NANO LETTERS
Fabrication of Hybrid Nanocapsules by Calcium Phosphate Mineralization of Shell Cross-Linked Polymer Micelles and Nanocages
2005 Vol. 5, No. 7 1457-1461
Kris K. Perkin,† Jeffrey L. Turner,‡ Karen L. Wooley,‡ and Stephen Mann*,† Centre for Organized Matter Chemistry, School of Chemistry, UniVersity of Bristol, Bristol BS8 1TS, U.K., and Center for Materials InnoVation and Department of Chemistry, CB 1134, Washington UniVersity in Saint Louis, Saint Louis, Missouri 63130-4899 Received May 3, 2005; Revised Manuscript Received June 10, 2005
ABSTRACT Self-assembled shell cross-linked poly(acrylic acid-b-isoprene) (PAA78-b-PI97) micelles or cross-linked PAA nanocages in aqueous solution were used as templates for the preparation of novel polymer−inorganic nanocapsules. The hybrid nanostructures were typically 50−70 nm in diameter and consisted of spherical polymer nanoparticles or nanocages enclosed within a continuous 10−20 nm thick surface layer of amorphous calcium phosphate. Nucleation of calcium phosphate specifically in association with the polymer nanoparticles was facilitated by low supersaturation levels and by sequestration of Ca2+ ions within the carboxylate-rich PAA domains prior to addition of HPO42-. Modifications in ionic concentrations were used to control the calcium phosphate surface layer thickness and prepare mineralized cross-linked PAA-b-PI micelles with variable shell permeability. The permeability of β-carotene into the hydrophobic PI core of mineralized shell cross-linked PAA-b-PI micelles was reduced by approximately 50 or 100% respectively for hybrid nanostructures enclosed within 10 or 20 nm thick calcium phosphate layers. Our results suggest that calcium phosphate−polymer cross-linked nanocapsules could have potential applications as pHresponsive biocompatible hybrid nanostructures for use in applications such as drug delivery, bioimaging, and therapeutics.
1. Introduction. The spontaneous self-assembly of coreshell spherical micelles from amphiphilic block copolymers is well documented.1 Such nanostructures often exhibit environmentally responsive properties2 and can be used to sequester guest molecules, for example, for drug delivery,3-5 or guest ions for the synthesis and spatial patterning of polymer-encapsulated metallic nanoparticles.6-9 In addition, low levels of covalent cross-linking of functional groups within the micelle corona result in structural stabilization without compromising the sequestration of molecules, such as drugs, pesticides, and fragrances, in the core.10-13 Recently, ligands for folate receptors14 and integrin Rvβ315 have been covalently coupled to functionalities of the cross-linked micelle shell for potential targeting to cancer cells, and DNA molecules have been packaged by electrostatic attraction to positively charged surface groups for potential gene therapy.16 Other studies have shown that selective chemical degradation of the core-forming segments can be used to produce discrete nanocages consisting of an intact porous shell of cross-linked coronal domains.17,18 * Corresponding author. E-mail
[email protected]. † University of Bristol. ‡ Washington University in Saint Louis. 10.1021/nl050817w CCC: $30.25 Published on Web 06/22/2005
© 2005 American Chemical Society
In this paper, we use shell cross-linked micelles and crosslinked nanocages, each derived from poly(acrylic acid-bisoprene) (PAA78-b-PI97), and produced by shell cross-linking amidation reactions or shell cross-linking amidation reactions followed by ozonolytic degradation of the PI core of the parent micelles, respectively, as templates for the fabrication of novel calcium phosphate-polymer nanostructures. Zeta potential measurements indicated that the polymer nanoparticles had a net surface charge of ca. -25 mV at pH 7 due to unmodified carboxylate groups in the PAA shell, and these were used to preorganize Ca2+ ions at the surface or within near-surface regions of the polymer nanostructures prior to addition of HPO42- ions and onset of calcium phosphate nucleation. Moreover, unlike previous studies, which showed that complex nanofilaments of calcium phosphate were formed by emergent processes in aqueous solutions containing metastable aggregates of a partially alkylated poly(ethylene oxide-b-methacrylic acid) block copolymer,19 the use of stable cross-linked polymer architectures as reported herein resulted in direct replication to produce intact hybrid nanocapsules. These inorganic-organic hybrid nanostructures, which consisted of spherical polymer nanoparticles or nanocages encapsulated within a continuous 10-20 nm thick
inorganic veneer, exhibited modified permeabilities to organic molecules and were stable in water over periods of several months. Our results suggest that as calcium phosphate is thermally and chemically stable, nontoxic, and biocompatible, and displays pH-dependent dissolution behavior,20 mineralized polymer micelles and nanocages could have important uses in diverse applications. 2. Experimental Section. 2.1. Preparation of Shell Cross-Linked PAA78-b-PI97 Micelles. PAA-b-PI diblock copolymer was synthesized according to previous methods.15 In brief, methane sulfonic acid was added (0.052 mL, 0.8 mM) to 250 mL of a 80:20 (v/v) toluene/acetic acid solution of PtBA-b-PI (PtBA, poly(tert-butyl acrylate) and the reaction mixture refluxed for 1.5 h. The solvent was removed with a rotary evaporator and the resulting solid dissolved in THF (100 mL). Deionized water (100 mL) was then added dropwise over 30 min, and the mixture dialyzed (MW cutoff, 3.5k) against water for 4 days, followed by lyophilization overnight to yield pure PAA-b-PI as a colorless solid. Micellization of PAA-b-PI was induced by dissolving the polymer (950 mg) in THF (475 mL), followed by addition of deionized water at a rate of ∼10 mL/h over a period of 2 days. Subsequently, 500 mL of deionized water was added, and the resulting solution stirred for 2 h followed by dialysis (MW cutoff, 3.5k) against water for 5 days. The PAA corona of PAA-b-PI core-shell micelles was cross-linked as described previously.15 Typically, 2,2′(ethylenedioxy)bis(ethylamine) was added dropwise to an aqueous micelle dispersion at a final amine/acrylate monomer molar ratio of 0.22, and the solution was left for 1 h to equilibrate before 1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide methiodide was added at 0.22 equiv of the acrylic acid monomer concentration. The resulting solution was stirred overnight under ambient conditions to facilitate condensation reactions between the acrylic acid residues in the micelle shell and the diamino cross-linker. The solution was then dialyzed against water (MW cutoff, 10k) for 1 week to remove any small molecule contaminants. The final concentration was typically 0.5 mg/mL. 2.2. Preparation of Cross-Linked PAA Nanocages. PAA nanocages were prepared by ozonolytic degradation of the hydrophobic PI core of shell cross-linked PAA-b-PI micelles using methods previously described.18 Briefly, ozone mixed with dried compressed air (Red Sea AquaZone 100 ozone generator) was delivered at a rate of 100 mg/h for 30 min into an aqueous dispersion of cross-linked PAA-b-PI micelles. Residual dissolved ozone was removed by sparging with ozone-free nitrogen for 10 min, and intermediate ozonides were reduced by addition of freshly prepared saturated aqueous sodium sulfite. The reaction solution was stirred overnight and dialyzed to remove low molecular weight impurities. 2.3. Calcium Phosphate Mineralization of Polymer Micelles and Nanocages. In general, successful mineralization experiments were undertaken at room temperature by adding aqueous Na2HPO4 (5-20 µL, 5-25 mM) to a stirred aqueous solution containing 5-10 µL of 5 mM CaCl2 and 1458
90 µL of 0.5 mg/mL shell cross-linked PAA-b-PI micelles or cross-linked PAA nanocages and allowing the reaction to proceed for 24 h. The CaCl2/polymer solutions were stirred overnight prior to addition of Na2HPO4. Typically, micelles/ nanocages comprising ultrathin mineralized coatings were prepared by using
