M. Brasch

Encapsulation of Phthalocyanine Supramolecular Stacks into Virus-like Particles

We report herein the encapsulation of a water-soluble phthalocyanine (Pc) into virus-like particles (VLPs) of two different sizes, depending on the conditions. At neutral pH, the cooperative encapsulation/templated assembly of the particles induces the formation of Pc stacks instead of Pc dimers, due to an increased confinement concentration. The Pc-containing VLPs may potentially be used as photosensitizer/vehicle systems for biomedical applications such as photodynamic therapy.

Virus-like Particles Templated by DNA Micelles: A General Method for Loading Virus Nanocarriers

DNA amphiphile particles template formation of virus capsids and enable their loading.

Generation-Dependent Templated Self-Assembly of Biohybrid Protein Nanoparticles around Photosensitizer Dendrimers

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.

Self-assembly and characterization of small and monodisperse dye nanospheres in a protein cage

Phthalocyanines (Pc) are dyes in widespread use in materials science and nanotechnology, with numerous applications in medicine, photonics, electronics and energy conversion. With the aim to construct biohybrid materials, we here prepared and analyzed the structure of two Pc-loaded virus-like particles (VLP) with diameters of 20 and 28 nm (i.e., T = 1 and T = 3 icosahedral symmetries, respectively). Our cryo-electron microscopy (cryo-EM) studies show an unprecedented, very high level of Pc molecule organization within both VLP. We found that 10 nm diameter nanospheres form inside the T = 1 VLP by self-assembly of supramolecular Pc stacks. Monodisperse, self-assembled organic dye nanospheres were not previously known, and are a consequence of capsid-imposed symmetry and size constraints. The Pc cargo also produces major changes in the protein cage structure and in the mechanical properties of the VLP. Pc-loaded VLP are potential photosensitizer/carrier systems in photodynamic therapy (PDT), for which their mechanical behaviour must be characterized. Many optoelectronic applications of Pc dyes, on the other hand, are dependent on dye organization at the nanoscale level. Our multidisciplinary study thus opens the way towards nanomedical and nanotechnological uses of these functional molecules.

Assembling Enzymatic Cascade Pathways inside Virus-Based Nanocages Using Dual-Tasking Nucleic Acid Tags

The packaging of proteins into discrete compartments is an essential feature for cellular efficiency. Inspired by Nature, we harness virus-like assemblies as artificial nanocompartments for enzyme-catalyzed cascade reactions. Using the negative charges of nucleic acid tags, we develop a versatile strategy to promote an efficient noncovalent co-encapsulation of enzymes within a single protein cage of cowpea chlorotic mottle virus (CCMV) at neutral pH. The encapsulation results in stable 21–22 nm sized CCMV-like particles, which is characteristic of an icosahedral T = 1 symmetry. Cryo-EM reconstruction was used to demonstrate the structure of T = 1 assemblies templated by biological soft materials as well as the extra-swelling capacity of these T = 1 capsids. Furthermore, the specific sequence of the DNA tag is capable of operating as a secondary biocatalyst as well as bridging two enzymes for co-encapsulation in a single capsid while maintaining their enzymatic activity. Using CCMV-like particles to mimic nanocompartments can provide valuable insight on the role of biological compartments in enhancing metabolic efficiency.

Self-assembly triggered by self-assembly: optically active, paramagnetic micelles encapsulated in protein cage nanoparticles

In this contribution, optically active and paramagnetic micelles of the ligand 1,4,7,10-tetraaza-1-(1-carboxymethylundecane)-4,7,10-triacetic acid cyclododecane (DOTAC10) have been incorporated inside capsids of the cowpea chlorotic mottle virus (CCMV) protein through a hierarchical process of self-assembly triggered by self-assembly. The DOTAC10 ligand was used to complex Gd(III), in order to form paramagnetic micelles, as well as to encapsulate an amphiphilic Zn(II) phthalocyanine (ZnPc) dye that optically confirmed the encapsulation of the micelles. The incorporation of ZnPc molecules in the paramagnetic micelles led to high capsid loading of both Gd(III) and ZnPc, as the micelles were stabilized by the amphiphilic dye encapsulation. The resulting protein cage nanoparticles (PCNs) show an improved r1 relaxivity, suggesting the possible use of these nanostructures as contrast agents (CAs) for magnetic resonance imaging (MRI). Since the encapsulated ZnPc dye also has a potential therapeutic value, the present results represent a first step towards the consecution of fully self-assembled PCNs for multimodal imaging and therapy.

Phototriggered cargo release from virus-like assemblies

There has been tremendous progress towards the development of responsive polymers that are programmed to respond to an external stimulus such as light, pH and temperature. The unique combination of molecular packaging followed by slow, controlled release of molecular cargo is of particular importance for self-healing materials and the controlled release of drugs. While much focus and progress remains centred around synthetic carriers, viruses and virus-like particles can be considered ideal cargo carriers as they are intrinsically designed to package, protect and deliver nucleic acid cargo to host cells. Here, we report the encapsulation of a stimuli-responsive self-immolative polymer within virus-like assemblies of Cowpea Chlorotic Mottle Virus. Upon photo-irradiation, the self-immolative polymer undergoes a head-to-tail depolymerization into its monomeric subunits, resulting in the slow release of the molecular cargo. We propose that the liberated monomers are small enough to diffuse through the pores of the virus capsid shell and offer an alternative strategy for the controlled loading and unloading of the molecular cargo using viruses as cargo carriers.

Quantum dot encapsulation in virus-like particles with tuneable structural properties and low toxicity

A simple method for the encapsulation of quantum dots (QDs) in virus-like particle (VLP) nanoassemblies with tuneable structural properties and enhanced biocompatibility is presented. Cowpea chlorotic mottle virus-based capsid proteins assemble around the carboxylated QDs to form QD/VLP nanoassemblies of different capsid size as a function of pH and ionic strength. Detailed structural characterizations verify that nanoassemblies with probably native capsid icosahedral symmetry (T = 3) are obtained at low pH and high ionic strength (pH 5.0, 1.0 M NaCl), whereas high pH and low ionic strength conditions (pH 7.5, 0.3 M NaCl) result in the formation of smaller assembly sizes similar to T = 1 symmetry. In vitro studies reveal that QD/VLP nanoassemblies are efficiently internalized by RAW 264.7 macrophages and HeLa cells with no signs of toxicity at QD concentrations exceeding the potentially-toxic levels. The presented route holds great promise for preparation of size-tuneable, robust, non-toxic luminescent probes for long term cellular imaging applications. Furthermore, thanks to the possibility of chemical and genetic manipulation of the viral protein shell encaging the QDs, the nanoassemblies have potential for in vivo targeting applications.

Chapter 8:Protein cages as a new tool in synthetic biology

Synthetic biology involves the design and construction (or reconstruction) of new biological components that exhibit non-natural functionality. The use of small proteins as natural building blocks for the assembly of highly ordered complex structures is an emerging field in synthetic biology. Here, we focus on the use of protein cages, viruses and bacteriophages, which have emerged as promising tools for the development of complex molecular components and ultimately, to the design of artificial organelles and to the creation artificial life.