Efficient pancreatic cancer detection through personalized protein corona of gold nanoparticles.

Characterization of the personalized protein corona (PC) that forms around nanomaterials upon exposure to human plasma is emerging as powerful technology for early cancer detection. However, low material stability and interbatch variability have limited its clinical application so far. Here, we present a nanoparticle-enabled blood (NEB) test that uses 120 nm gold nanoparticles (NPs) as the accumulator of blood plasma proteins. In the test, the personalized PC of gold NPs is characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. As a paradigmatic case study, pancreatic ductal adenocarcinoma (PDAC) was chosen due to the lack of effective detection strategies that lead to poor survival rate after diagnosis (<1 year) and extremely low 5-years survival rate (15-20%). Densitometric analysis of 75 protein patterns (28 from healthy subjects and 47 from PDAC patients) allowed us to distinguish nononcological and PDAC patients with good sensitivity (78.6%) and specificity (85.3%). The gold NEB test is completely aligned to affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end users criteria stated by the World Health Organization for cancer screening and detection. Thus, it could be very useful in clinical practice at the first level of investigation to decide whether to carry out more invasive analyses or not.

A protein corona sensor array detects breast and prostate cancers.

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.

Effect of Protein Corona on The Transfection Efficiency of Lipid-Coated Graphene Oxide-Based Cell Transfection Reagents.

Coating graphene oxide nanoflakes with cationic lipids leads to highly homogeneous nanoparticles (GOCL NPs) with optimised physicochemical properties for gene delivery applications. In view of in vivo applications, here we use dynamic light scattering, micro-electrophoresis and one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis to explore the bionano interactions between GOCL/DNA complexes (hereafter referred to as "grapholipoplexes") and human plasma. When exposed to increasing protein concentrations, grapholipoplexes get covered by a protein corona that evolves with protein concentration, leading to biocoronated complexes with modified physicochemical properties. Here, we show that the formation of a protein corona dramatically changes the interactions of grapholipoplexes with four cancer cell lines: two breast cancer cell lines (MDA-MB and MCF-7 cells), a malignant glioma cell line (U-87 MG) and an epithelial colorectal adenocarcinoma cell line (CACO-2). Luciferase assay clearly indicates a monotonous reduction of the transfection efficiency of biocoronated grapholipoplexes as a function of protein concentration. Finally, we report evidence that a protein corona formed at high protein concentrations (as those present in in vivo studies) promotes a higher capture of biocoronated grapholipoplexes within degradative intracellular compartments (e.g., lysosomes), with respect to their pristine counterparts. On the other hand, coronas formed at low protein concentrations (human plasma = 2.5%) lead to high transfection efficiency with no appreciable cytotoxicity. We conclude with a critical assessment of relevant perspectives for the development of novel biocoronated gene delivery systems.

Bone Marrow Stromal Cell-Derived IL-8 Upregulates PVR Expression on Multiple Myeloma Cells via NF-kB Transcription Factor.

Bone marrow stromal cells (BMSCs) strongly contribute to multiple myeloma (MM) progression, promoting the survival and growth of malignant plasma cells (PCs). However, the possible impact of these cells on the immune-mediated recognition of MM cells remains largely unknown. DNAM-1 activating receptor plays a prominent role in NK cell anti-MM response engaging the ligands poliovirus receptor (PVR) and nectin-2 on malignant PCs. Here, we analysed the role of MM patient-derived BMSCs in the regulation of PVR expression. We found that BMSCs enhance PVR surface expression on MM cells and promote their NK cell-mediated recognition. PVR upregulation occurs at transcriptional level and involves NF-kB transcription factor activation by BMSC-derived soluble factors. Indeed, overexpression of a dominant-negative mutant of IKBα blocked PVR upregulation. IL-8 plays a prominent role in these mechanisms since blockade of CXCR1/2 receptors as well as depletion of the cytokine via RNA interference prevents the enhancement of PVR expression by BMSC-derived conditioned medium. Interestingly, IL-8 is associated with stromal microvesicles which are also required for PVR upregulation via CXCR1/CXCR2 signaling activation. Our findings identify BMSCs as regulators of NK cell anti-MM response and contribute to define novel molecular pathways involved in the regulation of PVR expression in cancer cells.

Synergistic Analysis of Protein Corona and Haemoglobin Levels Detects Pancreatic Cancer.

Simultaneous detection of multiple analytes from a single biological sample is gaining more attention in the development of more reliable and point-of-care diagnostic devices. We developed a multiplexed strategy that combined outcomes of clinical biomarkers with analysis of the protein corona that forms around graphene oxide sheets upon exposure to patient's plasma. As a paradigmatic case study, we selected pancreatic ductal adenocarcinoma (PDAC), mainly because of the absence of effective detection strategies that resulted in an extremely low five-year survival rate after diagnosis (<10%). Association of protein corona analysis and haemoglobin levels discriminated PDAC patients from healthy volunteers in up to 90% of cases. If further confirmed in larger-cohort studies, this approach may be used in the detection of PDAC.

Converting the personalized biomolecular corona of graphene oxide nanoflakes into a high-throughput diagnostic test for early cancer detection.

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.

Microfluidic manufacturing of surface-functionalized graphene oxide nanoflakes for gene delivery.

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.

Protein Corona Fingerprints of Liposomes: New Opportunities for Targeted Drug Delivery and Early Detection in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth cause of cancer-related mortality in the Western world and is envisaged to become the second cause by 2030. Although our knowledge about the molecular biology of PDAC is continuously increasing, this progress has not been translated into better patients' outcome. Liposomes have been used to circumvent concerns associated with the low efficiency of anticancer drugs such as severe side effects and damage of healthy tissues, but they have not resulted in improved efficacy as yet. Recently, the concept is emerging that the limited success of liposomal drugs in clinical practice is due to our poor knowledge of the nano⁻bio interactions experienced by liposomes in vivo. After systemic administration, lipid vesicles are covered by plasma proteins forming a biomolecular coating, referred to as the protein corona (PC). Recent studies have clarified that just a minor fraction of the hundreds of bound plasma proteins, referred to as "PC fingerprints" (PCFs), enhance liposome association with cancer cells, triggering efficient particle internalization. In this study, we synthesized a library of 10 liposomal formulations with systematic changes in lipid composition and exposed them to human plasma (HP). Size, zeta-potential, and corona composition of the resulting liposome⁻protein complexes were thoroughly characterized by dynamic light scattering (DLS), micro-electrophoresis, and nano-liquid chromatography tandem mass spectrometry (nano-LC MS/MS). According to the recent literature, enrichment in PCFs was used to predict the targeting ability of synthesized liposomal formulations. Here we show that the predicted targeting capability of liposome⁻protein complexes clearly correlate with cellular uptake in pancreatic adenocarcinoma (PANC-1) and insulinoma (INS-1) cells as quantified by flow-assisted cell sorting (FACS). Of note, cellular uptake of the liposomal formulation with the highest abundance of PCFs was much larger than that of Onivyde®, an Irinotecan liposomal drug approved by the Food and Drug Administration in 2015 for the treatment of metastatic PDAC. Given the urgent need of efficient nanocarriers for the treatment of PDAC, we envision that our results will pave the way for the development of more efficient PC-based targeted nanomaterials. Here we also show that some BCs are enriched with plasma proteins that are associated with the onset and progression of PDAC (e.g., sex hormone-binding globulin, Ficolin-3, plasma protease C1 inhibitor, etc.). This could open the intriguing possibility to identify novel biomarkers.

Nanoparticle-biomolecular corona: A new approach for the early detection of non-small-cell lung cancer.

Lung cancer (LC) is the most common type of cancer and the second cause of death worldwide in men and women after cardiovascular diseases. Non-small-cell lung cancer (NSCLC) is the most frequent type of LC occurring in 85% of cases. Developing new methods for early detection of NSCLC could substantially increase the chances of survival and, therefore, is an urgent task for current research. Nowadays, explosion in nanotechnology offers unprecedented opportunities for therapeutics and diagnosis applications. In this context, exploiting the bio-nano-interactions between nanoparticles (NPs) and biological fluids is an emerging field of research. Upon contact with biofluids, NPs are covered by a biomolecular coating referred to as "biomolecular corona" (BC). In this study, we exploited BC for discriminating between NSCLC patients and healthy volunteers. Blood samples from 10 NSCLC patients and 5 subjects without malignancy were allowed to interact with negatively charged lipid NPs, leading to the formation of a BC at the NP surface. After isolation, BCs were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We found that the BCs of NSCLC patients was significantly different from that of healthy individuals. Statistical analysis of SDS-PAGE results allowed discriminating between NSCLC cancer patients and healthy subjects with 80% specificity, 80% sensitivity and a total discriminate correctness rate of 80%. While the results of the present investigation cannot be conclusive due to the small size of the data set, we have shown that exploitation of the BC is a promising approach for the early diagnosis of NSCLC.

Effect of Glucose on Liposome-Plasma Protein Interactions: Relevance for the Physiological Response of Clinically Approved Liposomal Formulations.

Recently, the concept is emerging that the reduced success of nanoparticles in clinical practice is due to the adsorption of the "biomolecular corona (BC)," which alters their biological identity. Apart from protein variations, alterations in the human metabolome may change the BC decoration, which has poorly been addressed so far. Here, glucose is used as a model metabolite and how the interactions between liposomes (as a model nanoparticle) and plasma proteins are influenced by normal and diabetic sugar blood levels is explored. As model liposomes, Doxoves and Onivyde are used that are used for the treatment of breast and metastatic pancreatic cancer, respectively. It is shown that glucose does affect the structure and composition of BC. The biological effects of liposome-BC complexes are investigated in MCF 7 and MDA-MB-231 breast cancer cells for Doxoves and in pancreatic adenocarcinoma (PANC-1) and insulinoma (INS-1) cells for Onivyde. In the presence of glucose, the cellular toxicity of liposome-protein complexes and uptake by human monocytic THP1 cell line increases. These results demonstrate that alterations in glucose concentration, and more generally changes in the human metabolome, may play a fundamental role in the biological identity of liposomes and, consequently, on their in vivo physiological readouts including therapeutic efficacy.

Brain Targeting by Liposome-Biomolecular Corona Boosts Anticancer Efficacy of Temozolomide in Glioblastoma Cells.

Temozolomide (TMZ) is the current first-line chemotherapy for treatment of glioblastoma multiforme (GBM). However, similar to other brain therapeutic compounds, access of TMZ to brain tumors is impaired by the blood-brain barrier (BBB) leading to poor response for GBM patients. To overcome this major hurdle, we have synthesized a set of TMZ-encapsulating nanomedicines made of four cationic liposome (CL) formulations with systematic changes in lipid composition and physical-chemical properties. The targeting nature of this nanomedicine is provided by the recruitment of proteins, with natural targeting capacity, in the biomolecular corona (BC) layer that forms around CLs after exposure to human plasma (HP). TMZ-loaded CL-BC complexes were thoroughly characterized by dynamic light scattering (DLS), electrophoretic light scattering (ELS), and nanoliquid chromatography tandem mass spectrometry (nano-LC MS/MS). BCs were found to be enriched of typical BC fingerprints (BCFs) (e.g., Apolipoproteins, Vitronectin, and vitamin K-dependent protein), which have a substantial capacity in binding to receptors that are overexpressed at the BBB (e.g., scavenger receptor class B, type I and low-density lipoprotein receptor). We found that the CL formulation exhibiting the highest levels of targeting BCFs had larger uptake in human umbilical vein endothelial cells (HUVECs) that are commonly used as an in vitro model of the BBB. This formulation could also deliver TMZ to the human glioblastoma U-87 MG cell line and thus substantially enhance their antitumor efficacy compared to corona free CLs. Thus, we propose that the BC-based nanomedicines may pave a more effective way for efficient treatment of GBM.

Human Biomolecular Corona of Liposomal Doxorubicin: The Overlooked Factor in Anticancer Drug Delivery.

More than 20 years after its approval by the Food and Drug Administration (FDA), liposomal doxorubicin (DOX) is still the drug of choice for the treatment of breast cancer and other conditions such as ovarian cancer and multiple myeloma. Yet, despite the efforts, liposomal DOX did not satisfy expectations at the clinical level. When liposomal drugs enter a physiological environment, their surface gets coated by a dynamic biomolecular corona (BC). The BC changes liposome's synthetic identity, providing it with a new one, referred to as "biological identity" (size, aggregation state, and BC composition). Today, the concept is emerging that specific BCs may determine either success (e.g., stealth effect and accumulation at the target site) or failure (e.g., rapid blood clearance and off-target interactions) of liposomal drugs. To get a comprehensive investigation of liposome synthetic identity, biological identity, and cellular response as a function of human plasma (HP) concentration, here we used a straightforward combination of quantitative analytical and imaging tools, including dynamic light scattering, microelectrophoresis, synchrotron small-angle X-ray scattering, transmission electron microscopy (TEM), fluorescence lifetime imaging microscopy (FLIM), nano-liquid chromatography tandem mass spectrometry/mass spectrometry (nano-LC-MS/MS), confocal microscopy, flow cytometry, and cell viability assays. Doxoves was selected as a reference. Following exposure to HP, Doxoves was surrounded by a complex BC that changed liposome's synthetic identity. Observations made with nano-LC-MS/MS revealed that the BC of Doxoves did not evolve as a function of HP concentration and was poorly enriched of typical "opsonins" (complement proteins, immunoglobulins, etc.). This provides a possible explanation for the prolonged blood circulation of liposomal DOX. On the other hand, flow cytometry showed that protein binding reduced the internalization of DOX in MCF7 and MDA-MB-435S human breast carcinoma. Combining FLIM and TEM experiments, we clarified that reduction in DOX intracellular content was likely due to the frequent rupture of the liposome membrane and consequent leakage of the cargo. In light of reported results, we are prompted to speculate that a detailed understanding of BC formation, composition, and effects on liposome stability and uptake is an indispensable task of future research in the field, especially along the way to clinical translation of liposomal drugs.

Tumor-Derived Microvesicles Modulate Antigen Cross-Processing via Reactive Oxygen Species-Mediated Alkalinization of Phagosomal Compartment in Dendritic Cells.

Dendritic cells (DCs) are the only antigen-presenting cells able to prime naïve T cells and cross-prime antigen-specific CD8+ T cells. Their functionality is a requirement for the induction and maintenance of long-lasting cancer immunity. Albeit intensively investigated, the in vivo mechanisms underlying efficient antigen cross-processing and presentation are not fully understood. Several pieces of evidence indicate that antigen transfer to DCs mediated by microvesicles (MVs) enhances antigen immunogenicity. This mechanism is also relevant for cross-presentation of those tumor-associated glycoproteins such as MUC1 that are blocked in HLA class II compartment when internalized by DCs as soluble molecules. Here, we present pieces of evidence that the internalization of tumor-derived MVs modulates antigen-processing machinery of DCs. Employing MVs derived from ovarian cancer ascites fluid and established tumor cell lines, we show that MV uptake modifies DC phagosomal microenvironment, triggering reactive oxygen species (ROS) accumulation and early alkalinization. Indeed, tumor MVs carry radical species and the MV uptake by DCs counteracts the chemically mediated acidification of the phagosomal compartment. Further pieces of evidence suggest that efficacious antigen cross-priming of the MUC1 antigen carried by the tumor MVs results from the early signaling induced by MV internalization and the function of the antigen-processing machinery of DCs. These results strongly support the hypothesis that tumor-derived MVs impact antigen immunogenicity by tuning the antigen-processing machinery of DCs, besides being carrier of tumor antigens. Furthermore, these findings have important implications for the exploitation of MVs as antigenic cell-free immunogen for DC-based therapeutic strategies.

Genotoxic stress modulates the release of exosomes from multiple myeloma cells capable of activating NK cell cytokine production: Role of HSP70/TLR2/NF-kB axis.

Exosomes are a class of nanovesicles formed and released through the late endosomal compartment and represent an important mode of intercellular communication. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Here, we investigated whether genotoxic stress could promote the release of exosomes from multiple myeloma (MM) cells and studied the immunomodulatory properties they exert on NK cells, a major component of the antitumor immune response playing a key role in the immunosurveillance of MM. Our findings show that melphalan, a genotoxic agent used in MM therapy, significantly induces an increased exosome release from MM cells. MM cell-derived exosomes are capable of stimulating IFNγ production, but not the cytotoxic activity of NK cells through a mechanism based on the activation of NF-κB pathway in a TLR2/HSP70-dependent manner. Interestingly, HSP70+ exosomes are primarily found in the bone marrow (BM) of MM patients suggesting that they might have a crucial immunomodulatory action in the tumor microenvironment. We also provide evidence that the CD56high NK cell subset is more responsive to exosome-induced IFNγ production mediated by TLR2 engagement. All together, these findings suggest a novel mechanism of synergism between chemotherapy and antitumor innate immune responses based on the drug-promotion of nanovesicles exposing DAMPs for innate receptors.

Influence of dynamic flow environment on nanoparticle-protein corona: From protein patterns to uptake in cancer cells.

The fast growing use of nanoparticles (NPs) in biotechnology and biomedicine raises concerns about human health and the environment. When introduced in physiological milieus, NPs adsorb biomolecules (especially proteins) forming the so-called protein corona (PC). As it is the PC that mostly interacts with biological systems, it represents a major element of the NPs' biological identity with impact on nanotoxicology, nanosafety and targeted delivery of nanomedicines. To date, NP-protein interactions have been largely investigated in vitro, but this condition is far from mimicking the dynamic nature of physiological environments. Here we investigate the effect of shear stress on PC by exposing lipid NPs with different surface chemistry (either unmodified and PEGylated) to circulating fetal bovine serum (FBS). PC formed upon in vitro incubation was used as a reference. We demonstrate that PC is significantly influenced by exposure to dynamic flow and that changes in PC composition are dependent on both exposure time and NP's surface chemistry. Notably, alterations induced by dynamic flow affected cellular uptake of lipid NPs in both human cervical cancer (HeLa) and human breast adenocarcinoma (MCF7) cell lines.

Manipulation of lipoplex concentration at the cell surface boosts transfection efficiency in hard-to-transfect cells.

To date, efficiency upon non-viral DNA delivery remains low and this implies the existence of unidentified transfection barriers. Here we explore the mechanisms of action of multicomponent (MC) cationic liposome/DNA complexes (lipoplexes) by a combination of reporter technologies, dynamic light scattering (DLS), synchrotron small angle X-ray scattering (SAXS), fluorescence activated cell sorting (FACS) analysis and laser scanning confocal microscopy (LSCM) in live cells. Lipofectamine - the gold standard among transfection reagents - was used as a reference. On the basis of our results, we suggest that an additional transfection barrier impairs transfection efficiency, that is: low lipoplex concentration at the cell surface. Based on the acquired knowledge we propose an optimized transfection protocol that allowed us to efficiently transfect DND41, JURKAT, MOLT3, P12-ICHIKAWA, ALL-SILL, TALL-1 human T-cell acute lymphoblastic leukemia (T-ALL) cell lines known to be difficult-to-transfect by using non-viral vectors and where LFN-based technologies fail to give satisfactory results.

Exploring Cellular Interactions of Liposomes Using Protein Corona Fingerprints and Physicochemical Properties.

To control liposomes fate and transport upon contact with biofluids, it is essential to consider several parameters affecting the synthetic and biological identity of liposomes, as well as liposome-protein corona (PC) aspects. As a powerful tool in this data mining adventure, quantitative structure-activity relationship (QSAR) approach is used to correlate physicochemical properties of liposomes and their PC fingerprints to multiple quantified biological responses. In the present study, the relationship between cellular interactions of a set of structurally diverse liposomal formulations and their physicochemical and PC properties has been investigated via linear and nonlinear QSAR models. Significant parameters affecting cellular uptake and cell viability of liposomes in two important cancer cell lines (PC3 and HeLa) have been identified. The developed QSARs have the capacity to be implemented in advanced targeted delivery of liposomal drugs.

Personalized liposome-protein corona in the blood of breast, gastric and pancreatic cancer patients.

When nanoparticles (NPs) are dispersed in a biofluid, they are covered by a protein corona the composition of which strongly depends on the protein source. Recent studies demonstrated that the type of disease has a crucial role in the protein composition of the NP corona with relevant implications on personalized medicine. Proteomic variations frequently occur in cancer with the consequence that the bio-identity of NPs in the blood of cancer patients may differ from that acquired after administration to healthy volunteers. In this study we investigated the correlation between alterations of plasma proteins in breast, gastric and pancreatic cancer and the biological identity of clinically approved AmBisome-like liposomes as determined by a combination of dynamic light scattering, zeta potential analysis, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) and semi-quantitative densitometry. While size of liposome-protein complexes was not significantly different between cancer groups, the hard corona from pancreatic cancer patients was significantly less negatively charged. Of note, the hard corona from pancreatic cancer patients was more enriched than those of other cancer types this enrichment being most likely due to IgA and IgG with possible correlations with the autoantibodies productions in cancer. Given the strict relationship between tumor antigen-specific autoantibodies and early cancer detection, our results could be the basis for the development of novel nanoparticle-corona-based screening tests of cancer.

Stealth effect of biomolecular corona on nanoparticle uptake by immune cells.

When injected in a biological milieu, a nanomaterial rapidly adsorbs biomolecules forming a biomolecular corona. The biomolecular corona changes the interfacial composition of a nanomaterial giving it a biological identity that determines the physiological response. Characterization of the biomolecular structure and composition has received increasing attention mostly due to its detrimental impact on the nanomaterial's metabolism in vivo. It is generally accepted that an opsonin-enriched biomolecular corona promotes immune system recognition and rapid clearance from circulation. Here we applied dynamic light scattering and nanoliquid chromatography tandem mass spectrometry to thoroughly characterize the biomolecular corona formed around lipid and silica nanoparticles (NPs). Incubation with human plasma resulted in the formation of NP-biomolecular coronas enriched with immunoglobulins, complement factors, and coagulation proteins that bind to surface receptors on immune cells and elicit phagocytosis. Conversely, we found that protein-coated NPs were protected from uptake by macrophage RAW 264.7 cells. This implies that the biomolecular corona formation provides a stealth effect on macrophage recognition. Our results suggest that correct prediction of the NP's fate in vivo will require more than just the knowledge of the biomolecular corona composition. Validation of efficient methods for mapping protein binding sites on the biomolecular corona of NPs is an urgent task for future research.

Transfected poly(I:C) activates different dsRNA receptors, leading to apoptosis or immunoadjuvant response in androgen-independent prostate cancer cells.

Despite the effectiveness of surgery or radiation therapy for the treatment of early-stage prostate cancer (PCa), there is currently no effective strategy for late-stage disease. New therapeutic targets are emerging; in particular, dsRNA receptors Toll-like receptor 3 (TLR3) and cytosolic helicases expressed by cancer cells, once activated, exert a pro-apoptotic effect in different tumors. We previously demonstrated that the synthetic analog of dsRNA poly(I:C) induces apoptosis in the androgen-dependent PCa cell line LNCaP in a TLR3-dependent fashion, whereas only a weak apoptotic effect is observed in the more aggressive and androgen-independent PCa cells PC3 and DU145. In this paper, we characterize the receptors and the signaling pathways involved in the remarkable apoptosis induced by poly(I:C) transfected by Lipofectamine (in-poly(I:C)) compared with the 12-fold higher free poly(I:C) concentration in PC3 and DU145 cells. By using genetic inhibition of different poly(I:C) receptors, we demonstrate the crucial role of TLR3 and Src in in-poly(I:C)-induced apoptosis. Therefore, we show that the increased in-poly(I:C) apoptotic efficacy is due to a higher binding of endosomal TLR3. On the other hand, we show that in-poly(I:C) binding to cytosolic receptors MDA5 and RIG-I triggers IRF3-mediated signaling, leading uniquely to the up-regulation of IFN-ß, which likely in turn induces increased TLR3, MDA5, and RIG-I proteins. In summary, in-poly(I:C) activates two distinct antitumor pathways in PC3 and DU145 cells: one mediated by the TLR3/Src/STAT1 axis, leading to apoptosis, and the other one mediated by MDA5/RIG-I/IRF3, leading to immunoadjuvant IFN-ß expression.