Loco-regional recurrences and distant metastases represent the main cause of head and neck squamous cell carcinoma(HNSCC) mortality. The overexpression of chemokine receptor 4(CXCR4) in HNSCC primary tumors associates...Loco-regional recurrences and distant metastases represent the main cause of head and neck squamous cell carcinoma(HNSCC) mortality. The overexpression of chemokine receptor 4(CXCR4) in HNSCC primary tumors associates with higher risk of developing loco-regional recurrences and distant metastases, thus making CXCR4 an ideal entry pathway for targeted drug delivery. In this context, our group has generated the self-assembling protein nanocarrier T22-GFP-H6, displaying multiple T22 peptidic ligands that specifically target CXCR4. This study aimed to validate T22-GFP-H6 as a suitable nanocarrier to selectively deliver cytotoxic agents to CXCR4^(+)tumors in a HNSCC model. Here we demonstrate that T22-GFP-H6 selectively internalizes in CXCR4^(+)HNSCC cells, achieving a high accumulation in CXCR4^(+)tumors in vivo, while showing negligible nanocarrier distribution in non-tumor bearing organs. Moreover, this T22-empowered nanocarrier can incorporate bacterial toxin domains to generate therapeutic nanotoxins that induce cell death in CXCR4-overexpressing tumors in the absence of histological alterations in normal organs. Altogether, these results show the potential use of this T22-empowered nanocarrier platform to incorporate polypeptidic domains of choice to selectively eliminate CXCR4^(+)cells in HNSCC. Remarkably, to our knowledge, this is the first study testing targeted proteinonly nanoparticles in this cancer type, which may represent a novel treatment approach for HNSCC patients.展开更多
Modular protein engineering is suited to recruit complex and multiple functionalities in single-chain polypeptides. Although still unexplored in a systematic way, it is anticipated that the positioning of functional d...Modular protein engineering is suited to recruit complex and multiple functionalities in single-chain polypeptides. Although still unexplored in a systematic way, it is anticipated that the positioning of functional domains would impact and refine these activities, including the ability to organize as supramolecular entities and to generate multifunctional protein materials. To explore this concept, we have repositioned functional segments in the modular protein T22-GFP-H6 and characterized the resulting alternative fusions. In T22-GFP-H6, the combination of T22 and H6 promotes selfassembling as regular nanoparticles and selective binding and internalization of this material in CXCR4-overexpressing tumor cells, making them appealing as vehicles for selective drug delivery. The results show that the pleiotropic activities are dramatically affected in module-swapped constructs, proving the need of a carboxy terminal positioning of H6 for protein self-assembling, and the accommodation of T22 at the amino terminus as a requisite for CXCR4^+ cell binding and internalization. Furthermore, the failure of self-assembling as regular oligomers reduces cellular penetrability of the fusions while keeping the specificity of the T22-CXCR4 interaction.All these data instruct how multifunctional nanoscale protein carriers can be designed for smart, protein-driven drug delivery, not only for the treatment of CXCR4^+ human neoplasias, but also for the development of anti-HIV drugs and other pathologies in which CXCR4 is a relevant homing marker.展开更多
Poly-histidine peptides such as H6(HHHHHH)are used in protein biotechnologies as purification tags,protein-assembling agents and endosomal-escape entities.The pleiotropic properties of such peptides make them appealin...Poly-histidine peptides such as H6(HHHHHH)are used in protein biotechnologies as purification tags,protein-assembling agents and endosomal-escape entities.The pleiotropic properties of such peptides make them appealing to design protein-based smart materials or nanoparticles for imaging or drug delivery to be produced in form of recombinant proteins.However,the clinical applicability of H6-tagged proteins is restricted by the potential immunogenicity of these segments.In this study,we have explored several humanized histidine-rich peptides in tumor-targeted modular proteins,which can specifically bind and be internalized by the target cells through the tumoral marker CXCR4.We were particularly interested in exploring how protein purification,self-assembling and endosomal escape perform in proteins containing the variant histidine-rich tags.Among the tested candidates,the peptide H5 E(HEHEHEHEH)is promising as a good promoter of endosomal escape of the associated fulllength protein upon endosomal internalization.The numerical modelling of cell penetration and endosomal escape of the tested proteins has revealed a negative relationship between the amount of protein internalized into target cells and the efficiency of cytoplasmic release.This fact demonstrates that the His-mediated,proton sponge-based endosomal escape saturates at moderate amounts of internalized protein,a fact that might be critical for the design of protein materials for cytosolic molecular delivery.展开更多
基金supported by Instituto de Salud CarlosⅢ(ISCIII,SpainCo-funding from FEDER,European Union)[PI18/00650,PIE15/00028,PI15/00378 and EU COST Action CA 17140 to Ramon Mangues,PI19/01661 to Xavier León,and PI17/00584 to Miquel Quer]+7 种基金Agencia Estatal de Investigación(AEI,Spain)and Fondo Europeo de Desarrollo Regional(FEDER,European Union)[grant BIO2016-76063-R,AEI/FEDER,UE to Antonio Villaverde and grant PID2019-105416RB-I00/AEI/10.13039/501100011033 to Esther Vazquez]CIBER-BBN(Spain)[CB06/01/1031 and 4NanoMets to Ramon Mangues,VENOM4CANCER to Antonio Villaverde,NANOREMOTE to Esther Vazquez,and NANOSCAPE to Ugutz Unzueta]AGAUR(Spain)2017-SGR865 to Ramon Mangues,and 2017SGR-229 to Antonio VillaverdeJosep Carreras Leukemia Research Institute(Spain)[P/AG to Ramon Mangues]supported by a predoctoral fellowship from AGAUR(Spain)(2020FI_B200168 and 2018FI_B2_00051)co-funded by European Social Fund(ESF investing in your future,European Union)supported by a postdoctoral fellowship from AECC(Spanish Association of Cancer Research,Spain)Antonio Villaverde received an Icrea Academia Award(Spain)supported by Grant PERIS SLT006/17/00093 from la Generalitat de Catalunya(Spain)and Miguel Servet fellowship(CP19/00028)from Instituto de Salud CarlosⅢ(Spain)co-funded by European Social Fund(ESF investing in your future,European Union)。
文摘Loco-regional recurrences and distant metastases represent the main cause of head and neck squamous cell carcinoma(HNSCC) mortality. The overexpression of chemokine receptor 4(CXCR4) in HNSCC primary tumors associates with higher risk of developing loco-regional recurrences and distant metastases, thus making CXCR4 an ideal entry pathway for targeted drug delivery. In this context, our group has generated the self-assembling protein nanocarrier T22-GFP-H6, displaying multiple T22 peptidic ligands that specifically target CXCR4. This study aimed to validate T22-GFP-H6 as a suitable nanocarrier to selectively deliver cytotoxic agents to CXCR4^(+)tumors in a HNSCC model. Here we demonstrate that T22-GFP-H6 selectively internalizes in CXCR4^(+)HNSCC cells, achieving a high accumulation in CXCR4^(+)tumors in vivo, while showing negligible nanocarrier distribution in non-tumor bearing organs. Moreover, this T22-empowered nanocarrier can incorporate bacterial toxin domains to generate therapeutic nanotoxins that induce cell death in CXCR4-overexpressing tumors in the absence of histological alterations in normal organs. Altogether, these results show the potential use of this T22-empowered nanocarrier platform to incorporate polypeptidic domains of choice to selectively eliminate CXCR4^(+)cells in HNSCC. Remarkably, to our knowledge, this is the first study testing targeted proteinonly nanoparticles in this cancer type, which may represent a novel treatment approach for HNSCC patients.
基金Agencia Estatal de Investigación and to Fondo Europeo de Desarrollo Regional (grant BIO2016-76063-R, AEI/FEDER, UE) to Villaverde A, AGAUR (2017SGR-229) to Villaverde A and 2017SGR-865 GRC to Mangues R CIBER-BBN (project NANOPROTHER) +6 种基金granted to Villaverde A and CIBER-BBN project 4Nano Mets to Mangues R ISCIII (PI15/00272 cofounding FEDER) to Vázquez E and ISCIII (Co-founding FEDER) PIE15//00028 and PI18/00650 to Mangues R, and to EU COST Action CA 17140indebted to the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) that is an initiative funded by the VI National R&D&I Plan 2008–2011Iniciativa Ingenio 2010, Consolider ProgramCIBER Actions and financed by the Instituto de Salud Carlos Ⅲ, with assistance from the European Regional Development FundSánchez-García L and López-Laguna H were supported by a predoctoral fellowship from AGAUR (2018FI_B2_00051 and 2019FI_B_00352) respectivelyUnzueta U by PERIS program from the Health Department of la Generalitat de Catalunya
文摘Modular protein engineering is suited to recruit complex and multiple functionalities in single-chain polypeptides. Although still unexplored in a systematic way, it is anticipated that the positioning of functional domains would impact and refine these activities, including the ability to organize as supramolecular entities and to generate multifunctional protein materials. To explore this concept, we have repositioned functional segments in the modular protein T22-GFP-H6 and characterized the resulting alternative fusions. In T22-GFP-H6, the combination of T22 and H6 promotes selfassembling as regular nanoparticles and selective binding and internalization of this material in CXCR4-overexpressing tumor cells, making them appealing as vehicles for selective drug delivery. The results show that the pleiotropic activities are dramatically affected in module-swapped constructs, proving the need of a carboxy terminal positioning of H6 for protein self-assembling, and the accommodation of T22 at the amino terminus as a requisite for CXCR4^+ cell binding and internalization. Furthermore, the failure of self-assembling as regular oligomers reduces cellular penetrability of the fusions while keeping the specificity of the T22-CXCR4 interaction.All these data instruct how multifunctional nanoscale protein carriers can be designed for smart, protein-driven drug delivery, not only for the treatment of CXCR4^+ human neoplasias, but also for the development of anti-HIV drugs and other pathologies in which CXCR4 is a relevant homing marker.
基金Agencia Estatal de Investigación (AEI) and to Fondo Europeo de Desarrollo Regional (FEDER) (BIO2016-76063-R, AEI/FEDER, UE) to Villaverde A, AGAUR (2017SGR-229) to Villaverde A and 2017SGR-865 GRCISCⅢ (PI15/00272 co-founding FEDER) to Vázquez E and ISCⅢ (Co-founding FEDER) PIE15//00028 and PI18/00650 to Mangues R, and to EU COST Action CA 17140+3 种基金funded by the Ⅵ National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actionsfinanced by the Instituto de Salud Carlos Ⅲ, with assistance from the European Regional Development Fundsupported by a predoctoral fellowship from AGAUR (2019 FI_B 00352)PERIS program from the Health Department of the Generalitat de Catalunya
文摘Poly-histidine peptides such as H6(HHHHHH)are used in protein biotechnologies as purification tags,protein-assembling agents and endosomal-escape entities.The pleiotropic properties of such peptides make them appealing to design protein-based smart materials or nanoparticles for imaging or drug delivery to be produced in form of recombinant proteins.However,the clinical applicability of H6-tagged proteins is restricted by the potential immunogenicity of these segments.In this study,we have explored several humanized histidine-rich peptides in tumor-targeted modular proteins,which can specifically bind and be internalized by the target cells through the tumoral marker CXCR4.We were particularly interested in exploring how protein purification,self-assembling and endosomal escape perform in proteins containing the variant histidine-rich tags.Among the tested candidates,the peptide H5 E(HEHEHEHEH)is promising as a good promoter of endosomal escape of the associated fulllength protein upon endosomal internalization.The numerical modelling of cell penetration and endosomal escape of the tested proteins has revealed a negative relationship between the amount of protein internalized into target cells and the efficiency of cytoplasmic release.This fact demonstrates that the His-mediated,proton sponge-based endosomal escape saturates at moderate amounts of internalized protein,a fact that might be critical for the design of protein materials for cytosolic molecular delivery.
