Chaperoned amyloid proteins for immune manipulation: a-Synuclein/Hsp70 shifts immunity toward a modulatory phenotype

α-Synuclein (αSyn) is a 140-residue amyloid-forming protein whose aggregation is linked to Parkinson's disease (PD). It has also been found to play a critical role in the immune imbalance that accompanies disease progression, a characteristic that has prompted the search for an effective αSyn-based immunotherapy. In this study, we have simultaneously exploited two important features of certain heat-shock proteins (HSPs): their classical “chaperone” activities and their recently discovered and diverse “immunoactive” properties. In particular, we have explored the immune response elicited by immunization of C57BL/6 mice with an αSyn/Hsp70 protein combination in the absence of added adjuvant. Our results show differential effects for mice immunized with the αSyn/Hsp70 complex, including a restrained αSyn-specific (IgM and IgG) humoral response as well as minimized alterations in the Treg (CD4+CD25+Foxp3+) and Teff (CD4+Foxp3−) cell populations, as opposed to significant changes in mice immunized with αSyn and Hsp70 alone. Furthermore, in vitro-stimulated splenocytes from immunized mice showed the lowest relative response against αSyn challenge for the “αSyn/Hsp70” experimental group as measured by IFN-γ and IL-17 secretion, and higher IL-10 levels when stimulated with LPS. Finally, serum levels of Th1-cytokine IFN-γ and immunomodulatory IL-10 indicated a unique shift toward an immunomodulatory/immunoprotective phenotype in mice immunized with the αSyn/Hsp70 complex. Overall, we propose the use of functional “HSP-chaperoned amyloid/aggregating proteins” generated with appropriate HSP-substrate protein combinations, such as the αSyn/Hsp70 complex, as a novel strategy for immune-based intervention against synucleinopathies and other amyloid or “misfolding” neurodegenerative disorders.España, Ministerio de Economía y Competitividad SAF-2012/39720Junta de Andalucía P10-CTS-6928Junta de Andalucía P11-CTS-816

Chaperoned amyloid proteins for immune manipulation: A-synuclein/hsp70 shifts immunity toward a modulatory phenotype

a-Synuclein (aSyn) is a 140-residue amyloid-forming protein whose aggregation is linked to Parkinson’s disease (PD). It has also been found to play a critical role in the immune imbalance that accompanies disease progression, a characteristic that has prompted the search for an effective aSyn-based immunotherapy. In this study, we have simultaneously exploited two important features of certain heat-shock proteins (HSPs): their classical ‘‘chaperone’’ activities and their recently discovered and diverse ‘‘immunoactive’’ properties. In particular, we have explored the immune response elicited by immunization of C57BL/6 mice with an aSyn/Hsp70 protein combination in the absence of added adjuvant. Our results show differential effects for mice immunized with the aSyn/Hsp70 complex, including a restrained aSyn-specific (IgM and IgG) humoral response as well as minimized alterations in the Treg (CD4 CD25 Foxp3 ) and Teff (CD4 Foxp3 ) cell populations, as opposed to significant changes in mice immunized with aSyn and Hsp70 alone. Furthermore, in vitro-stimulated splenocytes from immunized mice showed the lowest relative response against aSyn challenge for the ‘‘aSyn/Hsp70’’ experimental group as measured by IFN-g and IL-17 secretion, and higher IL-10 levels when stimulated with LPS. Finally, serum levels of Th1-cytokine IFN-g and immunomodulatory IL-10 indicated a unique shift toward an immunomodulato-ry/immunoprotective phenotype in mice immunized with the aSyn/Hsp70 complex. Overall, we propose the use of functional ‘‘HSP-chaperoned amyloid/ aggregating proteins’’ generated with appropriate HSP-substrate protein combinations, such as the aSyn/Hsp70 complex, as a novel strategy for immune-based intervention against synucleinopathies and other amyloid or ‘‘misfolding’’ neurodegenerative disorders.Financial support was provided by the Carlos III Institute of Health of Spain (Spanish Ministry of Economy and Competitiveness) according to the Strategic Action in Health (CP10/00527 to CR; PI14-01600 to DP) with co-funding by FEDER funds, the Spanish Ministry of Economy and Competitiveness (SAF-2012/39720 to CR), the Andalusian Ministry of Economy, Science and Innovation (P10-CTS-6928 and P11-CTS-8161 to DP) and the PAIDI Program from the Andalusian Government (CTS- 677 to DP). ALG holds a FPU Predoctoral Fellowship from the Spanish Ministry of Education (AP-2009/3816). The works of EJDG and CMD are supported by the Wellcome Trust, and the UK Medical, and Biotechnological and Biological Sciences Research Councils

Design of antibody-functionalized carbon nanotubes filled with radioactivable metals towards a targeted anticancer therapy

In the present work we have devised the synthesis of a novel promising carbon nanotube carrier for the targeted delivery of radioactivity, through a combination of endohedral and exohedral functionalization. Steam-purified single-walled carbon nanotubes (SWCNTs) have been initially filled with radioactive analogues (i.e. metal halides) and sealed by high temperature treatment, affording closed-ended CNTs with the filling material confined in the inner cavity. The external functionalization of these filled CNTs was then achieved by nitrene cycloaddition and followed by the derivatization with a monoclonal antibody (Cetuximab) targeting the epidermal growth factor receptor (EGFR), overexpressed by several cancer cells. The targeting efficiency of the so-obtained conjugate was evaluated by immunostaining with a secondary antibody and by incubation of the CNTs with EGFR positive cells (U87-EGFR+), followed by flow cytometry, confocal microscopy or elemental analyses. We demonstrated that our filled and functionalized CNTs can internalize more efficiently in EGFR positive cancer cells

Polymeric glabrescione B nanocapsules for passive targeting of Hedgehog-dependent tumor therapy in vitro

Aim: With the purpose of delivering high doses of glabrescione B (GlaB) to solid tumors after systemic administration, long-circulating GlaB-loaded oil-cored polymeric nanocapsules (NC-GlaB) were formulated. Materials & methods: Synthesis of GlaB and its encapsulation in nanocapsules (NCs) was performed. Empty and GlaB- loaded NCs were assessed for their physico-chemical properties, in vitro cytotoxicity and in vivo biodistribution. Results: GlaB was ef ciently loaded into NCs (~90%), which were small (~160 nm), homogeneous and stable upon storage. Further, GlaB and NC-GlaB demonstrated speci c activities against the cancer stem cells. Preliminary studies in tumor-bearing mice supported the ability of NC to accumulate in pancreatic tumors. Conclusion: This study provides early evidence that NC-GlaB has the potential to be utilized in a preclinical setting and justi es the need to perform therapeutic experiments in mice

Nanotechnology approaches for improved vasoactive intestinal peptide based-drug delivery systems

The specific purpose of this project is to study the feasibility as a proof of concept of different nanotechnology approaches in order to improve the drug delivery of the neuropeptide vasoactive intestinal peptide (VIP). Two main questions have been addressed using VIP engineered NPs: 1. VIP functionalized gold NPs. In this case, VIP could act as a drug/diagnostic molecule for theranostic applications. 2. VIP functionalised liposomes as a targeting agent to transport drugs to a specific tissue site. The rationale behind the first part of this study is to substantially increase the half-life of VIP in biological fluids to be employed in therapeutics. VIP-based drug design is hampered by the instability of the peptide and has limited bioavailability. For this reason VIP was functionalized to gold nanoparticles to investigate a potential protection from protease degradation and the mechanisms involved in this process. Remarkably, although it has been hypothesized that surface functionalization of proteins and bioactive peptides on noble metallic nanoclusters might protect from protease degradation, so far there are no formal proofs in this sense. Our aim is to prove that coating gold NPs with the neuropeptide VIP impairs the hydrolytic activity of extracellular proteases, leading to VIP-mediated functional responses after harsh conditions resembling the extracellular circulating proteases milieu. We used several experimental approaches combining physical and chemical characterization to determine size, dispersion and homogeneity of VIP AuNPs by AFM/TEM/DLS analysis as well as additional biochemical quantification of VIP-elicited cellular responses mediated by specific VIP receptors. Our study places the concept of surface functionalization in the broader perspective of proteins escaping from extracellular proteases, which could represent a major driven force and an added value to steer the research in the field of engineering NPs. In the second part of this study, VIP was used as a directing agent of a drug-loaded liposome to prostate cancer cells, and was compared with a non-targeted liposome in order to evaluate its therapeutic efficacy. The specific delivery of anticancer drugs to prostate cancer cells has important implications for diagnosis and therapy. Biomarkers that differentiate cancerous tissues from normal tissues can be used as targets for this purpose and one of these attractive molecular targets is VIP receptors which are overexpressed in human prostate cancer compared to normal prostate tissue. For this reason VIP liposomes have been synthesized to exploit VIP receptors to actively target carriers to PCa and therefore improve its therapy. The use of liposomes is recognized as a promising strategy for improving the delivery of anticancer drugs to tumours, leading to a reduction in drug toxicity and improving the therapeutic outcomes. Furthermore, VIP liposomes were used to encapsulate doxorubicin (an anticancer anthracycline antibiotic) to prostate cancer cells. The aim of this study was to assess the potential of VIP as a ligand for prostate cancer targeting by liposomal nanocarriers and evaluate the efficacy of the treatment. Moreover, we wanted to investigate the effect of a peptide coupling method on the cellular uptake, cytotoxicity and apoptosis of doxorubicin liposomal formulations. In addition, in vivo experiments were addressed in a preclinical setting in order to evaluate the VIP active driven targeting to the prostate cancer cells by liposomes.Peer Reviewe

Nanotechnology-based manipulation of dendritic cells for enhanced immunotherapy strategies

7 páginas, 2 figuras, 1 tabla.-- Potential Clinical Relevance.Dendritic cells (DCs) are potent antigen-presenting cells capable of initiating a primary immune response and possess the ability to activate T cells and stimulate the growth and differentiation of B cells. DCs provide a direct connection between innate and adaptive immune response, and arise from bone marrow precursors that are present in immature forms in peripheral tissues, where they are prepared to capture antigens. DCs migrate from the peripheral tissues to the closest lymph nodes through afferent lymphatic vessels to present the foreign antigens, stimulating T-cell activation and initiating a cellular immune response. Moreover, it is known that DCs have an important role in various diseases and conditions involving the immune system, particularly in cancer and autoimmune disorders. For these reasons, targeting nanoparticles (NPs) to DCs provides a promising strategy for developing an efficient balanced and protective immune response. NPs can modulate the immune response and might be potentially useful as effective vaccine adjuvants for infectious disease and cancer therapy. The objective of this review is to present the latest advances in NP delivery methods targeting DCs, the mechanisms of action, potential effects, and therapeutic results of these systems and their future applications, such as improved vaccination strategies, cancer immunotherapy, and immunomodulatory treatments. [From the Clinical Editor]: Dendritic cells (DCs) are potent antigen-presenting cells capable of initiating a primary immune response and activating T and B cells. The role of DC-s can be considered as a bridge between innate and adaptive immunity. Targeting nanoparticles (NPs) to DCs can modulate the immune response and might be useful as vaccine adjuvants in infectious disease and cancer therapy.The authors are grateful for financial support from the Spanish Ministry of Health (PI05/2056; PI06/1641; PS09/2252); the Andalusian Ministry of Health (PI0068), and the PAIDI Program from the Andalusian Government (BIO323).Peer reviewe