Evaluation of zeta potential of nanomaterials by electrophoretic light scattering: Fast field reversal versus Slow field reversal modes.

Zeta potential of nanomaterials designed to be used in nanomedicine is an important parameter to evaluate as it influences in vivo behaviour hence biological activity, efficacy and safety. As mentioned by the International Organization for Standardization (ISO), electrophoretic light scattering is a relevant method for evaluating zeta potential. The present work aimed to validate a new protocol based on the application of Fast Field Reversal mode and to explore its scope with nanomaterials investigated as nanomedicines. Its scope was then compared with that of an already validated protocol which uses both Fast Field Reversal and Slow Field Reversal modes. The new protocol was validated within the framework of the application of the Smoluchowski approximation. Its performances complied with the ISO standard. The protocol could be applied to evaluate mean zeta potential of soft nanomaterials including polymer-based nanoparticles and liposomes. However, it appeared unsuitable to evaluate zeta potential of dense nanomaterials including rutile titanium dioxide nanoparticles. Compared with the previously validated protocol which only applied to the determination of zeta potential of polymer nanoparticles, this new validated protocol gives access to the determination of zeta potential to a wider range of nanomedicines under conditions complying with quality control assessments.

Immunotoxicity of poly (lactic-co-glycolic acid) nanoparticles: influence of surface properties on dendritic cell activation.

Modified nanoparticles (NPs) can interact with the immune system by causing its activation to fight tumors or for vaccination. During this activation, dendritic cells (DCs) are effective in generating robust immune response. However, the effect of nanomaterials on dendritic cell (DC) maturation, and the associated adjuvant effect, should be assessed as a novel biocompatibility criteria for biomaterials since immune consequences may constitute potential complications in nanomedicine. Among emerging biomaterials, poly(lactic-co-glycolic acid) NPs (PLGA NPs) are widely explored for various applications in which the degree of desired adjuvant effect may vary. As contradictory results are reported regarding their effects on DCs, we aimed at clarifying this point with particular emphasis on the relative impact of particle surface properties. To that end, NP uptake and effects on the viability, phenotype, and secretory activity of DC primary cultures. Intracellular signaling pathways were explored and evaluated. Immature human and murine DCs were exposed to cationic, neutral, or anionic PLGA NPs. Particle uptake was assessed by both confocal microscopy and flow cytometry. Cell viability was then evaluated prior to the study of maturation by examination of both surface marker expression and cytokine release. Our results demonstrate that PLGA NPs are rapidly engulfed by DCs and do not exert cytotoxic effects. However, upon exposure to PLGA NPs, DCs showed phenotypes and cytokine secretion profiles consistent with maturation which resulted, at least in part, from the transient intracellular activation of mitogen-activated protein kinases (MAPKs). Interestingly, NP-specific stimulation patterns were observed since NP surface properties had a sensible influence on the various parameters measured.

Size of monodispersed nanomaterials evaluated by dynamic light scattering: Protocol validated for measurements of 60 and 203nm diameter nanomaterials is now extended to 100 and 400nm.

In vivo fate of nanomaterials is influenced by the particle size among other parameters. Thus, Health Agencies have identified the size of nanomaterial as an essential physicochemical property to characterize. This parameter can be explored by dynamic light scattering (DLS) that is described in the ISO standard 22412:2008(E) and is one of the methods recognized by Health Agencies. However, no protocol of DLS size measurement has been validated over a large range of size so far. In this work, we propose an extension of validation of a protocol of size measurement by DLS previously validated with certified reference materials (CRM) at 60 and 203nm. The present work reports robustness, precision and trueness of this protocol that were investigated using CRM at 100 and 400nm. The protocol was robust, accurate and consistent with the ISO standard over the whole range of size that were considered. Expanded uncertainties were 4.4 and 3.6% for CRM at 100 and 400nm respectively indicating the reliability of the protocol. The range of application of the protocol previously applied to the size measurement of liposomes and polymer nanoparticles was extended to inorganic nanomaterial including silica nanoparticles.

Freeze-drying of squalenoylated nucleoside analogue nanoparticles.

Nucleoside analogues are potent anticancer or antiviral agents that however display some limitations (rapid metabolism, induction of resistance). In order to overcome these drawbacks, we recently proposed new prodrugs, in which nucleoside analogues were covalently coupled to squalene (SQ). The resulting amphiphilic compounds spontaneously formed nanoparticles (NPs) and displayed a promising efficacy both in vitro and in vivo. Since long-term stability is essential for further clinical development we needed to develop a laboratory-scale freeze-drying protocol in order to improve the colloidal stability of those NPs. Squalenoylated gemcitabine (SQdFdC) has been successfully freeze-dried with trehalose (10%, w/w) as a cryoprotectant. Concentrations of SQdFdC up to 4mg/mL after freeze-drying and rehydration have been obtained, which is necessary for in vivo studies. Stability measurements by dynamic light scattering showed that trehalose had a stabilizing effect on SQdFdC NPs, and that freeze-dried SQdFdC NPs could be stored up to four months at room temperature before rehydration, without loss of stability. In vitro cytotoxicity studies on three murine cell lines showed that SQdFdC NPs retained their cytotoxic activity after freeze-drying. We showed that this freeze-drying protocol could also be applied to squalenoylated didanosine (SQddI) and zalcitabine (SQddC). Overall, these results allow for the use of freeze-dried NPs in upcoming preclinical trials of the different squalenoylated compounds developed in our laboratory.

Encapsulation of mono- and oligo-nucleotides into aqueous-core nanocapsules in presence of various water-soluble polymers.

In a previous study, we have shown that cidofovir (CDV) and azidothymidine-triphosphate (AZT-TP) were poorly encapsulated in poly(iso-butylcyanoacrylate) (PIBCA) aqueous-core nanocapsules. This was attributed to the rapid leakage of these small and hydrophilic molecules through the thin polymer wall of the nanocapsules. In the present study, we have selected various water-soluble polymers as increasing Mw adjuvants and investigated their influence on the entrapment of mononucleotides (CDV, AZT-TP) as well as of oligonucleotides (ODN) into these PIBCA aqueous-core nanocapsules. We show here that the presence of cationic polymers (i.e. poly(ethyleneimine) (PEI) or chitosan) in the nanocapsule aqueous compartment allowed successful encapsulation of AZT-TP and ODN.

Encapsulation of antiviral nucleotide analogues azidothymidine-triphosphate and cidofovir in poly(iso-butylcyanoacrylate) nanocapsules.

Nucleoside analogues are widely used in the treatment of various viral infections. However, the poor in vivo conversion of the nucleoside analogues like azidothymidine (AZT) into their active triphosphate nucleotide counterpart limits their pharmacological efficacy. This could be overcome by the direct administration of azidothymidine triphosphate (AZT-TP), but it requires an appropriate drug delivery approach. Besides nucleoside analogues, nucleotide analogues like cidofovir (CDV) are also used in the treatment of viral infections. CDV has raised recent interest because of its promising activity against smallpox, but its use is limited by its poor bioavailability and nephrotoxicity. Here again, a proper drug delivery system should address these issues. In this study, we investigated the encapsulation of the nucleotide analogues AZT-TP and CDV into poly(iso-butylcyanoacrylate) aqueous core nanocapsules, known to efficiently entrap oligonucleotides. We show here that the encapsulation of these mono-nucleotides is less efficient than with oligonucleotides and that a rapid release of AZT-TP from the nanocapsules occurred in vitro. This highlights the importance of the molecular weight of the entrapped molecules which, if they are too small, are diffusing through the thin polymer membrane of the nanocapsules. On the other hand, a good protection of the encapsulated AZT-TP was observed.