1) Developement of radio-labelled SNPs for the targeted detection and treatment of Her2-positive breast cancer.Aim of this work was to develop a SNP-based system loaded with radioactive/fluorescent probes and functionalized with the half-chain of a monoclonal antibody, Trastuzumab, which specifically recognizes the human epidermal growth factor receptor 2 (Her2), overexpressed in 25-30% of human breast tumours. The silica core was covalently functionalized with FITC, further protected by a 10 nm silica shell and stabilized in saline buffer by means of differently terminated PEGs (SNP). Such nanoparticles were then conjugated with Trastuzumab half-chain (SNP-TZ). Finally, both SNP and SNP-TZ were derivatized with nitrilo-triacetic acid and labelled with 99mTc-Tricarbonyl complex, giving rise to SNP-NTA and SNP-NTA-TZ NPs. The functionalization steps were monitored both by size and z-potential measurements and the impact of each chemical moietiy on the NP behaviour in cells was assessed in prelabeling in vitro experiments comparing SNP, SNP-NTA, SNP-TZ and SNP-NTA-TZ. Targeting specificity of TZ-functionalized or TZ-free SNPs was studied in in vitro, in vivo and ex vivo experiments, both employing fluorescence and radionuclide techniques. Our results suggested that active targeting provided higher efficiency and selectivity in tumor detection compared to passive diffusion, confirming that our synthetic strategy provided stable nanoconjugates and did not affect their binding efficiency to HER2 expressing cells.2) Development of doxorubicin-loaded nonporous SNPs. Nonporous SNPs were chosen as the starting point to produce different drug carriers, bearing the well-known anticancer drug doxorubicin. Different silica nanoformulations containing the well-known anticancer drug doxorubicin were compared: OuterDox NPs, in which doxorubicin was covalently linked on the silica surface, InnerDox NPs, in which the chemotherapeutic was covalently immobilized in the core of the same particles and DoubleDox NPs, containing the drug both externally and internally.The nanoformulations were studied in terms of carrier degradation and payload release in physiological conditions.The in vitro efficiency was also investigated.3)Development of glutathione-sensitive apoferritin NPs for the controlled delivery of luciferin.Although bioluminescence imaging has been successfully used in a variety of applications to obtain information regarding biological processes in vivo, the detection of photon emission is limited by the short half-life of luciferin (less than 30 minutes), its modest cell penetration and inhomogeneous diffusion into different tissues. In this context, we developed a glutathione-sensitive NP for stimuli-responsive release of luciferin within cancer cells. The nanoconjugate bears luciferin by means of a disulfide containing linker (Luc-linker), which, in the presence of a reducing agent, undergoes an intramolecular cyclization reaction that results in the release of free luciferin. The correct luciferin release mechanism was checked in cell-free in vitro bioluminescence tests: an abundant photon production was detected when Luc-linker was preincubated with DTT and then reacted with luciferase, while no light emission was seen without DTT pretreatment. Luc-linker was then attached to apoferritin (HFn) NP surface, exploiting the free thiol groups of cysteine residues, leading to Luc-linker@HFn NPs.After the conjugation, an HPLC method was developed for the quantification of conjugation efficiency and drug loading, requiring a preliminary separation of the linker from the hosting HFn NPs. The Luc-linker@HFn was then tested in vitro to initially elucidate the bioluminescent kinetics and compare the luminous signal to the one of nanoparticle-free luciferin.

1) Developement of radio-labelled SNPs for the targeted detection and treatment of Her2-positive breast cancer.Aim of this work was to develop a SNP-based system loaded with radioactive/fluorescent probes and functionalized with the half-chain of a monoclonal antibody, Trastuzumab, which specifically recognizes the human epidermal growth factor receptor 2 (Her2), overexpressed in 25-30% of human breast tumours. The silica core was covalently functionalized with FITC, further protected by a 10 nm silica shell and stabilized in saline buffer by means of differently terminated PEGs (SNP). Such nanoparticles were then conjugated with Trastuzumab half-chain (SNP-TZ). Finally, both SNP and SNP-TZ were derivatized with nitrilo-triacetic acid and labelled with 99mTc-Tricarbonyl complex, giving rise to SNP-NTA and SNP-NTA-TZ NPs. The functionalization steps were monitored both by size and z-potential measurements and the impact of each chemical moietiy on the NP behaviour in cells was assessed in prelabeling in vitro experiments comparing SNP, SNP-NTA, SNP-TZ and SNP-NTA-TZ. Targeting specificity of TZ-functionalized or TZ-free SNPs was studied in in vitro, in vivo and ex vivo experiments, both employing fluorescence and radionuclide techniques. Our results suggested that active targeting provided higher efficiency and selectivity in tumor detection compared to passive diffusion, confirming that our synthetic strategy provided stable nanoconjugates and did not affect their binding efficiency to HER2 expressing cells.2) Development of doxorubicin-loaded nonporous SNPs. Nonporous SNPs were chosen as the starting point to produce different drug carriers, bearing the well-known anticancer drug doxorubicin. Different silica nanoformulations containing the well-known anticancer drug doxorubicin were compared: OuterDox NPs, in which doxorubicin was covalently linked on the silica surface, InnerDox NPs, in which the chemotherapeutic was covalently immobilized in the core of the same particles and DoubleDox NPs, containing the drug both externally and internally.The nanoformulations were studied in terms of carrier degradation and payload release in physiological conditions.The in vitro efficiency was also investigated.3)Development of glutathione-sensitive apoferritin NPs for the controlled delivery of luciferin.Although bioluminescence imaging has been successfully used in a variety of applications to obtain information regarding biological processes in vivo, the detection of photon emission is limited by the short half-life of luciferin (less than 30 minutes), its modest cell penetration and inhomogeneous diffusion into different tissues. In this context, we developed a glutathione-sensitive NP for stimuli-responsive release of luciferin within cancer cells. The nanoconjugate bears luciferin by means of a disulfide containing linker (Luc-linker), which, in the presence of a reducing agent, undergoes an intramolecular cyclization reaction that results in the release of free luciferin. The correct luciferin release mechanism was checked in cell-free in vitro bioluminescence tests: an abundant photon production was detected when Luc-linker was preincubated with DTT and then reacted with luciferase, while no light emission was seen without DTT pretreatment. Luc-linker was then attached to apoferritin (HFn) NP surface, exploiting the free thiol groups of cysteine residues, leading to Luc-linker@HFn NPs.After the conjugation, an HPLC method was developed for the quantification of conjugation efficiency and drug loading, requiring a preliminary separation of the linker from the hosting HFn NPs. The Luc-linker@HFn was then tested in vitro to initially elucidate the bioluminescent kinetics and compare the luminous signal to the one of nanoparticle-free luciferin.

(2017). investigating the functionalization of colloidal nanoparticles with small molecules of biological interest. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).

investigating the functionalization of colloidal nanoparticles with small molecules of biological interest

RIVA, BENEDETTA
2017-03-13

Abstract

1) Developement of radio-labelled SNPs for the targeted detection and treatment of Her2-positive breast cancer.Aim of this work was to develop a SNP-based system loaded with radioactive/fluorescent probes and functionalized with the half-chain of a monoclonal antibody, Trastuzumab, which specifically recognizes the human epidermal growth factor receptor 2 (Her2), overexpressed in 25-30% of human breast tumours. The silica core was covalently functionalized with FITC, further protected by a 10 nm silica shell and stabilized in saline buffer by means of differently terminated PEGs (SNP). Such nanoparticles were then conjugated with Trastuzumab half-chain (SNP-TZ). Finally, both SNP and SNP-TZ were derivatized with nitrilo-triacetic acid and labelled with 99mTc-Tricarbonyl complex, giving rise to SNP-NTA and SNP-NTA-TZ NPs. The functionalization steps were monitored both by size and z-potential measurements and the impact of each chemical moietiy on the NP behaviour in cells was assessed in prelabeling in vitro experiments comparing SNP, SNP-NTA, SNP-TZ and SNP-NTA-TZ. Targeting specificity of TZ-functionalized or TZ-free SNPs was studied in in vitro, in vivo and ex vivo experiments, both employing fluorescence and radionuclide techniques. Our results suggested that active targeting provided higher efficiency and selectivity in tumor detection compared to passive diffusion, confirming that our synthetic strategy provided stable nanoconjugates and did not affect their binding efficiency to HER2 expressing cells.2) Development of doxorubicin-loaded nonporous SNPs. Nonporous SNPs were chosen as the starting point to produce different drug carriers, bearing the well-known anticancer drug doxorubicin. Different silica nanoformulations containing the well-known anticancer drug doxorubicin were compared: OuterDox NPs, in which doxorubicin was covalently linked on the silica surface, InnerDox NPs, in which the chemotherapeutic was covalently immobilized in the core of the same particles and DoubleDox NPs, containing the drug both externally and internally.The nanoformulations were studied in terms of carrier degradation and payload release in physiological conditions.The in vitro efficiency was also investigated.3)Development of glutathione-sensitive apoferritin NPs for the controlled delivery of luciferin.Although bioluminescence imaging has been successfully used in a variety of applications to obtain information regarding biological processes in vivo, the detection of photon emission is limited by the short half-life of luciferin (less than 30 minutes), its modest cell penetration and inhomogeneous diffusion into different tissues. In this context, we developed a glutathione-sensitive NP for stimuli-responsive release of luciferin within cancer cells. The nanoconjugate bears luciferin by means of a disulfide containing linker (Luc-linker), which, in the presence of a reducing agent, undergoes an intramolecular cyclization reaction that results in the release of free luciferin. The correct luciferin release mechanism was checked in cell-free in vitro bioluminescence tests: an abundant photon production was detected when Luc-linker was preincubated with DTT and then reacted with luciferase, while no light emission was seen without DTT pretreatment. Luc-linker was then attached to apoferritin (HFn) NP surface, exploiting the free thiol groups of cysteine residues, leading to Luc-linker@HFn NPs.After the conjugation, an HPLC method was developed for the quantification of conjugation efficiency and drug loading, requiring a preliminary separation of the linker from the hosting HFn NPs. The Luc-linker@HFn was then tested in vitro to initially elucidate the bioluminescent kinetics and compare the luminous signal to the one of nanoparticle-free luciferin.
PROSPERI, DAVIDE
1) Developement of radio-labelled SNPs for the targeted detection and treatment of Her2-positive breast cancer.Aim of this work was to develop a SNP-based system loaded with radioactive/fluorescent probes and functionalized with the half-chain of a monoclonal antibody, Trastuzumab, which specifically recognizes the human epidermal growth factor receptor 2 (Her2), overexpressed in 25-30% of human breast tumours. The silica core was covalently functionalized with FITC, further protected by a 10 nm silica shell and stabilized in saline buffer by means of differently terminated PEGs (SNP). Such nanoparticles were then conjugated with Trastuzumab half-chain (SNP-TZ). Finally, both SNP and SNP-TZ were derivatized with nitrilo-triacetic acid and labelled with 99mTc-Tricarbonyl complex, giving rise to SNP-NTA and SNP-NTA-TZ NPs. The functionalization steps were monitored both by size and z-potential measurements and the impact of each chemical moietiy on the NP behaviour in cells was assessed in prelabeling in vitro experiments comparing SNP, SNP-NTA, SNP-TZ and SNP-NTA-TZ. Targeting specificity of TZ-functionalized or TZ-free SNPs was studied in in vitro, in vivo and ex vivo experiments, both employing fluorescence and radionuclide techniques. Our results suggested that active targeting provided higher efficiency and selectivity in tumor detection compared to passive diffusion, confirming that our synthetic strategy provided stable nanoconjugates and did not affect their binding efficiency to HER2 expressing cells.2) Development of doxorubicin-loaded nonporous SNPs. Nonporous SNPs were chosen as the starting point to produce different drug carriers, bearing the well-known anticancer drug doxorubicin. Different silica nanoformulations containing the well-known anticancer drug doxorubicin were compared: OuterDox NPs, in which doxorubicin was covalently linked on the silica surface, InnerDox NPs, in which the chemotherapeutic was covalently immobilized in the core of the same particles and DoubleDox NPs, containing the drug both externally and internally.The nanoformulations were studied in terms of carrier degradation and payload release in physiological conditions.The in vitro efficiency was also investigated.3)Development of glutathione-sensitive apoferritin NPs for the controlled delivery of luciferin.Although bioluminescence imaging has been successfully used in a variety of applications to obtain information regarding biological processes in vivo, the detection of photon emission is limited by the short half-life of luciferin (less than 30 minutes), its modest cell penetration and inhomogeneous diffusion into different tissues. In this context, we developed a glutathione-sensitive NP for stimuli-responsive release of luciferin within cancer cells. The nanoconjugate bears luciferin by means of a disulfide containing linker (Luc-linker), which, in the presence of a reducing agent, undergoes an intramolecular cyclization reaction that results in the release of free luciferin. The correct luciferin release mechanism was checked in cell-free in vitro bioluminescence tests: an abundant photon production was detected when Luc-linker was preincubated with DTT and then reacted with luciferase, while no light emission was seen without DTT pretreatment. Luc-linker was then attached to apoferritin (HFn) NP surface, exploiting the free thiol groups of cysteine residues, leading to Luc-linker@HFn NPs.After the conjugation, an HPLC method was developed for the quantification of conjugation efficiency and drug loading, requiring a preliminary separation of the linker from the hosting HFn NPs. The Luc-linker@HFn was then tested in vitro to initially elucidate the bioluminescent kinetics and compare the luminous signal to the one of nanoparticle-free luciferin.
bionanotechnology,; colloidal; chemistry,; theranostics,; bioluminescence
bionanotechnology,; colloidal; chemistry,; theranostics,; bioluminescence
BIO/07 - ECOLOGIA
English
SCIENZE - 80R
29
2015/2016
(2017). investigating the functionalization of colloidal nanoparticles with small molecules of biological interest. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2017).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/153282
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