Optimal centrifugal isolating of liposome–protein complexes from human plasma

Digiacomo, Luca; Giulimondi, Francesca; Capriotti, Anna Laura; Piovesana, Susy; Montone, Carmela Maria; Chiozzi, Riccardo Zenezini; Laganà, Aldo; Pozzi, Daniela; Caracciolo, Giulio

doi:10.1039/d1na00211b

Cited by 18 publications

(18 citation statements)

References 56 publications

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“…For SDS-PAGE and proteomics, lipoplexes-protein complexes were isolated by centrifugation for 15 min at 14 000 rpm. The pellets were washed three times with PBS to remove unbound proteins obtaining the “hard corona” (further details can be found in ref ( 55 )).…”

Section: Methodsmentioning

confidence: 99%

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Opsonin-Deficient Nucleoproteic Corona Endows UnPEGylated Liposomes with Stealth Properties In Vivo

et al. 2022

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For several decades, surface grafted polyethylene glycol (PEG) has been a go-to strategy for preserving the synthetic identity of liposomes in physiological milieu and preventing clearance by immune cells. However, the limited clinical translation of PEGylated liposomes is mainly due to the protein corona formation and the subsequent modification of liposomes’ synthetic identity, which affects their interactions with immune cells and blood residency. Here we exploit the electric charge of DNA to generate unPEGylated liposome/DNA complexes that, upon exposure to human plasma, gets covered with an opsonin-deficient protein corona. The final product of the synthetic process is a biomimetic nanoparticle type covered by a proteonucleotidic corona, or “proteoDNAsome”, which maintains its synthetic identity in vivo and is able to slip past the immune system more efficiently than PEGylated liposomes. Accumulation of proteoDNAsomes in the spleen and the liver was lower than that of PEGylated systems. Our work highlights the importance of generating stable biomolecular coronas in the development of stealth unPEGylated particles, thus providing a connection between the biological behavior of particles in vivo and their synthetic identity.

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Section: Methodsmentioning

confidence: 99%

“…Finally, gel images were acquired with a ChemiDoc gel imaging system (Bio-Rad, CA) and processed by means of custom MatLab scripts (MathWorks, Natick, MA), as previously reported. 55 …”

Section: Methodsmentioning

confidence: 99%

Opsonin-Deficient Nucleoproteic Corona Endows UnPEGylated Liposomes with Stealth Properties In Vivo

et al. 2022

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“…Each sample was loaded on a gradient polyacrylamide gel stain-free (4–20% TGX precast gels, Bio-Rad, Hercules, CA, USA) and run at 100 V for 150 min. Finally, gel images were acquired with a ChemiDoc™ gel imaging system (Bio-Rad, Hercules, CA, USA) and processed using custom MATLAB scripts (MathWorks, Natick, MA, USA), as previously reported [ 17 ].…”

Section: Methodsmentioning

confidence: 99%

“…One-dimensional SDS-PAGE experiments were performed after protein corona isolation via centrifugation for 15 min at 4 • C, 21,400× g. Pellets were washed three times with PBS to remove unbound and loosely bound proteins and then resuspended in 20 mL of Laemmli Loading buffer 1× and boiled for 10 min at 100 • C. Each sample was loaded on a gradient polyacrylamide gel stain-free (4-20% TGX precast gels, Bio-Rad, Hercules, CA, USA) and run at 100 V for 150 min. Finally, gel images were acquired with a ChemiDoc™ gel imaging system (Bio-Rad, Hercules, CA, USA) and processed using custom MATLAB scripts (MathWorks, Natick, MA, USA), as previously reported [17].…”

Section: Sodium Dodecyl Sulphate-polyacrylamide Gel Electrophoresis (...mentioning

confidence: 99%

Magnetic Levitation Patterns of Microfluidic-Generated Nanoparticle–Protein Complexes

et al. 2022

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Magnetic levitation (MagLev) has recently emerged as a powerful method to develop diagnostic technologies based on the exploitation of the nanoparticle (NP)–protein corona. However, experimental procedures improving the robustness, reproducibility, and accuracy of this technology are largely unexplored. To contribute to filling this gap, here, we investigated the effect of total flow rate (TFR) and flow rate ratio (FRR) on the MagLev patterns of microfluidic-generated graphene oxide (GO)–protein complexes using bulk mixing of GO and human plasma (HP) as a reference. Levitating and precipitating fractions of GO-HP samples were characterized in terms of atomic force microscopy (AFM), bicinchoninic acid assay (BCA), and one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (1D SDS-PAGE), and nanoliquid chromatography–tandem mass spectrometry (nano-LC-MS/MS). We identified combinations of TFR and FRR (e.g., TFR = 35 μL/min and FRR (GO:HP) = 9:1 or TFR = 3.5 μL/min and FRR (GO:HP) = 19:1), leading to MagLev patterns dominated by levitating and precipitating fractions with bulk-like features. Since a typical MagLev experiment for disease detection is based on a sequence of optimization, exploration, and validation steps, this implies that the optimization (e.g., searching for optimal NP:HP ratios) and exploration (e.g., searching for MagLev signatures) steps can be performed using samples generated by bulk mixing. When these steps are completed, the validation step, which involves using human specimens that are often available in limited amounts, can be made by highly reproducible microfluidic mixing without any ex novo optimization process. The relevance of developing diagnostic technologies based on MagLev of coronated nanomaterials is also discussed.

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“…Consequently, contamination from biological nanoparticles was not examined and could not be excluded. However, previous studies indicated that it should produce minor, if any, effect on the size of liposome-protein complexes [16,17].…”

Section: Size and Zeta Potential Measurementsmentioning

confidence: 99%

A Proteomic Study on the Personalized Protein Corona of Liposomes. Relevance for Early Diagnosis of Pancreatic DUCTAL Adenocarcinoma and Biomarker Detection

Digiacomo

Giulimondi

Pozzi

et al. 2021

JNT

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Due to late diagnosis, high incidence of metastasis, and poor survival rate, pancreatic cancer is one of the most leading cause of cancer-related death. Although manifold recent efforts have been done to achieve an early diagnosis of pancreatic cancer, CA-19.9 is currently the unique biomarker that is adopted for the detection, despite its limits in terms of sensitivity and specificity. To identify potential protein biomarkers for pancreatic ductal adenocarcinoma (PDAC), we used three model liposomes as nanoplatforms that accumulate proteins from human plasma and studied the composition of this biomolecular layer, which is known as protein corona. Indeed, plasma proteins adsorb on nanoparticle surface according to their abundance and affinity to the employed nanomaterial, thus even small differences between healthy and PDAC protein expression levels can be, in principle, detected. By mass spectrometry experiments, we quantified such differences and identified possible biomarkers for PDAC. Some of them are already known to exhibit different expressions in PDAC proteomes, whereas the role of other relevant proteins is still not clear. Therefore, we predict that the employment of nanomaterials and their protein corona may represent a useful tool to amplify the detection sensitivity of cancer biomarkers, which may be used for the early diagnosis of PDAC, with clinical implication for the subsequent therapy in the context of personalized medicine.

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Optimal centrifugal isolating of liposome–protein complexes from human plasma

Abstract: In the past few years, characterization of the protein corona (PC) that forms around liposomal systems has gained increasing interest for the development of novel therapeutic and diagnostic technologies. At...

Cited by 18 publications

References 56 publications

Opsonin-Deficient Nucleoproteic Corona Endows UnPEGylated Liposomes with Stealth Properties In Vivo

Opsonin-Deficient Nucleoproteic Corona Endows UnPEGylated Liposomes with Stealth Properties In Vivo

Magnetic Levitation Patterns of Microfluidic-Generated Nanoparticle–Protein Complexes

A Proteomic Study on the Personalized Protein Corona of Liposomes. Relevance for Early Diagnosis of Pancreatic DUCTAL Adenocarcinoma and Biomarker Detection

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