Development of Tumor-Targeted Indocyanine Green-Loaded Ferritin Nanoparticles for Intraoperative Detection of Cancers

Sitia, Leopoldo; Sevieri, Marta; Bonizzi, Arianna; Allevi, Raffaele; Morasso, Carlo; Foschi, Diego; Corsi, Fabio; Mazzucchelli, Serena

doi:10.1021/acsomega.0c00244

Cited by 30 publications

(44 citation statements)

References 40 publications

(65 reference statements)

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“…To further assess if the specificity of the HFn binding (TfR1 mediated) was preserved, we performed a competition assay pre-incubating cells with an excess of non-labelled purified HFn. By doing so, subsequent binding with F-HFn was almost completely inhibited in HCC1937 and HT29 cells (with 92 and 96% binding inhibition) and significantly reduced in 4T1 cells (40% inhibition) ( Figure 6 e), in line with what we have previously observed with non-purified HFn [ 42 ]. These results confirm that cell binding is not influenced by the purification process and is still mediated by interaction with TfR1 surface receptors.…”

Section: Resultssupporting

confidence: 89%

Combined Method to Remove Endotoxins from Protein Nanocages for Drug Delivery Applications: The Case of Human Ferritin

et al. 2021

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Protein nanocages represent an emerging candidate among nanoscaled delivery systems. Indeed, they display unique features that proved to be very interesting from the nanotechnological point of view such as uniform structure, stability in biological fluids, suitability for surface modification to insert targeting moieties and loading with different drugs and dyes. However, one of the main concerns regards the production as recombinant proteins in E. coli, which leads to a product with high endotoxin contamination, resulting in nanocage immunogenicity and pyrogenicity. Indeed, a main challenge in the development of protein-based nanoparticles is finding effective procedures to remove endotoxins without affecting protein stability, since every intravenous injectable formulation that should be assessed in preclinical and clinical phase studies should display endotoxins concentration below the admitted limit of 5 EU/kg. Different strategies could be employed to achieve such a result, either by using affinity chromatography or detergents. However, these strategies are not applicable to protein nanocages as such and require implementations. Here we propose a combined protocol to remove bacterial endotoxins from nanocages of human H-ferritin, which is one of the most studied and most promising protein-based drug delivery systems. This protocol couples the affinity purification with the Endotrap HD resin to a treatment with Triton X-114. Exploiting this protocol, we were able to obtain excellent levels of purity maintaining good protein recovery rates, without affecting nanocage interactions with target cells. Indeed, binding assay and confocal microscopy experiments confirm that purified H-ferritin retains its capability to specifically recognize cancer cells. This procedure allowed to obtain injectable formulations, which is preliminary to move to a clinical trial.

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Section: Resultssupporting

confidence: 89%

Combined Method to Remove Endotoxins from Protein Nanocages for Drug Delivery Applications: The Case of Human Ferritin

et al. 2021

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“…To further study the suitability of HFn-ICG as tumor-targeted nanotracer for in vivo image-guided surgery [ 38 ], we decided to perform an ex vivo study on a syngeneic orthotopic murine model of breast cancer. Starting from the in vivo pilot experiment that provided evidence regarding the potential of HFn−ICG to target the tumor mass [ 38 ], in this study, we evaluated the tumor accumulation and the biodistribution of both free and nano-formulated ICG ex vivo with the aim to demonstrate the improved performances and the potential of HFn-ICG as a nano-tracer for fluorescence tumor detection.…”

Section: Resultsmentioning

confidence: 99%

“…(Rome, Italy). The ICG was nano-formulated exploiting the ability of HFn to disassemble and reassemble its quaternary structure in response to changes in the pH, as previously reported [ 38 ].…”

Section: Methodsmentioning

confidence: 99%

“…Several HFn-based nanodrugs have been proposed for drug delivery with excellent results in terms of specific tumor recognition and increased activity with lower side effects [ 33 , 34 , 35 ]; however, ferritin nanoparticles suggested for diagnostic purposes are restricted to magnetic resonance imaging [ 27 , 32 , 36 ] and there are only a few examples of optical imaging applications [ 37 ] that do not include FGS or ICG exploitation. In light of this, we have proposed ICG loaded HFn nanocages as an in vivo system for FGS that will allow the surgeon to perform a more accurate surgical resection of the tumor with the ultimate goal of improving surgical outcomes [ 38 ].…”

Section: Introductionmentioning

confidence: 99%

“…Preliminary studies have demonstrated in vitro the tumor-targeted recognition of ICG upon nanoformulation in HFn nanocages. These studies have demonstrated that nanoformulation also affects the fluorescence stability, improving it, and resulting in better fluorescence signal in tumors [ 38 ]. Here, we performed an ex vivo analysis of HFn-ICG tumor accumulation and biodistribution to better elucidate the differences in ICG behavior in vivo upon nanoformulation.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Tumor Accumulation and Off-Target Biodistribution of an Indocyanine-Green Fluorescent Nanotracer: An Ex Vivo Study on an Orthotopic Murine Model of Breast Cancer

Sevieri

Sitia

Bonizzi

et al. 2021

IJMS

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Indocyanine green (ICG) is a near infrared fluorescent tracer used in image-guided surgery to assist surgeons during resection. Despite appearing as a very promising tool for surgical oncology, its employment in this area is limited to lymph node mapping or to laparoscopic surgery, as it lacks tumor targeting specificity. Recently, a nanoformulation of this dye has been proposed with the aim toward tumor targeting specificity in order to expand its employment in surgical oncology. This nanosystem is constituted by 24 monomers of H-Ferritin (HFn), which self-assemble into a spherical cage structure enclosing the indocyanine green fluorescent tracer. These HFn nanocages were demonstrated to display tumor homing due to the specific interaction between the HFn nanocage and transferrin receptor 1, which is overexpressed in most tumor tissues. Here, we provide an ex vivo detailed comparison between the biodistribution of this nanotracer and free ICG, combining the results obtained with the Karl Storz endoscope that is currently used in clinical practice and the quantification of the ICG signal derived from the fluorescence imaging system IVIS Lumina II. These insights demonstrate the suitability of this novel HFn-based nanosystem in fluorescence-guided oncological surgery.

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Ferritin‐based disruptor nanoparticles: A novel strategy to enhance LDL cholesterol clearance via multivalent inhibition of PCSK9–LDL receptor interaction

Incocciati,

Cappelletti,

Masciarelli

et al. 2024

Protein Science

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Hypercholesterolemia, characterized by elevated low‐density lipoprotein (LDL) cholesterol levels, is a significant risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in cholesterol metabolism by regulating LDL receptor degradation, making it a therapeutic target for mitigating hypercholesterolemia‐associated risks. In this context, we aimed to engineer human H ferritin as a scaffold to present 24 copies of a PCSK9‐targeting domain. The rationale behind this protein nanoparticle design was to disrupt the PCSK9–LDL receptor interaction, thereby attenuating the PCSK9‐mediated impairment of LDL cholesterol clearance. The N‐terminal sequence of human H ferritin was engineered to incorporate a 13‐amino acid linear peptide (Pep2‐8), which was previously identified as the smallest PCSK9 inhibitor. Exploiting the quaternary structure of ferritin, engineered nanoparticles were designed to display 24 copies of the targeting peptide on their surface, enabling a multivalent binding effect. Extensive biochemical characterization confirmed precise control over nanoparticle size and morphology, alongside robust PCSK9‐binding affinity (KD in the high picomolar range). Subsequent efficacy assessments employing the HepG2 liver cell line demonstrated the ability of engineered ferritin's ability to disrupt PCSK9–LDL receptor interaction, thereby promoting LDL receptor recycling on cell surfaces and consequently enhancing LDL uptake. Our findings highlight the potential of ferritin‐based platforms as versatile tools for targeting PCSK9 in the management of hypercholesterolemia. This study not only contributes to the advancement of ferritin‐based therapeutics but also offers valuable insights into novel strategies for treating cardiovascular diseases.

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Development of Tumor-Targeted Indocyanine Green-Loaded Ferritin Nanoparticles for Intraoperative Detection of Cancers

Cited by 30 publications

References 40 publications

Combined Method to Remove Endotoxins from Protein Nanocages for Drug Delivery Applications: The Case of Human Ferritin

Combined Method to Remove Endotoxins from Protein Nanocages for Drug Delivery Applications: The Case of Human Ferritin

Tumor Accumulation and Off-Target Biodistribution of an Indocyanine-Green Fluorescent Nanotracer: An Ex Vivo Study on an Orthotopic Murine Model of Breast Cancer

Ferritin‐based disruptor nanoparticles: A novel strategy to enhance LDL cholesterol clearance via multivalent inhibition of PCSK9–LDL receptor interaction

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