Polyion Complex Micelles Possessing Thermoresponsive Coronas

Polyion Complex Micelles Possessing Thermoresponsive Coronas

(Parte 1 de 4)

Polyion Complex Micelles Possessing Thermoresponsive Coronas and Their Covalent Core Stabilization via “Click” Chemistry

Jingyan Zhang,†,‡ Yueming Zhou,‡ Zhiyuan Zhu,‡ Zhishen Ge,‡ and Shiyong Liu*,‡

School of Materials and Chemical Engineering, Anhui UniVersity of Architecture, Hefei, Anhui 230022, China, and Joint Laboratory of Polymer Thin Films and Solution, Department of Polymer Science and Engineering, Hefei National Laboratory for Physical Sciences at the Microscale, UniVersity of Science and Technology of China, Hefei, Anhui 230026, China

ReceiVed October 2, 2007; ReVised Manuscript ReceiVed December 10, 2007

ABSTRACT: Two oppositely charged graft ionomers, P(MAA-co-AzPMA)-g-PNIPAM and P(QDMA-co- AzPMA)-g-PNIPAM, containing thermosensitive PNIPAM graft chains were successfully synthesized via a combination of atom transfer radical polymerization (ATRP) and “click” reactions, where PAzPMA, PMAA, PNIPAM,and PQDMAare poly(3-azidopropylmethacrylate),poly(methacrylicacid),poly(N-isopropylacrylamide), and poly(2-(dimethylamino)ethyl methacrylate) (PDMA) fully quaternized with methyl iodide, respectively. In aqueous solution, polyelectrolyte complexation between negatively charged backbone of P(MAA-co-AzPMA)- g-PNIPAM and positively charged backbone of P(QDMA-co-AzPMA)-g-PNIPAM leads to the formation of polyion complex (PIC) micelles consisting of polyion complex cores and thermoresponsive PNIPAM coronas. Upon addition of a difunctional cross-linker, propargy ether, PIC micelles can be facilely cross-linked via “click” reactions. The obtained covalently core-stabilized PIC micelles exhibit permanent stability against the addition of NaCl and pH changes, which are drastically different from that of non-cross-linked PIC micelles. Moreover, these novel types of stable PIC micelles exhibit thermoinduced dispersion/aggregation due to the presence of PNIPAM coronas, suggesting that their physical affinity to external substrates can be tuned with temperature. They might act as stable nanocarriers of charged compounds or highly efficient nanoreactors of polar compounds in the field of pharmaceutical formulation or biotechnology.

Introduction

In aqueous solution, double hydrophilic block copolymers

(DHBCs) can self-assemble into mesophases with varying morphologies such as micelles and vesicles, upon selectively rendering one of the blocks water-insoluble under proper external stimuli such as pH, temperature, ionic strength, light irradiation, and electric field.1-10 Polyelectrolyte complexation between two oppositely charged blocks can also be utilized to actuate the self-assemblyof block copolymers,taking advantage of the fact that polyion complex (PIC) is insoluble at charge neutralization and can thus provide an alternate driving force for the micellization of neutral-polyelectrolyte block copolymers.1-21

Original examples were reported by Kataoka et al.14 and

Kabanov et al.15 in 1995, employing two oppositely charged neutral-polyelectrolyte block copolymers. In aqueous solution, the polyelectrolytecomplexationbetweencomplementaryblocks resultsin the formationof PIC micelles.Followingthis principle, various charged species such as ions, proteins, and nucleic acids can be encapsulated within the PIC micelle cores. Thus, everincreasing attention has been paid to this research area due to their potential applications as delivery systems of protein drugs or therapeutic DNA. The presence of water-soluble neutral coronas can endow PIC micelles with enhanced chemical and structural stability during circulation, as compared to polyelectrolyte complexes prepared from protein or DNA and cationic liposomes or homopolycations.15,2,23 On the other hand, the polar PIC micelle cores can act as segregated nanoreactors for certainchemicalreactionssuch as reductionof metal ions.18,24-26 It should be noted that the neutral water-soluble coronas, such as poly(ethyleneoxide)(PEO),can also be replacedwith stimuliresponsive ones, such as thermoresponsive poly(2-isopropyl- 2-oxazoline), leading to the formation of environmentally sensitive PIC micelles for site-specific drug nanocarriers due to tunable affinity of substance affinity.27,28

However, the above practical applications of PIC micelles are usually associated with large dilution (e.g., blood circulation after administration) and/or the presence of high concentration of salts. It has been well-proved that both cases might disintegrate PIC micelles. They will dissociate into unimer chains when the concentration falls below the critical micelle concentration (cmc). The presence of salts can screen electrostatic interactions between oppositely charged blocks and can eventually lead to the decomposition of PIC micelles.29 Similar to those methodologies employed for the covalent stabilization of block copolymer micelles, such as shell cross-linking (SCL) or core cross-linking(CCL), PIC micellescan also be covalently stabilized to enhance their structural stability against changes of external conditions.24,29-3

Kataoka et al.29 prepared PIC micelles from poly(R,â-aspartic acid) (PAsp) and poly(ethylene oxide)-b-poly(L-lysine) (PEO- b-PLys) with PLys block being derivatized with thiol groups. The subsequent reversible core stabilization and dissociation can be achieved via the well-controlled formation and breakage of disulfide bonds via oxidation and the addition of excess of small molecule thiol compounds such as dithiothreitol (DTT). They also reported the stabilization of PIC micelles of PEO- b-PAsp and trypsin via the Schiff base formation upon addition of a difunctional reagent, glutaraldehyde.24 Bronich and coworkers also reported that PIC micelles formed from poly- (ethyleneoxide)-b-poly(sodiummethacrylate)(PEO-b-PNaMA)

* To whom correspondence should be addressed. E-mail: sliu@ ustc.edu.cn.

† Anhui University of Architecture. ‡ University of Science and Technology of China.

10.1021/ma702199f C: $40.75 © 2008 American Chemical Society Published on Web 01/25/2008

in the presence of CaCl2 can also be covalently stabilized via reaction with 1,2-ethylenediamine.30

Recently, “click” chemistry has been proved to be a highly efficient and quantitative reaction for the formation of stable covalent linkages between azido and alkynyl groups in the presence of copper(I) catalysts.34-38 Just recently, Wooley et al.39 reported the “click” core cross-linking of micelles selfassembled from amphiphilic polystyrene-b-poly(acrylic acid) (PS-b-PAA) block copolymer with the PS block employing alkynyl residues in the presence of dendrimers surface-functionalized with azide moieties as cross-linking agents.

In the current investigation, we utilized “click” reactions for the covalent stabilization of PIC micelles. Recently, Matyjaszewski et al.36 and Sumerlin et al.37 reported that azide moieties are compatible with controlled radical polymerizations (CRPs) such as reversible addition-fragmentation chain transfer polymerization (RAFT) or atom transfer radical polymerization (ATRP), whereas the CRP of alkynyl-containing monomers typically results in extensive gelation.38 Thus, azide-containing monomer was incorporated into two oppositely charged backbones of two graft ionomers bearing well-known thermoresponsive poly(N-isopropylacrylamide)(PNIPAM)40 graft chains (Schemes 1 and 2). The self-assembledPIC micelles in aqueous solution were subsequently core-stabilized via “click” reactions upon addition of a difunctional reagent, propargyl ether. The structuralstabilityand thermosensitiveaggregationof non-crosslinked and cross-linkedPIC micelles were thoroughlycharacterized by laser light scattering (LLS) and temperature-dependent turbidimetry. To the best of our knowledge, the current study represents the first example of “click” stabilization of PIC micelles with thermosensitive coronas.

Experimental Section

Materials. N-Isopropylacrylamide (NIPAM, 97%, Tokyo Kasei

Kagyo Co.) was purified by recrystallization from a mixture of benzene and n-hexane (1/3, v/v). tert-Butyl methacrylate (tBMA, 98%, TCI) and 2-(dimethylamino)ethyl methacrylate (DMA, 9%,

Acros) were vacuum-distilledover CaH2. 3-Azidopropylmethacrylate (AzPMA) was prepared by the esterification reaction of

3-azidopropanol and methacryloyl chloride according to literature procedures.36 All purified monomers were stored at -20 °C prior to use. Tris(2-(dimethylamino)ethyl)amine (Me6TREN) was prepared from tris(2-aminoethyl)amine (96%, Acros) following lit- erature procedures.41 Tetrahydrofuran (THF), methylene chloride

(CH2Cl2), isopropyl alcohol (IPA), and N,N-dimethylformamide (DMF) were distilled just prior to use. N,N′-Dicyclohexylcarbodim- ide (DCC), 4-(dimethylamino)pyridine (DMAP), 2-propanol, ethyl 2-bromoisobytyrate (EBIB, 9%, Avocado), copper(I) chloride (CuCl, 9.95+%, Aldrich), copper(I) bromide (CuBr, 9.9%, Aldrich),N,N,N′,N′′,N′′-pentamethyldiethylenetriamine(PMDETA, 9%, Aldrich),propargyether (9%, Aldrich),and all other reagents were commercially available and used without further purification.

Sample Preparation. Preparation of Propargyl 2-Chloropropionate (PCP). The ATRP initiator, PCP, was prepared by the esterification reaction of propargyl alcohol with 2-chloropropionic acid in the presence of DCC and DMAP. A 250 mL round-bottom flask was charged with 2-chloropropionic acid (10.85 g, 0.10 mol),

DCC (2.70 g, 0.1 mol), and CH2Cl2 (120 mL). The reaction mixture was cooled to 0 °Ci na ni ce-water bath, and a solution of propargyl alcohol (5.61 g, 0.10 mol), DMAP (0.5 g), and CH2-

Cl2 (30 mL) was added dropwise over a period of 1 h under magnetic stirring. After the addition was completed, the reaction mixture was stirred at 0 °C for 1 h and then at room temperature for 12 h. After removing the insoluble N,N′-dicyclohexylurea by suction filtration, the filtrate was concentrated and then was further purified by silica gel column chromatography using CH2Cl2 as the

Scheme 1. Synthetic Routes for the Preparation of P(MAA-co-AzPMA)-g-PNIPAM Anionic Graft Ionomer Macromolecules, Vol. 41, No. 4, 2008 Polyion Complex Micelles 1445

eluent. After removing the solvents by rotary evaporator, the obtained residues were distilled under reduced pressure. A colorless

Synthesis of Monoalkyne-Terminated PNIPAM (Alkyne-

PNIPAM).42,43 The general procedure employed for the preparation of monoalkyne-terminated PNIPAM was as follows. The mixture containing NIPAM (9.05 g, 80 mmol), Me6TREN (527 mg, 2.3 mmol), and IPA (18.10 g) was deoxygenated by bubbling with nitrogen for at least 30 min. CuCl (227 mg, 2.3 mmol) was introduced under the protection of N2 flow. The reaction mixture was stirred for 10 min to allow the formation of CuCl/Me6TREN complex. PCP (335 mg, 2.3 mmol) was then added via a microliter syringe to start the polymerization. The reaction was carried out at

25 °C and allowed to stir under a N2 atmosphere for 5 h. Polymerization was terminated by the addition of a few drops of saturated CuCl2 solution in IPA. The mixture was precipitated into an excess of n-hexane. The sediments were collected and redis- solved in CH2Cl2 and then passed througha neutralaluminacolumn using CH2Cl2 as the eluentto removecoppercatalysts.The collected eluents were concentrated and precipitated into an excess of anhydrous diethyl ether. This purification cycle was repeated for three times. After drying in a vacuum oven overnight at room temperature, white solids were obtained with an overall yield of 74%. The molecular weight and molecular weight distribution of alkyne-PNIPAM homopolymerwere determinedby GPC using

DMF as eluent: Mn ) 3800, Mw/Mn ) 1.09. The degree of polymerization (DP) was determined to be 3 by 1H NMR analysis

ATRP Synthesis of P(tBMA-co-AzPMA) Statistical Copolymer

(Scheme 1). EBIB (49 mg, 0.25 mmol), PMDETA (43 mg, 0.25 mmol), tBMA (4.97 g, 35 mmol), and AzPMA (0.84 g, 5 mmol) were charged into 20 mL glass ampule containing 6 mL of IPA. The ampulewas degassedvia two freeze-thaw-pumpcycles.After freezing the mixture in liquid N2, CuCl (25 mg, 0.25 mmol) was introduced. The ampule was further degassed via two freeze- pump-thaw cycles and flame-sealed under vacuum. It was then immersed into an oil bath thermostated at 30 °C to start the polymerization. After 10 h, the ampule was broken and the viscous reaction mixture was exposed to air. After diluting with CH2Cl2, the solution was precipitated into an excess of MeOH/H2O (1/1, v/v) mixture. After filtration, the residues was redissolved in CH2- Cl2 and passed through a mixed column of silica gel and neutral alumina using CH2Cl2 as the eluent to remove copper catalysts. The combined eluents were concentrated and precipitated into an excess of MeOH/H2O (1/1, v/v). This purification cycle was repeated two times. After drying overnight in a vacuum oven at room temperature,whitesolidswere obtainedwith a yieldof 7%. The molecular weight and molecular weight distribution of P(t- BMA-co-AzPMA) statistical copolymer were determined by GPC using DMF as the eluent: Mn ) 13 700, Mw/Mn ) 1.10. The AzPMA content of P(tBMA-co-AzPMA) was determined to be 14 mol%b y 1H NMR analysis in CDCl3. The overall DP of the obtained statistical copolymer was calculated to be 94 on the basis of GPC and NMR results.

Synthesis of P(tBMA-co-AzPMA)-g-PNIPAM Graft Copolymer

Via Click Chemistry (Scheme 1). A mixed solution of P(tBMA-co- AzPMA) (1.0 g, 0.96 mmol azido moieties, 1.0 equiv) and alkyne- PNIPAM (1.08 g, 0.28 mmol, 0.29 equiv) in 12 mL of DMF was degassed via two freeze-thaw-pump cycles. CuBr (40 mg, 0.28 mmol, 0.29 equiv) was introduced into the glass ampule under a

N2 atmosphere.After stirring for 24 h at 30 °C, the reaction mixture was exposed to air. After removing all the solvents under reduced pressure, the residues were dissolved in CH2Cl2 and precipitated into an excess of n-hexane. The sediments were collected and redissolved in CH2Cl2 and then passed through a neutral alumina column using CH2Cl2 as the eluent to remove copper catalysts. The

Scheme 2. Synthetic Routes Employed for the Preparation of P(QDMA-co-AzPMA)-g-PNIPAM Cationic Graft Ionomer 1446 Zhang et al. Macromolecules, Vol. 41, No. 4, 2008 combined eluents were concentrated using a rotary evaporator and precipitated into an excess of n-hexane. This purification cycle was repeated three times. After drying overnight in a vacuum oven at room temperature, slightly yellowish solids were obtained with a yield of 95%. The molecular weight and molecular weight distribution of P(tBMA-co-AzPMA)-g-PNIPAM were determined by GPC using DMF as the eluent: Mn ) 34,200, Mw/Mn ) 1.21. The NIPAM content in P(tBMA-co-AzPMA)-g-PNIPAM was determined to be 57 mol % by 1H NMR analysis in CDCl3.O n average, there are 3.8 PNIPAM chains per graft copolymer chain.

SelectiVe Hydrolysis of P(tBMA-co-AzPMA)-g-PNIPAM Graft Copolymer (Scheme 1). Into a solution of P(tBMA-co-AzPMA)- g-PNIPAM (1.0 g, 2.8 mmol tBMA residues) in 10 mL of CH2Cl2 was slowly added TFA (1.0 mL, 13.5 mmol) at 0 °C under vigorous stirring. The reaction mixture was allowed to stir at 0 °Cf or3h and then at room temperature for 8 h. During hydrolysis, the crude products progressively precipitated out. After removing all the solvents under reduced pressure, the residues were dissolved in alkalinesolution(pH 9) and dialyzed(MW cutoff, 7000 Da) against deionized water to remove the possible presence of unreacted alkyne-PNIPAM in the previous “click” grafting reaction. After acidification, the anionic graft ionomer, P(MAA-co-AzPMA)-g- PNIPAM, was obtained as white powders (0.71 g) by freeze-drying and subsequent drying in a vacuum oven at room temperature for 12 h.

ATRP Synthesis of P(DMA-co-AzPMA) Statistical Copolymer.

EBIB (50 mg, 0.26 mmol), PMDETA (4 mg, 0.26 mmol), DMA (5.50 g, 35 mmol), and AzPMA (0.85 g, 5 mmol) were charged into a 20 mL reaction flask containing 6 mL of IPA. The flask was degassed via three freeze-thaw-pump cycles and backfilled with

N2. After thermostated at 30 °C, CuCl (26 mg, 0.26 mmol) was then introduced into the reaction flask under protection of N2 flow to start the polymerization. After 5 h, the resulting viscous reaction mixture was exposed to air and diluted with CH2Cl2. After passing through a neutral alumina column using CH2Cl2 as the eluent to remove the copper catalysts and precipitation into an excess of n-hexane, the obtained white solids ( 79% yield) were dried overnight in a vacuum oven at room temperature. The molecular weight and molecular weight distribution of P(DMA-co-AzPMA) statistical copolymer were determined by GPC using DMF as eluent: Mn ) 30 200, Mw/Mn ) 1.12. The AzPMA content of P(DMA-co-AzPMA) was determined to be 12 mol % by 1H NMR analysis in CDCl3, and the overall DP of P(DMA-co-AzPMA) was calculated to be 190.

Synthesis of P(DMA-co-AzPMA)-g-PNIPAM Graft Copolymer Via ClickChemistry. A solutionof P(DMA-co-AzPMA)(1.0 g, 0.76 mmol of -N3 moieties, 1.0 equiv) and alkyne-PNIPAM (0.75 g, 0.19 mmol, 0.25 equiv) in 12 mL of DMF was degassed via two freeze-thaw-pump cycles. CuBr (27 mg, 0.19 mmol, 0.25 equiv) was then introduced into the reaction flask to start the “click” grafting reaction at 30 °C under a N2 atmosphere. After stirring for 24 h, the reaction mixture was exposed to air. After removing all the solvents under reduced pressure, the obtained polymer was dissolved in CH2Cl2 and precipitated into an excess of n-hexane. The sediments were redissolved in CH2Cl2 and then passed through a neutral alumina column using CH2Cl2 as the eluent to remove copper catalysts. The combined eluents were concentrated and precipitated into an excess of n-hexane. This purification cycle was repeated three times. After drying in a vacuum oven overnight at room temperature,whitesolidswere obtainedwith a yieldof 95%. The molecularweightand molecularweightdistributionof P(DMA- co-AzPMA)-g-PNIPAM graft copolymer were determined by GPC using DMF as eluent: Mn ) 56 400, Mw/Mn ) 1.19. The NIPAM content of P(DMA-co-AzPMA)-g-PNIPAM was determined to be

50 mol % by 1H NMR analysis in CDCl3. On average, there are 5.8 grafted PNIPAM chains per graft copolymer chain.

Quaternization of P(DMA-co-AzPMA)-g-PNIPAM Graft Copolymer. The quaternization of DMA residues was carried out at room temperature by reacting methyl iodide (150 íL, 2.4 mmol) with P(DMA-co-AzPMA)-g-PNIPAM (0.5 g, 1.6 mmol DMA residues) in THF (35 mL). The reaction was allowed to stir for 1 ha t2 0 °C. The precipitated residues were collected and thoroughly washed with THF to remove unreacted methyl iodide. It was dissolved in water and thoroughly dialyzed against deionized water (MW cutoff, 7000 Da) to remove impurities and any unreacted alkyne-PNIPAM. After lyophilization, the cationic graft ionomer, P(QDMA-co-AzPMA)-g-PNIPAM,was obtainedas whitepowders with a yield of 95% after drying overnight in a vacuum oven.

Preparation of Polyion Complex (PIC) Micelles. Predetermined amounts of P(MAA-co-AzPMA)-g-PNIPAM and P(QDMA-co- AzPMA)-g-PNIPAM were separately dissolved in water at a concentration of 1.0 g/L; the solution pH was adjusted to 8.0 with aqueous NaOH solution. After filtrationthrough a 0.4 ím Millipore nylon filter, PIC micelles were prepared by mixing these two solutions at varying molar ratios of MAA to QDMA residues, [MAA]:[QDMA].The molecularweightsof the two target polyions were calculated on the basis of Mn values of statistical polymers determined by DMF-GPC analysis and grafted PNIPAM contents determined by 1H NMR analysis. A bluish tinge was typically observed, indicating the formation of micellar aggregates. The aqueous dispersion of PIC micelles were equilibrated overnight at room temperature before subsequent “click” cross-linking or characterization.

“Click” Core Cross-Linking of PIC Micelles. To 1.0 g/L degassed aqueous solution of the above-prepared PIC micelles at moieties), CuSO4â5H2O (15 mg, 60 ímol), propargyl ether (3 íL, 30 ímol), and sodium ascorbate (1 mg, 60 ímol) were charged.4

The molar ratio of propargyl ether to that of total AzPAM residues was kept constant at 1:2. The reaction mixture was stirred at 25 °C for 48 h and then dialyzed (MW cutoff, 14 0 Da) against deionized water for 2 days to remove copper catalysts. The final dispersion exhibits a bluish tinge characteristic of micellar aggregates even in the presence of 1.0 M NaCl, indicating successful “click” core cross-linking reaction.

Characterization. Gel Permeation Chromatography (GPC).

(Parte 1 de 4)

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