Letter pubs.acs.org/NanoLett
Generation-Dependent Templated Self-Assembly of Biohybrid Protein Nanoparticles around Photosensitizer Dendrimers Francesca Setaro,† Melanie Brasch,‡ Uwe Hahn,†,§ Melissa S. T. Koay,‡ Jeroen J. L. M. Cornelissen,‡ Andrés de la Escosura,*,† and Tomás Torres*,† †
Departamento de Química Orgánica (C−I), Universidad Autónoma de Madrid/IMDEA Nanociencia (TT), Cantoblanco, 28049 Madrid, Spain ‡ Department of Biomolecular Nanotechnology, MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands § Laboratoire de Chimie des Matériaux Moléculaires, Université de Strasbourg et CNRS (UMR 7509), Ecole Européenne de Chimie, Polymères et Matériaux (ECPM), 25 rue Becquerel, 67087 Strasbourg Cedex 2, France S Supporting Information *
ABSTRACT: In this article, we show the great potential of dendrimers for driving the self-assembly of biohybrid protein nanoparticles. Dendrimers are periodically branched macromolecules with a perfectly defined and monodisperse structure. Moreover, they allow the possibility to incorporate functional units at predetermined sites, either at their core, branches, or surface. On these bases, we have designed and synthesized negatively charged phthalocyanine (Pc) dendrimers that behave as photosensitizers for the activation of molecular oxygen into singlet oxygen, one of the main reactive species in photodynamic therapy (PDT). The number of surface negative charges depends on dendrimer generation, whereas Pc aggregation can be tuned through the appropriate choice of the Pc metal center and its availability for axial substitution. Remarkably, both parameters determine the outcome and efficiency of the templated self-assembly process by which a virus protein forms 18 nm virus-like particles around these dendritic chromophores. Protein−dendrimer biohybrid nanoparticles of potential interest for therapeutic delivery purposes are obtained in this way. Biohybrid assemblies of this kind will have a central role in future nanomedical and nanotechnology applications. KEYWORDS: Biohybrid protein nanoparticles, phthalocyanine, dendrimers, photosensitizer
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with different shapes and sizes, generally through electrostatic interactions between the negatively charged cargo surface and the positively charged protein N-terminus. A common feature that all these template species share is their nondiscrete, illdefined size and number of surface negative charges,22,23 which makes difficult to understand and control the specific interactions between template and protein. Dendrimers, which are periodically branched macromolecules with a perfectly defined and monodisperse structure, would be ideal templates for such a goal. Dendrimers allow the possibility of incorporating functional units at predetermined sites, either at their core, branches, or surface. They have actually proven to be useful for multivalent binding of different biomolecules, including peptides,24,25 or between core−shell tecto(dendrimers),26 acting as a supramolecular glue toward complex hierarchical assemblies.27−32 In contrast, they were never employed to template the self-assembly of well-defined
rotein self-assembly processes are ubiquitious in Nature, whereas in nanotechnology, they provide robust and reliable strategies for creating biohybrid materials with novel physicochemical properties.1 Both natural and synthetic protein assemblies are mostly templated by macromolecules such as nucleic acids, polypeptides, and synthetic polymers.2 Singlestranded (ss)RNA virus capsids are a prominent example of closed-shell cages assembled from multiple copies of a coat protein (CP).3−8 Although the detailed assembly mechanism remains unsolved for any virus capsid, in native conditions it is known to be templated by the viral genome. In recent years, there has emerged a need for new applications research toward the encapsulation of other cargo in these protein cages. Such research lines are fundamental in the field of biomedicine, given that virus capsids and other protein cages are considered promising nanocarriers for drug delivery, allowing the possibility to target certain malignant tissues while reducing immune responses and toxicity.9 Toward this end, non-native nucleic acids,10−14 inorganic nanoparticles,15,16 polymers,17,18 micelles,19 and organic dye nanospheres20,21 have been used to template the production of functional virus-like particles (VLP) © 2015 American Chemical Society
Received: November 17, 2014 Revised: January 20, 2015 Published: January 23, 2015 1245
DOI: 10.1021/nl5044055 Nano Lett. 2015, 15, 1245−1251
Letter
Nano Letters Chart 1. Structures of Pc dendrimers ZnPc1, ZnPc2, RuPc1, and RuPc2
(SiPc)52−55 and ruthenium (RuPc)56,57 as central metal atoms have shown good PDT activity. Only a few of them were dendritic and anionic in nature, with the dendron substituents placed at peripheral positions of the Pc ring.48,49,51 Such studies revealed a strong correlation between the degree of dendritic environment (i.e., dendrimer generation), the extent of Pc aggregation and the Pc ability to photogenerate 1O2. Four different Pc dendrimers have been tested as templates for the self-assembly of the CCMV protein into VLP, namely, ZnPc1, ZnPc2, RuPc1, and RuPc2 (Chart 1). ZnPc1 and ZnPc2 are zero- and first-generation ZnPc dendrimers with four Fréchet-type dendritic wedges at the periphery of the Pc macrocycle. Due to the presence of 8 and 16 surface carboxylate groups, respectively, they are water-soluble and, as most ZnPc derivatives in aqueous solution, they present a strong tendency to aggregate by π−π stacking and hydrophobic interactions.58 In contrast, the analogous zero- and firstgeneration dendrimers RuPc1 and RuPc2 present axial substitution, which prevents aggregation, as shown for other RuPc derivatives.59,60 Furthermore, due to the heavy-atom effect, RuPcs give rise to long-lived triplet excited states, potentially favoring the activation of molecular oxygen. In spite of such advantages for the PDT field, only one case of RuPc dendrimers has been previously described,57 and systems bearing terminal carboxylates were never reported, probably because they imply a higher synthetic difficulty.
protein cages. Herein, we have studied the capacity of photoactive dendrimers, decorated with surface negative charges, as templates for driving the self-assembly of the Cowpea Chlorotic Mottel Virus (CCMV)33,34 CP into 18 nm VLP, and how the assembly efficiency is determined by dendrimer generation. Our experiments demonstrate the delicate interplay of electrostatic interactions that drive VLP self-assembly processes. We also show that the synthesized dendritic compounds are good photosensitizers for singlet oxygen generation. Overall, these results highlight the great potential of combining dendrimers and virus proteins for the construction of functional biohybrid nanoparticles. The photosensitizing ability of the presented dendritic templates relies on their core phthalocyanine (Pc) macrocycle. Pcs possess outstanding optoelectronic properties, which arise from their inner 18 π-electron aromatic ring.35,36 Pcs are also very stable and present a strong optical absorption from 630 to 750 nm, which makes them valuable in different scientific and technological areas.37−42 Photodynamic therapy (PDT), a medical technology that uses photosensitive compounds to generate singlet oxygen (1O2) for the localized treatment of cancer and other diseased tissues, or for the inactivation of microorganisms, has grown in popularity during the last decades.43−45 The use of Pcs in PDT requires their adequate functionalization or incorporation into nanocarriers, in order to render them water-soluble and biocompatible.46 In this respect, a number of Pc derivatives with zinc (ZnPc),47−51 silicon 1246
DOI: 10.1021/nl5044055 Nano Lett. 2015, 15, 1245−1251
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Nano Letters
Figure 1. (a) UV−vis spectra of RuPc1 (in blue) and RuPc2 (in red) in DMSO (solid lines) and water (dashed lines). (b) UV−vis spectra of ZnPc1 (in blue) and ZnPc2 (in red) in DMSO (solid lines) and water (dashed lines). For the spectra in DMSO, a small proportion of water was necessary (
