Overcoming immunogenicity issues of HIV p24 antigen by the use of innovative nanostructured lipid carriers as delivery systems: evidences in mice and non-human primates.

HIV is one of the deadliest pandemics of modern times, having already caused 35 million deaths around the world. Despite the huge efforts spent to develop treatments, the virus cannot yet be eradicated and continues to infect new people. Spread of the virus remains uncontrolled, thus exposing the worldwide population to HIV danger, due to the lack of efficient vaccines. The latest clinical trials describe the challenges associated with developing an effective prophylactic HIV vaccine. These immunological obstacles will only be overcome by smart and innovative solutions applied to the design of vaccine formulations. Here, we describe the use of nanostructured lipid carriers (NLC) for the delivery of p24 protein as a model HIV antigen, with the aim of increasing its immunogenicity. We have designed vaccine formulations comprising NLC grafted with p24 antigen, together with cationic NLC optimized for the delivery of immunostimulant CpG. This tailored system significantly enhanced immune responses against p24, in terms of specific antibody production and T-cell activation in mice. More importantly, the capacity of NLC to induce specific immune responses against this troublesome HIV antigen was further supported by a 7-month study on non-human primates (NHP). This work paves the way toward the development of a future HIV vaccine, which will also require the use of envelope antigens.

Synthetic ability of dinuclear mesocates containing 1,3-bis(diazinecarboxamide)benzene bridging ligands to form complexes of increased nuclearity. Crystal structures, magnetic properties and theoretical studies.

Triple stranded Ni-metallacyclic complexes Na2.5[Ni2(bpcb)3]·0.5OH·18.5H2O (1) and Na2[Ni2(bpzcb)3]·16H2O (2), and double stranded Cu-metallacyclic complexes [Cu2(bpcb)2(H2O)2]·8H2O (3) and [Cu2(bpzcb)2(H2O)2]·4H2O (4) have been assembled from the tailored bisbidentate bridging ligands, 1,3-bis(pyrimidine-2-carboxamide)benzene (H2bpcb) and 1,3-bis(pyrazine-2-carboxamide)benzene (H2bpzcb), and the corresponding nitrate salts of the metal ions. Following the "complex as ligand" strategy, 1 can be assembled with either Ni2+, Co2+ ions or the [Mn(acen)Cl] complex to afford unique, neutral, bent trinuclear molecules [MIINi(bpcb)3]·xH2O (5 and 6) and the 2D honeycomb-like complex (PPh4){[Ni2(bpcb)3]2[Mn(acen)]3} (7), respectively. In these cases, the Ni2 units are linked to the corresponding metal ions through amidate oxygen atoms and the outward nitrogen atom of one of the pyrimidine rings of the bcpb ligand. The assembly of 2 with Ln3+ ions (Ln3+ = Tb, Gd) leads to one dimensional complexes of formula [{[Ni2(bpzcb)3]Tb(H2O)5}(CF3SO3)·THF·5H2O]n (8) and [{[Ni2(bpzcb)3]Ln(H2O)4(NO3)}·2THF·nH2O]n (9 and 10) (Ln3+ = Gd and Tb), where the dinuclear Ni2 units are joined to two Ln3+ ions exclusively through amidate oxygen atoms of two different ligands. The analyses of the magnetic data indicate that 1-4 exhibit intradinuclear ferromagnetic interactions between the metal ions through a spin polarisation mechanism, as supported by DFT calculations. Trinuclear complexes 5 and 6 show predominant antiferromagnetic coupling, which is a result of an antiferromagnetic interaction between one of the Ni2+ ions of the Ni2 unit and the M2+ ion through the pyrimidine bridging fragment that is stronger than the polarised ferromagnetic interaction between the Ni2+ ions through the bpcb ligand in the dinuclear [Ni2(bpcb)3]2- moiety. Complex 7 shows a dominant antiferromagnetic interaction between the Ni2+ and Mn2+, whereas the Ni2Ln (Ln3+ = Gd, Tb) chain complexes present ferromagnetic interactions inside the Ni2 mesocate unit as well as between the Ni2+ ions of the Ni2 unit and the Ln3+ ions. The magnetic exchange interactions in these new materials have been experimentally analysed and supported by theoretical DFT studies.

Small rigid platforms functionalization with quaternary ammonium: targeting extracellular matrix of chondrosarcoma.

This work takes place in the "cartilage targeting strategy", consisting in using the quaternary ammonium (QA) function as a vector to proteoglycans (PGs) of extracellular matrix (ECM). The objective was to demonstrate that QA could address gadolinium based small rigid platforms (SRP) to PG-rich tumors. SRP were functionalized with QA, radiolabeled with (111)Indium and evaluated for biodistribution in vivo, respectively to non functionalized SRP, in two experimental models: (i) the HEMCSS human xenograft model; (ii) the Swarm rat chondrosarcoma (SRC) orthotopic model. The contribution of cellular uptake to tumoral accumulation of nano-objects was also determined from in vitro binding. In the SRC model expressing a highly and homogeneously distributed PG content, tumor accumulation and retention of SRP@QA were increased by 40% as compared to non-functionalized SRP. When considering the radiosensitizing potential of gadolinium based SRP, these results provide hopes for the radiobiological approach of highly resistant tumor such as chondrosarcoma. FROM THE CLINICAL EDITOR: In this study, gadolinium-based complexing DOTA-surfaced small polysiloxane nanoparticles were functionalized with quaternary ammonium derivatives that target the extracellular matrix of chondrosarcoma. The authors demonstrate in a rat model that the use of these constructs results in a 40% increase of tumor accumulation and retention compared to non-functionalized (and otherwise same) platforms. Similar approaches would be welcome additions to the clinical armamentarium addressing chondrosarcoma.

Functionalization of small rigid platforms with cyclic RGD peptides for targeting tumors overexpressing αvß3-integrins.

Gadolinium based Small Rigid Plaforms (SRPs) have previously demonstrated their efficiency for multimodal imaging and radiosensitization. Since the RGD sequence is well-known to be highly selective for αvß3 integrins, a cyclic pentapeptide containing the RGD motif (cRGDfK) has been grafted onto the SRP surface. An appropriate protocol led to the grafting of two targeting ligands per nano-object. The resulting nanoparticles have demonstrated a strong association with αvß3 integrins in comparison with cRADfK grafted SRPs as negative control. Flow cytometry and fluorescence microscopy have also been used to highlight the ability of the nanoparticles to target efficiently HEK293(ß3) and U87MG cells. Finally the grafted radiosensitizing nanoparticles were intravenously injected into Nude mice bearing subcutaneous U87MG tumors and the signal observed by optical imaging was twice as high for SRP-cRGDfK compared to their negative analogue.

In vivo evidence of the targeting of cartilaginous tissue by pyridinium functionalized nanoparticles.

Ultrasmall gadolinium based particles have been functionalized with positively charged pyridinium quaternary ammonium and labelled with (111)In. Evidence of their active targeting properties towards proteoglycans has been demonstrated in vivo after intravenous injection into rats opening thus a route to cancer imaging and therapy.

Development of gadolinium based nanoparticles having an affinity towards melanin.

Small Rigid Platforms (SRPs) are sub-5 nanometre gadolinium based nanoparticles that have been developed for multimodal imaging and theranostic applications. They are composed of a polysiloxane network surrounded by gadolinium chelates. A covalent coupling with quinoxaline derivatives has been performed. Such derivatives have proven their affinity for melanin frequently expressed in primary melanoma cases. Three different quinoxaline derivatives have been synthesised and coupled to the nanoparticles. The affinity of the grafted nanoparticles for melanin has then been shown in vitro by surface plasmon resonance on a homemade melanin grafted gold chip.