Periodic Reporting for period 1 - Glyco-NPs (Glycans coated nanoparticles as a biocompatible shield to increase nanoparticles colloidal stability and modulate their immunological response.)
Reporting period: 2020-09-01 to 2022-08-31
•What is the problem/issue being addressed?
Nanomedicine offers great opportunities for cancer treatment using a number of nanoparticle (NP) types that, upon surface functionalization, can overcome the limitations associated with conventional therapy. However, NP efficacy is highly impacted by many factors, including the composition complexity of physiological environments that contain thousands of biomolecules, which can adsorb on the surface of NPs, forming what is called protein corona (PC). This PC can alter NP colloidal characteristics, including NPs colloidal stability, and/or hydrodynamic diameter, which may influence the biological fate of NPs. In addition, the PC can affect the active targeting of conjugated NPs as it can change the NP surface composition. There is a need to develop new approaches to conjugate the NP with targeted ligands with the correct orientation that would be accessible in the complex fluid. This project proposed a solution to overcoming the abovementioned barriers by decorating the NPs’ surface with synthetic and naturally occurring glycans purified from blood plasma. The obtained results indicated the ability of developed glyco-coated NPs to increase their biocompatibility, colloidal stability, and biological targeting with an increased NP circulation half-life, allowing their binding site to be accessible for desirable cellular recognition.
•Why is it important for society?
The results of the Glyco-NPs project, if adequately transferred, can help in speeding up the transition of nanomedicine to the clinic, which can overcome the limitations associated with conventional therapy (chemotherapy & radiotherapy) and improve the quality of patients’ life, reducing the time consumed in hospitals, and thereby, reducing the total cost of treatment. Hence, the project can have a widespread impact by limiting the human suffering associated with cancer, decreasing cancer-related deaths, and reducing the economic burden on society.
•What are the overall objectives
Developing a new generation of biocompatible NPs by functionalizing their surface with biocompatible and immuno-silent sugars in order to i) increase the NPs’ colloidal stability, ii) regulate/ attenuate protein corona formation on NPs, iii) increase the NP circulation half-life in the bloodstream, and iv) enhance the targeting efficiency and cellular uptake.
Nanomedicine offers great opportunities for cancer treatment using a number of nanoparticle (NP) types that, upon surface functionalization, can overcome the limitations associated with conventional therapy. However, NP efficacy is highly impacted by many factors, including the composition complexity of physiological environments that contain thousands of biomolecules, which can adsorb on the surface of NPs, forming what is called protein corona (PC). This PC can alter NP colloidal characteristics, including NPs colloidal stability, and/or hydrodynamic diameter, which may influence the biological fate of NPs. In addition, the PC can affect the active targeting of conjugated NPs as it can change the NP surface composition. There is a need to develop new approaches to conjugate the NP with targeted ligands with the correct orientation that would be accessible in the complex fluid. This project proposed a solution to overcoming the abovementioned barriers by decorating the NPs’ surface with synthetic and naturally occurring glycans purified from blood plasma. The obtained results indicated the ability of developed glyco-coated NPs to increase their biocompatibility, colloidal stability, and biological targeting with an increased NP circulation half-life, allowing their binding site to be accessible for desirable cellular recognition.
•Why is it important for society?
The results of the Glyco-NPs project, if adequately transferred, can help in speeding up the transition of nanomedicine to the clinic, which can overcome the limitations associated with conventional therapy (chemotherapy & radiotherapy) and improve the quality of patients’ life, reducing the time consumed in hospitals, and thereby, reducing the total cost of treatment. Hence, the project can have a widespread impact by limiting the human suffering associated with cancer, decreasing cancer-related deaths, and reducing the economic burden on society.
•What are the overall objectives
Developing a new generation of biocompatible NPs by functionalizing their surface with biocompatible and immuno-silent sugars in order to i) increase the NPs’ colloidal stability, ii) regulate/ attenuate protein corona formation on NPs, iii) increase the NP circulation half-life in the bloodstream, and iv) enhance the targeting efficiency and cellular uptake.
W1. NPs’ synthesis and functionalization
The AuNPs were obtained in two shapes, spherical and nanorods, having different sizes, 25, 55, and 100 nm for spherical NPs, while 15 x 55 and 15 x 100 nm, for nanorods. The obtained NPs were coated with a negatively charged polymer shell (PMA coating) and used as a platform for particle decoration with glycopeptides (A2G2S2) through EDC/NHS coupling reaction. The characterisation of the physico-chemical properties of the AuNPs after each step of the surface modification indicated obtaining high-quality, monodisperse, colloidally stable, and well-characterized glycan-NPs.
W2: Optimization of developed glycol-NPs and extracellular studies.
Here, we studied the binding accessibility of glyco-NPs in serum-free and protein-rich media using Sambucus Nigra Lectin (SNA), which is a protein bind preferentially to sialic acid attached to terminal galactose in the α-2,6 linkage of A2G2S2. The data indicated a clear binding of SNA to glyco-NPs, which confirmed its biological accessibility. The colloidal stability of the NPs (i.e. on their agglomeration behaviors) was studied in different physiological conditions (cell media, and different pH) using UV/Vis spectroscopy and DLS. Data showed that all the AuNPs samples exhibited high colloidal stability in complete cell media (DMEM, 10% FBS, 1% P/S) for at least 24 h and at a wide range of pH values (pH= 5 - 12).
W3: Protein corona evaluation and intracellular studies
To study the protein corona, the obtained NPs were exposed to increasing concentrations of blood plasma and Fetal bovine serum in order to mimic in vitro / in vivo conditions and, following incubation at 37°C, the corona-NP complexes were isolated and characterized by different techniques, including UV-vis spectroscopy, DLS, DCS, and SDS-PAGE. Overall, the SDS-PAGE showed comparable protein patterns, indicating that the surface modification had a low influence on the corona formation. However, the data obtained from DLS and DCS (differences in the hydrodynamic size of NPs and their relative apparent particle diameter) suggests that the glycan-coated NPs might reduce the corona formation compared to the PMA-NPs. Toxicity and cellular uptake studies were performed using hepatocyte (HepG2) and Raw264.7 cell lines. The data from the LDH assay showed no acute toxicity features after 24 h of exposure to different concentrations of AuNPs. Cellular uptake studies by flow cytometry and ICP-MS indicated less uptake of glycan-conjugated NPs by both types of cells compared to PMA-NPs, which indicates the ability of the glycan-coated NPs to escape from macrophage recognition.
Dissemination
•The results of this project were disseminated to the scientific community in the form of a poster and oral presentation at two international conferences.
•The results were communicated to the public society through a blog post at the European research night, at TCD (Dublin/ Ireland) on 30 Sep 2022.
•Jennifer Fernandez Alarcon*, Mahmoud Soliman*, Tanja Ludtke, Eva Clemente, Marko Dobricic, Martina B. Violatto, Alessandro Corbelli, Fabio Fiordaliso1, Chiara Cordiglieri, Laura Talamini, Giovanni Sitia, Sergio Moya, Paolo Bigini and Marco P. Monopoli, Long-term retention of gold nanoparticles in the liver is not affected by their physicochemical characteristics. The manuscript is submitted to the Journal of Nanobiotechnology.
Exploitation
The obtained results will provide (upon publication) a solid reference to scientific research groups working on synthesizing NPs with enhanced physical-chemical properties, and scientists in nanomedicine-related disciplines such as targeting, drug delivery & toxicology. It will also push all other nanomaterials to a novel strategy developed during this fellowship toward the progress of clinical translation of nanomedicine. The raw data obtained during this project will be also available via a public repository e.g. OpenAIRE, having first taken care to protect IP and any sensitive information.
The AuNPs were obtained in two shapes, spherical and nanorods, having different sizes, 25, 55, and 100 nm for spherical NPs, while 15 x 55 and 15 x 100 nm, for nanorods. The obtained NPs were coated with a negatively charged polymer shell (PMA coating) and used as a platform for particle decoration with glycopeptides (A2G2S2) through EDC/NHS coupling reaction. The characterisation of the physico-chemical properties of the AuNPs after each step of the surface modification indicated obtaining high-quality, monodisperse, colloidally stable, and well-characterized glycan-NPs.
W2: Optimization of developed glycol-NPs and extracellular studies.
Here, we studied the binding accessibility of glyco-NPs in serum-free and protein-rich media using Sambucus Nigra Lectin (SNA), which is a protein bind preferentially to sialic acid attached to terminal galactose in the α-2,6 linkage of A2G2S2. The data indicated a clear binding of SNA to glyco-NPs, which confirmed its biological accessibility. The colloidal stability of the NPs (i.e. on their agglomeration behaviors) was studied in different physiological conditions (cell media, and different pH) using UV/Vis spectroscopy and DLS. Data showed that all the AuNPs samples exhibited high colloidal stability in complete cell media (DMEM, 10% FBS, 1% P/S) for at least 24 h and at a wide range of pH values (pH= 5 - 12).
W3: Protein corona evaluation and intracellular studies
To study the protein corona, the obtained NPs were exposed to increasing concentrations of blood plasma and Fetal bovine serum in order to mimic in vitro / in vivo conditions and, following incubation at 37°C, the corona-NP complexes were isolated and characterized by different techniques, including UV-vis spectroscopy, DLS, DCS, and SDS-PAGE. Overall, the SDS-PAGE showed comparable protein patterns, indicating that the surface modification had a low influence on the corona formation. However, the data obtained from DLS and DCS (differences in the hydrodynamic size of NPs and their relative apparent particle diameter) suggests that the glycan-coated NPs might reduce the corona formation compared to the PMA-NPs. Toxicity and cellular uptake studies were performed using hepatocyte (HepG2) and Raw264.7 cell lines. The data from the LDH assay showed no acute toxicity features after 24 h of exposure to different concentrations of AuNPs. Cellular uptake studies by flow cytometry and ICP-MS indicated less uptake of glycan-conjugated NPs by both types of cells compared to PMA-NPs, which indicates the ability of the glycan-coated NPs to escape from macrophage recognition.
Dissemination
•The results of this project were disseminated to the scientific community in the form of a poster and oral presentation at two international conferences.
•The results were communicated to the public society through a blog post at the European research night, at TCD (Dublin/ Ireland) on 30 Sep 2022.
•Jennifer Fernandez Alarcon*, Mahmoud Soliman*, Tanja Ludtke, Eva Clemente, Marko Dobricic, Martina B. Violatto, Alessandro Corbelli, Fabio Fiordaliso1, Chiara Cordiglieri, Laura Talamini, Giovanni Sitia, Sergio Moya, Paolo Bigini and Marco P. Monopoli, Long-term retention of gold nanoparticles in the liver is not affected by their physicochemical characteristics. The manuscript is submitted to the Journal of Nanobiotechnology.
Exploitation
The obtained results will provide (upon publication) a solid reference to scientific research groups working on synthesizing NPs with enhanced physical-chemical properties, and scientists in nanomedicine-related disciplines such as targeting, drug delivery & toxicology. It will also push all other nanomaterials to a novel strategy developed during this fellowship toward the progress of clinical translation of nanomedicine. The raw data obtained during this project will be also available via a public repository e.g. OpenAIRE, having first taken care to protect IP and any sensitive information.
The Glyco-NPs project proposed a solution to overcome the limitations associated with the current approaches of NP surface modifications, including lack of selectivity and rapid clearance by the mononuclear phagocyte system (MPS). The obtained results indicated the ability of the glyco-coated NPs to i) offer high colloidal stability and biocompatibility ii) increase the blood circulation lifetime and iii) keep the targeting properties of NPs in a complex biological medium and increase desirable cellular uptake. Therefore, the results obtained in this project, if adequately exploited and transferred, can push all other nanomaterials to this new technology and thus attract new partners to join new technological enterprises. With this, new markets are opening up, adequate jobs will be created for skilled researchers, and new technologies can be transferred to our life (economically and healthily). Here, the community can understand the importance of science and technology and how it can help in improving our lives and make them more comfortable in other ways.