RESUMEN
Stealth nanocarriers are a promising technology for the treatment of diseases. However, the preparation and characterization of well-defined soft nanoparticulate systems remain challenging. Here we describe a platform of amphiphilic graft copolymers leading to nanoparticles with multiple morphologies and the role of the hydrophilic backbone in their interaction with a model protein. The amphiphilic graft copolymers platform was composed of hydrophilic backbone poly(2-methyl-2-oxazoline-co-2-pentyl-2-oxazoline) (P(MeOx-co-PentOx)), prepared via cationic ring-opening polymerization. Hydrophobic poly(d,l-lactide) (PLA) chains were grafted on the backbone via Huisgen 1,3-dipolar cycloaddition. The "click" copper-catalyzed cycloaddition reactions of azides with alkynes (CuAAC) were successfully carried out, and a series of amphiphilic copolymers were prepared containing a backbone with a number-average molecular weight of 14.2 × 103 g mol-1 and different hydrophobic PLA grafts with various molecular weights (2.8 × 103-12.4 × 103 g mol-1). These original architectures of copolymers, when nanoprecipitated in water, the backbone-selective solvent, allowed us to obtain various structures of nanoparticles with a hydrodynamic diameter in the range of 65-99 nm. More interestingly, a plurality of morphologies going from unilamellar, multilamellar, and large compound vesicles to core-shell nanoparticles and depending on the PLA molecular weights were evidenced by combining cryo-transmission electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS) studies. A first evaluation of their stealthiness by studying the stability and the interaction of these nano-objects with a model protein revealed the role played by the P(MeOx-co-PentOx) in these interactions, demonstrating the utility of this amphiphilic graft copolymers platform with well-defined architectures for the design of nanocarriers in drug delivery applications.
RESUMEN
Cell-free hemoglobin (Hb)-based oxygen carriers have long been proposed as blood substitutes but their clinical use remains tricky due to problems of inefficiency and/or toxicity. Conjugation of Hb with the biocompatible polymer poly(ethylene glycol) (PEG) greatly improved their performance. However, physiological data suggested a polymer molecular weight (Mw) threshold of about 10 kDa, beyond which the grafting of two PEG chains no longer improves efficiency and nontoxicity of diPEG/Hb conjugates. We used small-angle neutron scattering and contrast variation, which are the only techniques able to probe separately the conformation of PEG chains and Hb protein within the complex, to investigate the role of PEG chain conformation in diPEGylated Hb conjugates as a function of the polymer Mw. We found out that the structure of Hb tetramer is not modified by the polymer grafting. Similarly, with a constant grafting of two chains per protein, there is no significant change of the Gaussian conformation between free and grafted PEG below â¼10 kDa, the complex being well described by the "dumbbell" model. However, beyond that threshold, the radius of gyration of grafted PEG is significantly smaller than that of the free polymer, showing a compaction of the PEG chains, either in the "dumbbell" model or in the "shroud" one. In the latter model, the polymer may be wrapped on the surface of the protein spreading a protective "shielding" effect over a larger fraction of the protein. Both proposed models are in good agreement with the physiological data reported in the literature.
Asunto(s)
Hemoglobinas/química , Polietilenglicoles/química , Humanos , Modelos Moleculares , Conformación Molecular , Difracción de Neutrones , Dispersión del Ángulo PequeñoRESUMEN
This Letter reports on the remarkable selectivity of capsid proteins for packaging synthetic polyelectrolytes in viruslike particles. By applying the contrast variation method in small-angle neutron scattering, we accurately estimated the mean mass of packaged polyelectrolytes ⟨Mp⟩ and that of the surrounding capsid ⟨Mcap⟩. Remarkably, the mass ratio ⟨Mp⟩/⟨Mcap⟩ was invariant for polyelectrolyte molecular weights spanning more than 2 orders of magnitude. To do so, capsids either packaged several chains simultaneously or selectively retained the shortest chains that could fit the capsid interior. Our data are in qualitative agreement with theoretical predictions based on free energy minimization and emphasize the importance of protein self-energy. These findings may give new insights into the nonspecific origin of genome selectivity for a number of viral systems.
Asunto(s)
Proteínas de la Cápside/química , Electrólitos/química , Virión/química , Cápside/química , Modelos Químicos , Modelos Moleculares , Difracción de Neutrones , ARN Viral/química , Dispersión del Ángulo PequeñoRESUMEN
In the Caliciviridae family of nonenveloped, positive-stranded RNA viruses, Noroviruses are major causes of human and animal gastroenteritis worldwide. The Norovirus T=3 icosahedral capsid is made of 180 copies of the VP1 protein, as exemplified in the crystal structure of the virus-like particle (VLP) of the human Norwalk virus (NV). It was previously shown that the ca 40-nm recombinant NV VLP can be disassembled and reassembled in vitro. Here we report on the disassembly and self-assembly properties for the related (VP1 sequence identity of 50%) bovine Newbury2 Norovirus (NB2) VLP. Using a panel of biophysical techniques, we show that while the NB2 VLP displays disassembly properties similar to the NV VLP, NB2-VP1 shows remarkable self-assembly properties heretofore unreported for NV-VP1 or any other calicivirus capsid protein. These properties include the capabilities of self-assembling not only into regular T=3 capsids but also into larger VLP (up to 76 nm in diameter) and of tolerating substitution of the spike domain for that of a distantly related Calicivirus. In conditions favoring the natural, T=3 capsid, NB2-VP1 reproducibly assembles by an apparent two-phase process. Our results establish a robust new system with which to probe the dynamics of viral capsid self-assembly.
Asunto(s)
Norovirus/química , Virión/química , Virión/ultraestructura , Ensamble de Virus , Cristalización/métodos , Dimerización , Conformación ProteicaRESUMEN
HYPOTHESIS: We describe the possibility of using the same block copolymer carriers prepared by PISA for in situ drug encapsulation or drug conjugation. EXPERIMENTS: Block copolymers containing poly((ethylene glycol) methacrylate)-co-poly(pentafluorophenyl methacrylate)-b-poly(hydroxypropyl methacrylate) (P((PEGMA-co-PFBMA)-b-PHPMA)) were synthesized at 10 wt% using PISA. The first approach involved in situ Doxorubicin (DOX) loading during PISA, while the second exhibited surface functionalization of PISA-made vesicles with dual drug therapies, N-acetyl cysteine (NAC) and DOX using para-fluoro-thiol reaction (PFTR) and carbodiimide chemistry, respectively. Cytotoxicity, cell uptake, and cell apoptosis were assessed on MDA-MB-231 cell lines. FINDINGS: P((PEGMA-co-PFBMA)-b-PHPMA) nanocarriers were prepared, showing size and shape transformations from spheres, cylinders to raspberry-forming vesicles. DOX was readily loaded into NPs during PISA with relatively high encapsulation efficiency of 70 %, whereas the plain PISA-made vesicles could be functionalized with NAC and DOX at high yields. DOX-free NPs showed biocompatibility, whilst DOX-conjugated NPs imparted a concentration-dependent cytotoxicity, as well as an enhanced cell uptake compared to free DOX. The results demonstrated that the same PISA-derived self-assemblies enabled either in situ drug encapsulation, or post-polymerization surface engineering with useful functionalities upon tuning the macro-CTA block, thus holding promises for future drug delivery and biomedical applications.