catalysis.1-4 Polymeric micelles seem to be versatile and useful for drug delivery systems.5-7 The essential requirement for drug delivery system is biocompatibility, defined as “the quality of not having toxic or injurious effects on biological systems”.8 Poly(ethylene glycol) (PEG) is a popular choice since it is water-soluble, nontoxic, non-immunogenic, non-antigenic nature and has FDA approval.9 In polymeric aggregates, PEG can form a protective layer of water through hydration so that opsonin binding can be inhibited.10-14 Bile acids are a group of natural compounds that exist in most animals, a metabolite of cholesterol in mammals.15,16 Their structural rigidity, facial amphiphilicity, and the ease of functionalization of the hydroxyl and carboxylic groups give rise to a diverse range of bile acid-based materials,17-23 some of which may serve as drug delivery systems.24,25 These materials can maintain some properties of bile acids, including biocompatibility, high stability of the steroid skeleton, amphiphilicity, chirality, and self-assembling capacity.26 Therefore, bile acids in combination with PEG would be useful and attractive for drug delivery systems and biomaterials. Some PEGylated bile acid derivatives were used as anticancer drug delivery systems. These polymers formed nano-carriers with excellent stability and efficient drug loading in vitro and in vivo.25 In our previous study, the empty micelles formed by polynorbornenes consisting of oligo(ethylene glycol) and cholic acid (the most abundant bile acid in humans and many other species) pendants showed the cell viability of ∼100%, supporting that micelles possess good biocompatibility. Paclitaxel (PTX)-loaded polymer micelles showed apparent antitumor efficacy toward the ovarian cancer cells and the half maximal inhibitory concentrations (IC50) were particularly low, at 27.4 and 40.2 ng/mL, respectively.27 However, the carboxylic acid group of cholic acid was modified to couple with the polymer chain, resulting in the loss of its pH-responsiveness. Free carboxylic acid groups may help to trigger the release of drugs upon variation of the pH values in the body so that a controlled release of drugs may be achieved. In this work, we chose to modify the hydroxyl group of cholic acid on position 3 in the synthesis of the monomer and synthesized both block and random copolymers via ring-opening metathesis polymerization (ROMP) through a protection-deprotection scheme (Scheme 1). The 2


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self-assembly and responsiveness of such copolymers toward temperature and pH have been studied. The micelles formed by these copolymers were loaded with a model hydrophobic compound, Nile Red (NR). The controlled release of NR upon pH variation has been studied in an effort to demonstrate the application of such systems, especially as a potential drug delivery device.

Experimental Section Materials. All reagents were purchased from Aldrich and were used without further purification unless otherwise stated. Water and dichloromethane (DCM) were treated with a Milli-Q system and a Glass Contour system, respectively. The Grubbs’ catalyst (third generation) was prepared as described previously.28 Endo-exo-bicyclo-hept-5-2,3-dicarboxylic acid bis(tetraethyleneglycol monomethyl ether) ester (NPEG) was prepared according to a previous report.27,29 Characterization. The experimental conditions of the characterization methods, including 1H and

13

C spectroscopy, molecular weights and dispersity measurements, dynamic light scattering

(DLS), cloud point (CP) measurements (with an Agilent Cary 5000 UV-vis-NIR spectrophotometer), fluorescence spectroscopy and transmission electron microscopy (TEM) can be found in a previous report.27 Synthesis of cholic acid-based norbornene monomer (NMC). The general synthetic procedure was shown in Scheme 1A. 3-Amino-methyl cholate (600 mg, 1.43 mmol), prepared as described previously,30 5-norbornene-2-carboxylic acid (197 mg, 1.43 mmol) and HOBt (28 mg, 0.207 mmol) were placed in a round-bottom flask equipped with a stir bar. 4 mL dry DMF was added to the flask under nitrogen atmosphere. A clear solution was obtained and degassed with nitrogen for 10 min at 0 °C. EDC (353 mg, 1.78 mmol) was then slowly added into solution above until it was dissolved completely. After 1 h, the mixture was allowed to warm up to room temperature and stirred for 24 h. The reaction was quenched by pouring the mixture into a beaker filled with crushed ice. The crude product was collected by air suction and purified by column chromatography over silica gel using ethyl acetate as the eluent to obtain 517 mg of white solid noted as NMC (Scheme 1A, 67% yield). 1H NMR (CDCl3, 400 MHz): δ (ppm) = 6.21 (dd, 1H), 5.94 (m, 1H), 5.57 (dd, 1H), 4.05 (m, 1H), 3.93 (m, 1H), 3.82 (m, 1H), 3.62 (s, 3H), 3.09 (s, 1H), 2.89 (s, 1H), 2.84 (m, 1H), 2.48 (m, 1H), 0.94 (d, 3H), 0.91 (d, 3H), 0.65 (d, 3H). 13C NMR (100 3



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MHz, CDCl3): δ (ppm) = 174.75, 173.48, 137.88, 137.75, 132.49, 132.24, 77.35, 77.23, 77.03, 76.71, 72.90, 68.25, 51.53, 50.17, 50.14, 47.18, 46.54, 46.43, 46.41, 45.17, 45.08, 45.05, 42.83, 42.79, 41.95, 39.48, 37.80, 35.19, 35.17, 34.36, 33.59, 31.37, 31.04, 30.87, 30.45, 30.22, 28.59, 27.44, 26.08, 24.63, 23.43, 23.19, 17.32, 12.56. HRMS (ESI Pos): found for C33H52NO5 [M + H]+: 542.3853 m/z, calculated 542.3840 m/z. Polymerization. A representative block copolymer PNPEG30-b-PNMC7 (B4:1) was obtained by the polymerization of NPEG (290 mg, 0.51 mmol) and NMC (69.7 mg, 0.13 mmol) in the presence of the third generation Grubbs’ catalyst (15.0 mg, 1.7 × 10-2 mmol) in DCM. The polymerization conditions were the same as reported previously.27 Selective hydrolysis. All the polymers were hydrolyzed to restore the carboxylic acid group of the cholic acid residues under the same conditions.31 For example, the block copolymer B4:1 (150 mg) was dissolved in 4 ml methanol followed by the addition of aqueous solution of lithium hydroxide (0.1 N, 1.5 equivalents to the amount of NMC units). The mixture was stirred for 12 h at 22 °C. A 0.1 N hydrochloric acid solution was added dropwise until complete acidification (pH = 2). The copolymer was obtained by removing methanol under reduced pressure followed by dialysis (COMW = 3500) to separate the copolymer from salts and solvents for 3 days. The aqueous solution was freeze-dried and the resulting copolymer is noted as PNPEG30-b-PNCA7 (B4:1-H). Loading and release of Nile Red (NR). To 4 mL of B4:1-H solution (1.0 g/L, equal volume of THF and water), 1.0 mg NR was added under magnetic stirring. The mixed dispersion was stirred for 10 min, sonicated for another 15 min at 22 °C and then stirred overnight at 22 °C. THF was then removed by rotary evaporation, and 2 mL water was added dropwise under vigorous stirring. The dispersion was filtered through a Millipore 0.45 µm PTFE membrane to remove the unloaded NR precipitate. To determine the loading capacity, the aqueous solution of the copolymer with NR was freeze-dried and 4 mL methanol was added. The concentration of NR in the solution was determined by UV-vis spectrophotometer. 2 mL NR-loaded micelles (2.0 g/L) was first incubated at 22 °C for 20 min and the solution was adjusted to pH 4.0 by adding 2 mL of 100 mM pH 4.0 acetate buffer. The absorbance at 568 nm was recorded at time 0 and also measured at certain time points. Before each UV-vis measurement, the released NR precipitates were removed by filtering through a 0.45 µm PTFE 4


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membrane. Percent cumulative release of NR was calculated according to the formula (A0−A)/A0⁎100%, where A0 and A denote the absorbance at time 0 and a specific incubation time point, respectively. The data of NR release at other pH values were obtained in a similar way.

Results and discussion Synthesis of copolymers. To couple norbornene through an amide bond on position 3 of cholic acid, the carboxylic acid group of cholic acid was first protected and then restored (Scheme 1) for pH-responsiveness of the polymer. Two block copolymers, B4:1 and B6:1 in which the ratios denote the molar ratios of the norbornene comonomers bearing PEG and cholic acid pendants in the blocks, were synthesized via ROMP of NMC with a hydrophilic comonomer NPEG (Scheme 1B). Two random copolymers, R4:1 and R6:1 were also prepared under the same conditions. ROMP in the presence of the third generation Grubbs’ catalyst shows the “living” polymerization characteristics.32 The molecular weights of the copolymers prepared are similar (26-30 kDa) and their molecular weight distributions range from 1.08 to 1.24. The composition and characteristics of the copolymers made are listed in Table 1.

Table 1. Composition and characteristics of block and random copolymers. Copolymersa

DId

Ratio in

Yield

Mn,GPC

polymersb

(%)c

(g/mol)d

PNPEG30-b-PNMC5 (B6:1)

6.7 : 1

95.0

26 100

1.08

PNPEG30-b-PNMC7 (B4:1)

4.7 : 1

94.7

28 400

1.17

P(NPEG-r-NMC)6:1 (R6:1)

6.5 : 1

90.6

25 800

1.24

P(NPEG-r-NMC)4:1 (R4:1)

4.4 : 1

93.2

29 600

1.24

a

Molar ratio of NPEG : NMC in the feed. bMolar ratio of NPEG : NMC estimated from NMR. cYield calculated

from the insoluble portion in cold hexane. d Molecular weight and dispersity index (DI) determined by SEC.

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Scheme 1. Synthesis of (A) the cholic acid-based norbornene monomer (NMC) and (B) the block copolymers PNPEG-b-PNCA. A

HCl Methanol

OH O

OH OH

O O

OH OH

95%

HO O

O S

O 74%

18

5-Norbornene-2carboxylic acid EDC/HOBt

21

19

O O N H

O

OH OH

67%

PPh3 THF/H2O

O O

OH OH

H 2N

O

70%

O

OH OH

N3

b

b

O

O

O

n

m

m O

NaN3 DMF

NMC

B O

O

OH

85%

OH

OH

O

4-Toluenesulfonyl chloride Pyridine

O

O

O O O

O O

n

m O

O

O O O

HN

O HN

3rd generation O

O O

O

Grubbs' catalyst

O

O

O O

NMC

O

O

LiOH

19

HO HO

O O

O

O 19

O O

HO HO

18

O

O

O

O

O

O

O O

NPEG

PNPEG

O

O

O O

21

18

O

O O

O

O O

21

O OH

PNPEG-b-PNMC

PNPEG-b-PNCA

(B4:1 and B6:1)

(B4:1-H and B6:1-H)

Figure 1． 1H NMR spectrum of B4:1 in CDCl3 and the assignments of related peaks. 6


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The 1H NMR spectrum of B4:1 (Figure 1) shows the disappearance of the peak at 6.3-5.8 ppm (double bonds of the two monomers in Figure S1), suggesting that the comonomers were completely consumed. The molar ratio of NMC to NPEG can be calculated from the integration ratio of peaks “c” to peak “18”. In all cases, the content of NPEG units in the copolymers is slightly higher than that in the feed (Table 1). Meanwhile, the molar ratios of NMC to NPEG in the random copolymers are somewhat higher than in their block copolymer counterparts. These may be caused by the steric hindrance of NMC which lowers its reactivity during polymerization, especially in its homopolymerization. The carboxylic acid groups of cholic acid residues were restored via the selective hydrolysis of the methyl ester protective groups (Scheme 1B), as evidenced by the 1H NMR spectrum in Figure S2. Thermo- and pH-responsive properties. The aqueous solutions of the block and random copolymers after the selective hydrolysis (B4:1-H and R4:1-H) show sharp CPs at 58.3 and 51.1

°C, respectively at pH 4.0 (Figure 2 and Table 2). These values are comparable to those of their precursors before hydrolysis (B4:1 and R4:1) at 61.7 and 56.6 °C, respectively (Figure S4). It is well-known that the thermo-sensitivity of the polymers in aqueous solutions can be adjusted by changing of the hydrophilicity-hydrophobicity balance.33,34 The slightly higher molar ratio of NPEG to NCA in B4:1-H (Table 1) results in a higher hydrophilicity and also a higher CP. The random distribution of the carboxylic acid and/or amide groups on R4:1-H may also facilitate the intra/intermolecular hydrogen bonding, resulting in a lower CP. For each type of the copolymers, the CPs show an increasing trend with increasing NPEG contents (Figure S5), consistent with our pervious results.27 The same polymer displays different phase transition temperatures as a function of the degree of deprotonation at different pH values. The CP of B4:1-H (Figure 2A and Table 2) at pH 4.0 is observed at 58.3 °C and shifts to 58.6 and 64.1 °C at the pH 5.0 and 6.0, respectively. The pKa value of the micellar cholic acid is about 5.3.35 Below the pKa, pH variation (from pH 4.0 to 5.0) does not change the degree of deprotonation to any significant extent and the CP manifests only a small change of 0.3 °C. In contrast, above the pKa an increase in pH can cause increases in CP. Therefore, the CP of B4:1-H increased first by 5.5 and then by 5.3 °C, respectively, when the pH increased from 5.0 to 6.0 and then to 7.4. Similar behavior is also observed for the random copolymer R4:1-H (Figure 2B and Table 2). 7



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Figure 2. The transmittance variation of aqueous solutions at various pH values of selected copolymers (1.0 g/L) in as a function of temperature observed at 400 nm. (A) block copolymer B4:1-H; (B) random copolymer R4:1-H. Note that simply increasing the environmental pH results in the increase of CP.

Table 2. CPs at various pHs, micellar size and CMC of the copolymers B4:1-H and R4:1-H in water (1.0 g/L). CPs at various pHs (°C)a

Copolymers

Micellar size

CMC at pH b

at pH 4.0 (nm)

4.0 (mg/L)c

4.0

5.0

6.0

7.4

B4:1-H

58.3

58.6

64.1

69.4

61

4.5

R4:1-H

51.1

52.1

58.4

66.3

18

6.3

a

Determined by UV-vis-NIR spectrophotometer. bDetermined by DLS. cDetermined by fluorescence

spectrophotometer.

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Aggregation of copolymers. Amphiphilic copolymers are capable of forming micelles in water.36 A pyrene probe was used to estimate the CMC values of both B4:1-H and R4:1-H (Table 2 and Figure S6).37 The ratio of excitation intensities at 336 and 333 nm (I336/I333) can be related to the environment of pyrene.38,39 The peak at 333 nm on the excitation spectra of pyrene showed a red shift as increasing concentrations of copolymer. B4:1-H and R4:1-H showed similar CMC values, 4.5 and 6.3 mg/L at pH 4, respectively. Figures 3A and 3B show unimodal distributions and the Dh of the aggregates formed by B4:1-H and R4:1-H. The micelles formed by the former, i.e., block copolymer (BC)-based micelles, seem to be larger in size (61 nm) and more uniform than those formed by the latter, i.e., random copolymer (RC)-based micelles (18 nm). Both showed an increase in size, in comparison to their precursors before hydrolysis (46 and 14 nm, respectively, Figure S7). Furthermore, the TEM images (Figures 4A and 4B) confirm the formation of micelles for both types of copolymers. The more regular morphology of the micelles formed by B4:1-H on TEM images also agrees well with its narrower size distribution shown by the DLS measurements. The BC-based micelles may be stabilized by the relatively thick hydrophilic shell in aqueous solutions (Scheme 2) and with a narrower size distribution of the micelles as shown by both TEM and DLS measurements. The diameter obtained with DLS is slightly larger than that obtained with TEM, likely caused by a size shrinkage during the sample preparation for TEM.40 For the RC-based micelles, the PEG shell may not be thick enough to stabilize the individual micelles, leading to the formation of micellar clusters. This results in a broader size distribution (DLS measurements) and morphological variation of the aggregates (TEM images). This is consistent with the dynamic nature of the RC-based micelles observed in our previous study, where the aggregation number of the BC-based micelles remained constant with increasing temperature, whereas that of the RC-based micelles increased under the same conditions.27 Figure 3C shows that both hydrodynamic diameter and size distribution of the BC-based micelles remains unchanged as the pH increases from 4.0 to 5.0. When the pH further increases from 5.0 to 6.0 and to 7.4, the hydrodynamic diameter shows a small and gradual increase from 61 to 65 and to 70 nm, respectively. Meanwhile, the size distribution of these micelles is also broadened significantly at pH 7.4 (an increase from 0.10 at pH 6.0 to 0.28 at pH 7.4), indicating micellar clusters, rather than simple core-shell structured micelles, begin to form. The 9



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deprotonation of carboxylic acid groups results in charge repulsion. Consequently, the core of micelles may become swollen due to the ionized carboxylic acid groups on the cholic acid residues at pH 7.4.35 The micellar clusters or large aggregates may form when the core is not stabilized as at lower pH by the shell. This dependence on pH may be explored conveniently for the controlled release of the hydrophobic guests from the micelles.

Figure 3．Intensity-average size distribution of the aggregates formed by copolymers (1.0 g/L) (A) B4:1-H, (B) R4:1-H before and after loading Nile Red (NR) at pH 4.0 and (C) B4:1-H at various pHs.

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Figure 4．Representative TEM images of the aggregates formed by copolymers (A) B4:1-H and (B) R4:1-H (1.0 g/L, 25 °C and pH 4.0) before and after (C and D) loading Nile Red, respectively.

Scheme 2. Representation of the amphiphilic block and random copolymer assembly responding toward pH stimuli.

Loading and controlled release of Nile Red. Nile Red (NR) is known to be poorly soluble in aqueous media (water solubility in the order of 80 µg/L),41 even though it has some polar moiety 11



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in its structure (Scheme 2). The purple color of the solutions (Figure S8) indicates the dissolution of NR into the micelles. Dh of the NR-loaded BC micelles shows an increase from 61 to 63 nm but a large increase in size distribution (from 0.10 to 0.30) (Figure 3A). The RC aggregates are and exhibit a multimodal distribution (Figure 3B). Table 3. Loading capacities and cumulative release of Nile Red from the aggregates formed by block and random copolymers. Copolymers

B4:1-H

R4:1-H

Cumulative releaseb

Loading capacity (mg/L)a

pH

21.9

4.0

7.88

36

7.4

12.3

56

4.0

6.23

75

7.4

8.05

97

8.3

(mg/L)

(%)

a

Determined by UV-vis-NIR spectrophotometer. bAt the release point of 96 h.

The TEM images also confirm the size increase of micelles formed by both B4:1-H and R4:1-H upon loading with NR, both up to ca. 70 nm (Figures 4C and 4D). The shell structures of the micelles formed by B4:1-H may be clearly observed in the insert of Figure 4C. The micelles formed by B4:1-H have a thicker shell than those formed by R4:1-H. The loading capacity of the micelles formed by B4:1-H (pH 4.0) is also larger (21.9 vs 8.3 mg/L for R4:1-H in Table 3). The more stable micelles with a thicker shell formed by B4:1-H seem to help in the solubilization of NR (Scheme 2 and Figure 4C insert). This result is different from our previous study for other polymers, where 79.1 and 88.8% of paclitaxel were loaded by the BC- and RC-based micelles, respectively.27 This difference may be attributed to the electrostatic interaction between the amino group of NR (Scheme 2) and the carboxylic acid group of the cholic acid monomers as well as the hydrophobic interactions between NR and micellar cores.42 The electrostatic interaction in the core of the BC micelles may be enhanced by the high density of carboxylic acid groups in the polymer. In general, B6:1-H and R6:1-H also show lower loading capacities

than

B4:1-H and

R4:1-H,

respectively,

probably

due

to

their

lower

hydrophobicity/hydrophilicity ratios.43

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Figure 5. Nile Red release profiles from the micelles of B4:1-H (squares) and R4:1-H (circles) in aqueous solutions at pHs of 4.0 (solid symbols) and 7.4 (open symbols) and 25 °C ([polymer] = 1.0 g/L).

The release of encapsulated molecules can be triggered by adjusting the pHs for the pH-responsive polymers.44,45 Figure 5 compares the release profiles of NR-loaded micelles formed by B4:1-H and R4:1-H at pHs of 4.0 and 7.4. In both cases, the cumulative release of NR at pH 7.4 is faster than at pH 4.0 due to the deprotonation at a higher pH value. The micelles formed by R4:1-H have a faster cumulative release than that formed by B4:1-H under the same conditions (Table 3). The thicker hydrophilic shell formed by B4:1-H may stabilize the micelles to slow down the diffusion-controlled release. It should be noted that the micelles formed by B4:1-H have a higher loading capacity than R4:1-H, and thus the cumulative amount released (12.3 mg/L) is still higher than that for R4:1-H (8.05 mg/L), even though the percentage released is lower (56 vs 96%). Conclusions We have prepared norbornene-based copolymers containing biocompatible PEG and cholic acid pendants with restored carboxylic acid groups. These copolymers are all thermo- and pH-responsive and their phase transition temperatures may be tuned by varying the monomer ratio and the pH of the media. The micelles self-assembled by the block and random copolymers all show pH-dependent swelling property due to the deprotonation of the carboxylic acid groups, even disintegration at pH 7.4 in water. The block copolymer-based micelles showed a higher NR loading capacity and a slower cumulative release rate in percentage than those formed by the 13



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random copolymer. The release of hydrophobic NR from the micelles may be triggered by pH changes. Therefore, such micelles may be useful in controlled drug delivery and biotherapeutics. Issues regarding the cytotoxicity, targeting effect and improvement of drug efficiency are subject of further studies. Supporting Information 1H NMR spectra of the monomers NMC and NPEG and of B4:1-H; SEC curves of homopolymer PNPEG30 and block copolymer B4:1; comparison of the intensity-average size distribution (Dh) and transmittance of B4:1 and R4:1 as a function of temperature; variation of the transmittance of the aqueous solution of B6:1-H and R6:1-H as a function of temperature; the intensity ratio in the fluorescence excitation spectra of pyrene as a function of concentration of copolymers; intensity-average size distribution (Dh) of the micelles formed by B4:1 and R4:1; UV/vis absorbance spectra of NR stabilized by micelles at pH 4.0. Corresponding Author *E-mail: [email protected]. Acknowledgments We gratefully acknowledge the financial support from NSERC of Canada, FQRNT of Quebec and the Canada Research Chairs program. The authors are members of GRSTB funded by FRSQ and CSACS funded by FQRNT. We wish to thank Dr. M. Veerapandian and Mr. Pierre Ménard-Tremblay for their help with the TEM and SEC measurements. References: (1) Mai, Y.; Eisenberg, A. Selective Localization of Preformed Nanoparticles in Morphologically Controllable Block Copolymer Aggregates in Solution. Acc. Chem. Res. 2012, 45, 1657-1666. (2) Tanner, P.; Baumann, P.; Enea, R.; Onaca, O.; Palivan, C.; Meier, W. Polymeric Vesicles: From Drug Carriers to Nanoreactors and Artificial Organelles. Acc. Chem. Res. 2011, 44, 1039-1049 (3) Kita-Tokarczyk, K.; Junginger, M.; Belegrinou, S.; Taubert, A. Amphiphilic Polymers at Interfaces. Adv. Polym. Sci. 2010, 242, 151-201. (4) Gaucher, G.; Marchessault, R. H.; Leroux, J. C. Polyester-Based Micelles And Nanoparticles for the Parenteral Delivery of Taxanes. J. Control. Release 2010, 143, 2-12. (5) Vonarbourg, A.; Passirani, C.; Saulnier, P.; Benoit, J.-P. Parameters Influencing the 14


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Thermo- and pH-responsive copolymers bearing cholic acid and oligo(ethylene glycol) pendants: self-assembly and pH-controlled release Yong-Guang Jia, X. X. Zhu*

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Thermo- and pH-Responsive Copolymers Bearing Cholic Acid and

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