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Among the various types of pancreatic cancers, pancreatic ductal adenocarcinoma (PDAC) is the most lethal and aggressive, due to its tendency to metastasize quickly and has a particularly low five-year survival rate. Carbohydrate antigen 19-9 (CA 19-9) is the only biomarker approved by the Food and Drug Administration for PDAC and has been a focal point in diagnostic strategies, but its sensitivity and specificity are not sufficient for early and accurate detection. To address this issue, we introduce a synergistic approach combining CA 19-9 levels with a graphene oxide (GO)-based blood test. This non-invasive technique relies on the analysis of personalized protein corona formed on GO sheets once they are embedded in human plasma. Pairing CA 19-9 values with GO protein patterns from N = 106 donors significantly improved the ability to differentiate between non-oncological and PDAC patients (up to 92%), also boosting the classification of PDAC subjects by 50% compared to CA 19-9 testing alone. Overall, this study sought to bridge the existing gaps in PDAC detection by exploiting the complementary strengths of conventional biomarkers and cutting-edge nanotechnology. Exploration of this combined strategy holds promise for advancing the early detection of PDAC, ultimately contributing to improved patient prognosis and treatment outcomes.

Pollution from micro- and nanoplastics (MNPs) has long been a topic of concern due to its potential impact on human health. MNPs can circulate through human blood and, thus far, have been found in the lungs, spleen, stomach, liver, kidneys and even in the brain, placenta, and breast milk. While data are already available on the adverse biological effects of pristine MNPs ( e.g. oxidative stress, inflammation, cytotoxicity, and even cancer induction), no report thus far clarified whether the same effects are modulated by the formation of a protein corona around MNPs. To this end, here we use pristine and human-plasma pre-coated polystyrene (PS) nanoparticles (NPs) and investigate them in cultured breast cancer cells both in terms of internalization and cell biochemical response to the exposure. It is found that pristine NPs tend to stick to the cell membrane and inhibit HER-2-driven signaling pathways, including phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways, which are associated with cancer cell survival and growth. By contrast, the formation of a protein corona around the same NPs can promote their uptake by endocytic vesicles and final sequestration within lysosomes. Of note is that such intracellular fate of PS-NPs is associated with mitigation of the biochemical alterations of the phosphorylated AKT (pAKT)/AKT and phosphorylated ERK (pERK)/ERK levels. These findings provide the distribution of NPs in human breast cancer cells, may broaden our understanding of the interactions between NPs and breast cancer cells and underscore the crucial role of the protein corona in modulating the impact of MNPs on human health.

New technologies with the capacity to tune immune system activity are highly desired in clinical practice and disease management. Here we demonstrate that nanoparticles with a protein corona enriched with gelsolin (GSN), an abundant plasma protein that acts as a modulator of immune responses, are avidly captured by human monocytic THP-1 cells in vitro and by leukocyte subpopulations derived from healthy donors ex vivo . In human monocytes, GSN modulates the production of tumor necrosis factor alpha (TNF-α) in an inverse dose-dependent manner. Overall, our results suggest that artificial coronas can be exploited to finely tune the immune response, opening new approaches for the prevention and treatment of diseases.

The supramolecular organization of Doxorubicin (DOX) within the standard Doxoves® liposomal formulation (DOX®) is investigated using visible light and phasor approach to fluorescence lifetime imaging (phasor-FLIM). First, the phasor-FLIM signature of DOX® is resolved into the contribution of three co-existing fluorescent species, each with its characteristic mono-exponential lifetime, namely: crystallized DOX (DOX c , 0.2 ns), free DOX (DOX f , 1.0 ns), and DOX bound to the liposomal membrane (DOX b , 4.5 ns). Then, the exact molar fractions of the three species are determined by combining phasor-FLIM with quantitative absorption/fluorescence spectroscopy on DOX c , DOX f , and DOX b pure standards. The final picture on DOX® comprises most of the drug in the crystallized form (∼98%), with the remaining fractions divided between free (∼1.4%) and membrane-bound drug (∼0.7%). Finally, phasor-FLIM in the presence of a DOX dynamic quencher allows us to suggest that DOX f is both encapsulated and non-encapsulated, and that DOX b is present on both liposome-membrane leaflets. We argue that the present experimental protocol can be applied to the investigation of the supramolecular organization of encapsulated luminescent drugs/molecules all the way from the production phase to their state within living matter.

Following exposure to human plasma (HP), nanoparticles (NPs) are coated with a biomolecular layer referred to as a protein corona. We recently revealed that characterizing the protein coronas of various NPs may provide a unique opportunity for cancer identification and discrimination. In other words, protein corona profiles of several NPs, when being analyzed using classifiers, would provide a unique "fingerprint" for each type of disease. Here, we probed the capacity of the protein corona for the identification and discrimination of breast and prostate cancer patients from healthy individuals. Using three lipid NP formulations with distinct physical-chemical properties as a cross-reactive sensor array and a supervised random forest classifier, we identified a set of proteins that showed a significant difference in cancer patients and control subjects. Our data show that many of the corona proteins with the highest discrimination ability between oncological patients and healthy individuals are related to cellular and molecular aspects of breast and prostate cancers.

Advances in nanotechnology are introducing the exciting possibility of cancer identification at early stages via analysis of the personalized biomolecular corona (BC), i.e. the dynamic "halo" of proteins that adsorbs onto NPs following exposure to patients' plasma. In this study, we develop a blood test for early cancer detection based on the characterization of the BC that forms around Graphene Oxide (GO) nanoflakes. Among its elective properties, GO binds low amounts of albumin, the most abundant protein in the blood and one of the most enriched proteins in the BC of many nanomaterials. This unique property of GO allows strong adsorption of poorly concentrated plasma proteins without abundant protein depletion. In our study, GO nanometric flakes have been used to analyze BCs from 50 subjects, half of them diagnosed with pancreatic cancer and half of them being healthy volunteers. Pancreatic cancer was chosen as the model of a high mortality disease with poor survival rates due to its delayed diagnosis. The receiver operating characteristic (ROC) curve analysis was applied to measure the diagnostic accuracy of the BC-based test. We obtained an area under the curve (AUC) of 0.96 and the test discriminated cancer patients from healthy subjects with a sensitivity of 92%. Finally, a double-blind validation was made using a second test dataset (10 healthy subjects + 10 pancreatic cancer patients) and it confirmed the results obtained on the first training dataset. Being highly accurate, fast, inexpensive and easy to perform, we believe that the BC-enabled blood test has the potential to become a turning point in early detection of cancer and other diseases.

Graphene oxide (GO) is a single-atomic-layered material made of a sheet of oxidized carbon atoms arranged in a honeycomb structure. Thanks to the notable physical and chemical properties of GO, GO-based nanomaterials have applications in many fields of research, including gene delivery. It has been reported that pristine GO can absorb single-stranded DNA and RNA through π-π stacking, which cannot be used as a gene carrier because it is hard to load double-stranded DNA (dsDNA). To tackle this issue, this work was aimed at developing a hybrid nanoparticle (NP) system made of GO coated with cationic lipids (hereafter referred to as GOCL) with suitable physical-chemical properties for gene delivery applications. To this end, nanosized GO flakes (nGO) were coated with the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) by microfluidic mixing. Comprehensive characterization of GOCL NPs was performed by a combination of dynamic light scattering (DLS), micro-electrophoresis and atom force microscopy (AFM). Our results show that GOCL NPs exhibit adequate size (<150 nm) and surface charge (ξ = +15 mV) for gene delivery purposes. Complexes made of GOCL NPs and plasmid DNA (pDNA) were used to transfect human cervical cancer cells (HeLa) and human embryonic kidney (HEK-293) cells. Pristine nGO and DOTAP cationic liposomes were used as a reference. GOCL NPs exhibited a similar TE but a much higher cell viability compared with DOTAP cationic liposomes. Confocal fluorescence microscopy provided a reasonable explanation for the superior performance of GOCL/DNA complexes showing that they are much more numerous, regular in size and homogeneously distributed than DOTAP/DNA complexes, thus splitting their gene payload over the entire cell population. Because of the imperative demand for efficient and safe nanocarriers, this study will contribute to the development of novel surface-functionalized GO-based hybrid gene vectors.

Following exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biological fluids, and target cells, respectively. By conducting confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liquid chromatography tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP-cell interactions and biological responses and set the basis for a rational exploitation of the BC for targeted delivery.

Today, liposomes are an advanced technology of drug carriers with a dozen drugs in clinical practice and many more in clinical trials. A bottleneck associated with the clinical translation of liposomes has long been 'opsonization', i.e. the adsorption of plasma proteins at the liposome surface resulting in their rapid clearance from circulation. For decades, the most popular way to avoid opsonization has been grafting polyethylene glycol (PEG) onto the liposome surface. Recent studies have clarified that grafting PEG onto the liposome surface reduces, but does not completely prevent protein binding. In this work, we employed dynamic light scattering, zeta-potential analysis, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE), semi-quantitative densitometry and cell imaging to explore the bio-nano-interactions between human plasma (HP) and Onivyde, a PEGylated liposomal drug that has recently been approved by the Food and Drug Administration (FDA) for the treatment of metastatic pancreatic ductal adenocarcinoma (PDAC). To properly evaluate the role of PEGylation, an unPEGylated variant of Onivyde was used as a reference. Collectively, our findings suggest that: (i) although PEGylated, Onivyde is not "stealth" in HP; (ii) surface chemistry is more important than PEGylation in controlling the bio-nano-interactions between Onivyde and plasma components. Of note is that the PC was found to boost the cellular uptake of Onivyde in the pancreas ductal adenocarcinoma cell line (PANC-1) thus suggesting its prominent role in its indication for PDAC treatment. Relevant implications for drug delivery and drug design are discussed.

Pancreatic cancer is a very aggressive malignancy that is often diagnosed in the advanced stages, with the implication that long-term survivors are extremely rare. Thus, developing new methods for the early detection of pancreatic cancer is an urgent task for current research. To date, nanotechnology offers unprecedented opportunities for cancer therapeutics and diagnosis. The aim of this study is the development of a new pancreatic cancer diagnostic technology based on the exploitation of the nano-bio-interactions between nanoparticles and blood samples. In this study, blood samples from 20 pancreatic cancer patients and 5 patients without malignancy were allowed to interact with designed lipid nanoparticles, leading to the formation of a hard "protein corona" at the nanoparticle surface. After isolation, the protein patterns were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). We found that the protein corona of pancreatic cancer patients was much more enriched than that of healthy individuals. Statistical analysis of SDS-PAGE results allowed us to discriminate between healthy and pancreatic cancer patients with a total discriminate correctness rate of 88%.

In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a "protein corona" layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø≈ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.

When nanoparticles come into contact with biological media, they are covered by a biomolecular 'corona', which confers a new identity to the particles. In all the studies reported so far nanoparticles are incubated with isolated plasma or serum that are used as a model for protein adsorption. Anyway, bodily fluids are dynamic in nature so the question arises on whether the incubation protocol, i.e. dynamic vs. static incubation, could affect the composition and structure of the biomolecular corona. Here we let multicomponent liposomes interact with fetal bovine serum (FBS) both statically and dynamically, i.e. in contact with circulating FBS (≈40 cm s(-1)). The structure and composition of the liposome-protein corona, as determined by dynamic light scattering, electrophoretic light scattering and liquid chromatography tandem mass spectrometry, were found to be dependent on the incubation protocol. Specifically, following dynamic exposure to FBS, multicomponent liposomes were less enriched in complement proteins and appreciably more enriched in apolipoproteins and acute phase proteins (e.g. alpha-1-antitrypsin and inter-alpha-trypsin inhibitor heavy chain H3) that are involved in relevant interactions between nanoparticles and living systems. Supported by our results, we speculate that efficient predictive modeling of nanoparticle behavior in vivo will require accurate knowledge of nanoparticle-specific protein fingerprints in circulating biological media.

When nanoparticles (NPs) enter a physiological environment, medium components compete for binding to the NP surface leading to formation of a rich protein shell known as the "protein corona". Unfortunately, opsonins are also adsorbed. These proteins are immediately recognized by the phagocyte system with rapid clearance of the NPs from the bloodstream. Polyethyleneglycol (PEG) coating of NPs (PEGylation) is the most efficient anti-opsonization strategy. Linear chains of PEG, grafted onto the NP surface, are able to create steric hindrance, resulting in a significant inhibition of protein adsorption and less recognition by macrophages. However, excessive PEGylation can lead to a strong inhibition of cellular uptake and less efficient binding with protein targets, reducing the potential of the delivery system. To reach a compromise in this regard we employed a multi-component (MC) lipid system with uncommon properties of cell uptake and endosomal escape and increasing length of PEG chains. Nano liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) analysis allowed us to accurately determine the corona composition showing that apolipoproteins are the most abundant class in the corona and that increasing the PEG length reduced the protein adsorption and the liposomal surface affinity for apolipoproteins. Due to the abundance of apolipoproteins, we exploited the "protein corona effect" to deliver cationic liposome-human plasma complexes to human prostate cancer PC3 cells that express a high level of scavenger receptor class B type 1 in order to evaluate the cellular uptake efficiency of the systems used. Combining laser scanning confocal microscopy with flow cytometry analysis in PC3 cells we demonstrated that MC-PEG2k is the best compromise between an anti-opsonization strategy and active targeting and could be a promising candidate to treat prostate cancer in vivo.