Communication
Intramolecular Click Cycloaddition: An Efficient Room-Temperature Route towards Bioconjugable Polymeric Nanoparticlesa Alaitz Ruiz de Luzuriaga, Nerea Ormategui, Hans J. Grande, Ibon Odriozola, Jose´ A. Pomposo, Iraida Loinaz*
A highly efficient room-temperature synthetic route to bioconjugable polymeric nanoparticles in the 5–20 nm size range based on single-chain intramolecular click cycloaddition is described. It is illustrated by preparing single-chain cross-linked polymeric NPs from poly[MMA-co(3-azidopropyl methacrylate)-co-(3-trimethylsilyl-propyn1-yl methacrylate)] terpolymers using a one-pot procedure and a continuous addition technique. For polymeric NPs with an excess of azide groups, aminoacid/PMMA NPs were easily obtained by performing a second click reaction with propargyl glycine. This versatile and general method opens the way to the synthesis of other kinds of polymeric and bioconjugated NPs beyond those reported in this communication.
Introduction Nanoparticles (NPs) are currently ubiquitous in many research fields, including physics, chemistry, biology and medicine, due to new and promising properties of nano-objects compared to bulk materials. In spite of this general interest, synthetic routes to single-chain crosslinked polymeric NPs in the 5–20 nm size range are scarce. The main approaches employed are: (i) collapse and intramolecular coupling of single-polymer chains at ultradiA. R. de Luzuriaga, N. Ormategui, H. J. Grande, I. Odriozola, J. A. Pomposo, I. Loinaz New Materials Department, CIDETEC, Centre for Electrochemical Technologies, Parque Tecnolo´gico de San Sebastia´n, Paseo Miramo´n 196, Donostia-San Sebastia´n E-20009, Spain Fax: þ34 943 30 9136; E-mail:
[email protected] a
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: Supporting information for this article is available at the bottom of the article’s abstract page, which can be accessed from the journal’s homepage at http://www.mrc-journal.de, or from the author.
Macromol. Rapid Commun. 2008, 29, 1156–1160 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
luted (ca. 105–106 M) reaction conditions,[1] (ii) thermally activated benzocyclobutene-coupling of individual chains by means of a continuous addition technique[2] and (iii) microwave-assisted surfactant-free emulsion polymerization (SFEP) in the presence of bifunctional crosslinkers with enhanced reactivity.[3] Often, poorly defined materials (and in some cases even gels) are obtained using the first strategy. The second route requires high temperature conditions (250 8C) leading to undesirable side reactions (i.e., oligomerization). Moreover, it cannot be employed with polymers that are not stable at high temperatures such as acrylic or methacrylic (bio)polymers. Finally, the microwave-assisted SFEP methodology requires the use of a specific equipment to guarantee a high stability of the microwave power in the reactor and hence a narrow NP size distribution. Click chemistry,[4] and specifically the CuI-catalyzed [3 þ 2] cycloaddition of alkynes and azides,[5] is a selective and very efficient kind of reaction that can be performed under extremely mild conditions with high yields, good functional group tolerance and negligible byproducts.
DOI: 10.1002/marc.200700877
Intramolecular Click Cycloaddition: An Efficient Room-Temperature Route . . .
Among other applications, click chemistry is attracting great interest[6] for easy and almost quantitative functionalization of synthetic polymers (e.g., copolymers,[7] dendrimers[8]), biomolecules (e.g., amino acids,[9] glycopeptides[10]) and NPs (e.g., gold,[11] luminescent CdSe particles,[12] silica NPs[13] and magnetic oxide NPs[14]) as well as macromolecular cyclization[15] and step-growth coupling of polymers.[16] Recently, click strategies have been extended to functionalization and shell-stabilization of amphiphilic block copolymer micelles (35 nm in size) using multifunctional dendritic cross-linkers.[17] Herein, we report a general and efficient room temperature synthetic route to bioconjugable polymeric NPs based on single-chain intramolecular click (CuIcatalyzed azide alkyne 1,3-dipolar) cycloaddition. The main challenges for a versatile synthetic route to single-chain cross-linked polymeric NPs are: (i) easy incorporation of coupling precursors into the individual polymer chains, (ii) highly efficient and selective crosslinking (coupling) reaction and (iii) appropriate room temperature reaction conditions favoring intramolecular coupling versus intermolecular cross-linking. In this communication, we demonstrate that single-chain intramolecular click cycloaddition meets all the above requirements and furthermore allows the obtention of functionalized polymeric NPs for bioconjugation applications.
Experimental Part Materials Unless otherwise stated, all the chemicals used in the syntheses were purchased from Sigma-Aldrich (reagent grade) and used without further purification. Methyl methacrylate (1) was distilled prior to use. 2-Phenyl-2-yl dithiobenzoate (cumyl dithiobenzoate, CDB),[18] 3-azidopropanol,[19a] 3-azidopropylmethacrylate[19a] (2) and 2-methyl-acrylic acid 3-trimethylsilanyl-prop-2-ynyl ester[19b] (3) were synthesized and purified as reported in the literature. Silica gel for flash chromatography was Merck Kieselgel 60 (0.040– 0.063 mm) and aluminium oxide was Fluka-Brockmann Activity I.
Preparation of Poly[(methyl methacrylate)co-(3-azidopropyl methacrylate)co-(3-trimethylsilyl-propyn-1-yl methacrylate)] Terpolymers In a typical reaction, a Schlenk flask containing 1 (0.690 g, 6.89 mmol), 2 (0.144 g, 0.84 mmol), 3 (0.166 g, 0.85 mmol), CDB (0.010 g, 0.04 mmol) and 2,20 -azo(2-methylpropionitrile) (AIBN) (0.0025 g, 0.015 mmol) dissolved in 4 mL of acetone was degassed using five freeze/pump/thaw cycles. The reaction mixture was placed in a preheated (65 8C) oil bath and stirred for 16 h. The reaction mixture was then cooled to room temperature and purified by precipitation into Macromol. Rapid Commun. 2008, 29, 1156–1160 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
hexane to yield 4a as a slightly pink solid (0.89 g, yield: 89%). 1 H NMR (CDCl3): d ¼ 4.61 (s, CH2O alkyne), 4.06 (s, CH2O azide), 3.61 (s, CH3O), 3.45 (t, CH2N3, J ¼ 5.59, 11.18 Hz,) and 1.91–2.02 (m, CCH2C); 0.20 [s, Si(CH3)3]. FTIR (neat): 3 423, 2 994, 2 651, 2 180, 2 099, 1 730, 1 484, 1 448, 1 362, 1 243, 1147, 1061, 1 033, 846, 750 cm1. Size exclusion chromatography (SEC) in THF: Mn ¼ 31 800 and Mw ¼ 41 000 g mol1 (PDI ¼ 1.29). Mn (1H NMR) ¼ 33 000 g mol1.
General Procedure for Intramolecular Click Cycloaddition Leading to Single-Chain Polymeric Nanoparticles from Poly[(methyl methacrylate)-co-(3-azidopropyl methacrylate)-co-(3-trimethylsilyl-propyn-1-yl methacrylate)] Terpolymers In a typical reaction, CuBr (0.4 g, 2.79 mmol), 2,20 -bipyridil (bipy) (0.4 g, 2.56 mmol) and tetrabutylammonium fluoride (TBAF) (3 mL, 0.6 mmol, 0.2 M in DMF) were dissolved under N2 in previously degassed DMF (100 mL). To this solution, a solution of 4a (200 mg) in DMF (10 mL) was added via a syringe pump (LHS 300/600 Brand GmbH) at a rate of 2 mL h1 under N2. After addition was complete, the solution was stirred at room temperature for 2 h. Then, CH2Cl2 was poured and the organic layer was washed multiple times with saturated aqueous NH4Cl, dried over MgSO4 and concentrated in vacuo. The crude product was purified via flash column chromatography (SiO2, CH2Cl2) to remove the excess of TBAF. A solid was precipitated from the resulting solution with hexane (169 mg, yield: 85%). 1 H NMR (CDCl3): d ¼ 5.36 (s, 1H, NCHC), 3.60 (s, CH3O) and 1.12–2.02 broad signal (m, CCH2C). IR (neat): 3 366, 2 948, 1 728, 1 448, 1 242, 1 147 cm1. NP diameter by transmission electron microscopy (TEM): 6.5 1.4 nm. NP diameter by dynamic light scattering (DLS) in THF: 5.5 1.2 nm.
Preparation of Aminoacid/PMMA Nanoparticles Nanoparticles7preparedfrompoly[(methylmethacrylate0.71)co-(3-azidopropyl methacrylate0.24)-co-(3-trimethylsilylpropyn-1-yl methacrylate0.05)] (5) with an excess of azide functional groups (75 mg), CuBr (0.1 g, 0.69 mmol), bipy 2,20 -bipyridil (0.08 g, 0.51 mmol) and L-C propargyl glycine, 8, (0.017 g, 0.15 mmol) were dissolved in DMF (5 mL) under N2. The reaction was allowed to proceed at room temperature for 5 h. The resulting solution was then precipitated in a saturated aqueous NH4Cl solution. The solid was finally separated by centrifugation after washing with distilled water several times (65 mg, yield: 87%).
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1
H NMR (CDCl3) d ¼ 5.36, (bs, CH – – C), 4.93 (bs, CO2H – – CH NH2). FTIR (neat): 3 432, 3 147, 3 050, 2 950, 2 850, 1 730, 1 627, 1 439, 1 405, 1 264, 1 150 cm1. NP diameter by DLS in THF: 6.4 1.8 nm. Scheme 1. Synthesis of poly[(methyl methacrylatex)-co-(3-azidopropyl methacrylatey)co-(3-trimethylsilyl-propyn-1-yl methacrylatez)] terpolymers by RAFT polymerization.
Measurements and Analysis
performed by polymerization of methyl methacrylate (1) in the presence of small stoichiometric amounts of azide- and protected alkyne-containing methacrylate co-monomers at three different concentrations (4, 7 and 10 mol-%) using controlled/living radical polymerization techniques.[20] Hence, terpolymers of 1, 3-azidopropyl methacrylate (2) and 3-(trimethylsilyl)propyn-1-yl methacrylate (3) were prepared under reversible addition fragmentation chain transfer (RAFT) conditions[21] (see Scheme 1 and Table 1). RAFT polymerization allowed the synthesis of terpolymers with good control over molecular weight and polydispersity, as determined by 1H NMR spectroscopy and SEC measurements (see Supporting Information). In a second step, one-pot deprotection of the propargyl monomer units in the terpolymer was performed[19b] followed by single-chain intramolecular CuI-catalyzed azide alkyne 1,3-dipolar cycloaddition at room temperature using a CuI salt and a continuous addition technique[3] (see Figure 1a). Quantitative intramolecular coupling leading to new 1,2,3-triazole units was confirmed by FTIR data, which showed the disappearance of the azide (2 102 cm1) and protected alkyne (2 181 cm1) peaks (see Supporting Information). Further evidence was gained from 1H NMR measurements showing a broadening of methyl and methylene peaks upon NP formation, the presence of a new peak at 5.36 ppm from triazole units and the lack of the Me3Si-peak at 0.2 ppm (see Figure 1b).
Size exclusion chromatography measurements were carried out at room temperature on a Shimadzu SCL 10 AVP apparatus connected to three 5 mm-PLgel columns ´˚ using a RID-10A with pore sizes of 102, 103 and 104 A detector and a LC-10ADVP pump. THF was used as an eluent with a flow rate of 1 mL min1. The molecular weights of the polymers were calculated relative to linear polystyrene standards. Particle size measurements were performed on a DLS Beckman Coulter N5 Submicron Particle Size Analyzer at a scattering angle of 908. TEM analysis was performed using a JEOL JEM-1011 microscope operated at 100 kV. Samples were prepared by depositing dilute particle solutions onto 400 mesh carbon-coated copper grids and staining with OsO4. 1H NMR (500 MHz) spectra were recorded with a Bruker Avance spectrometer at room temperature using CDCl3 as solvent. FTIR spectra were recorded on a Nicolet Avatar 360 spectrophotometer.
Results and Discussion As an illustrative example of the versatility of the intramolecular click cycloaddition route reported in this communication, we have selected the room temperature preparation of single-chain cross-linked poly(methyl methacrylate) (PMMA) NPs. In a first step, introduction of coupling precursors into the PMMA chains was simply
Table 1. Reaction yield, composition and molecular weight of poly[(methyl methacrylatex)-co-(3-azidopropyl methacrylatey)co-(3-trimethylsilyl-propyn-1-yl methacrylatez)] terpolymers synthesized by RAFT polymerization.
Yielda)
xb)
yb)
zb)
Mn c)
Mn d)
%
mol-%
mol-%
mol-%
g molS1
g molS1
4a
89
0.8
0.1
0.1
33 000
31 800
1.29
4b
94
0.86
0.07
0.07
34 000
34 200
1.22
4c
91
0.92
0.04
0.04
31 600
30 600
1.27
5
86
0.71
0.24
0.05
32 100
31 900
1.25
Terpolymer
a)
Yield (%) ¼ (grams of terpolymer/grams of monomers) 100; As determined by SEC referred to polystyrene standards.
PDId)
b)
As determined by 1H NMR; c)Absolute Mn determined by 1H NMR;
d)
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DOI: 10.1002/marc.200700877
Intramolecular Click Cycloaddition: An Efficient Room-Temperature Route . . .
formation of well-defined spherical NPs was confirmed by TEM. As an example, NPs 6a prepared from terpolymer 4a showed a diameter of 6.5 1.4 nm by TEM, in good agreement with the hydrodynamic size determined by DLS in THF (5.5 1.2 nm). NPs 6b and 6c, prepared from terpolymers of similar molecular weights containing 7 and 4 mol-% of reactive co-monomers, respectively, showed NP sizes of 6.0 1.3 and 6.0 1.6 nm. This result illustrates the high efficiency of the intramolecular click cycloaddition reaction, since NP formation was achieved with only 4 mol-% of reactive functional groups in the terpolymer. With minor changes, the intramolecular click cycloaddition approach was easily adapted to the synthesis of amino acid-substituted polymeric NPs (Figure 3). Hence, terpolymers containing an excess of azide groups (over-stoichiometric protected alkyne moieties) were prepared by RAFT polymerization such as poly[(methyl methacrylate0.71 ) - co - ( 3 - azidopropyl methacrylate0.24 )-co-(3-trimethylsilylpropyn-1-yl methacrylate0.05)] (5). Upon intramolecular click cycloaddiFigure 1. (a) Schematic illustration of the preparation of single-chain cross-linked polymeric tion of 5, unreacted azide groups were NPs by intramolecular click cycloaddition of poly[(methyl methacrylate)-co-(3-azidopropyl found in the cross-linked NPs (7). Click methacrylate)-co-(3-trimethylsilyl-propyn-1-yl methacrylate)] terpolymers. (b) 1H NMR chemistry was performed over the free (500 MHz) spectra of terpolymer 4a and NPs 6a. azide groups of NPs 7 by reacting them with propargyl glycine (8) biomolecules. Successful formation of amino acid-substituted polymeric NP (9) was confirmed Intramolecular click cycloaddition induces an intramoby FTIR and 1H NMR spectroscopies (see Supporting lecular collapse of the linear chains to individual NPs. This results in a significant reduction of the hydrodynamic Information). volume and hence the apparent molecular weight (40–46%) with no significant change in polydispersity was clearly observed by SEC (see Table 2 and Figure 2). The Table 2. Mn , Mw and polydispersity values obtained for corresponding NPs synthesized by click-cycloaddition.
Nanoparticle
PDI
Mn
Mw
g molS1
g molS1
6a
17 300
19 400
1.12
6b
19 300
21 600
1.12
6c
18 600
22 300
1.22
Macromol. Rapid Commun. 2008, 29, 1156–1160 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. (a) SEC chromatograms of terpolymer 4a and singlechain cross-linked NPs 6a prepared thereof by intramolecular click cycloaddition showing an increase in retention time due to a reduction in hydrodynamic volume upon NP formation. (b) TEM micrograph of single-chain polymeric NPs 6a.
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Figure 3. Preparation of bioconjugated single-chain cross-linked PMMA NPs 9.
Conclusion In summary, a highly efficient room temperature synthetic route towards bioconjugable polymeric NPs in the 5– 20 nm size range has been described based on single-chain intramolecular click cycloaddition. This new synthetic route has been demonstrated by preparing neat PMMA NPs as well as amino acid/PMMA NPs. We anticipate that this versatile and general method will be very valuable for the synthesis of other kind of polymeric and bioconjugated NPs beyond those reported in this communication.
Acknowledgements: We thank the Basque Government and Diputacio´n de Guipuzkoa for financial support of this work though CIC Biomagune - Etortek research program and Spanish MEC (NAN2004-09415-C05-05). Received: December 19, 2007 Revised: January 18, 2008; Accepted: January 29, 2008; DOI: 10.1002/marc.200700877 Keywords: click chemistry; functionalization; polymeric nanoparticles; reversible addition fragmentation chain transfer (RAFT); TEM
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DOI: 10.1002/marc.200700877
