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.

Cationic lipid/DNA complexes manufactured by microfluidics and bulk self-assembly exhibit different transfection behavior.

Recent advances in biochemical and biophysical research have been achieved through the employment of microfluidic devices. Microfluidic mixing of therapeutic agents with biomaterials yields systems with finely tuned physical-chemical properties for applications in drug and gene delivery. Here, we investigate the role of preparation technology (microfluidic mixing vs. bulk self-assembly) on the transfection efficiency (TE) and cytotoxicity of multicomponent cationic liposome/DNA complexes (lipoplexes) in live Chinese hamster ovarian (CHO) cells. Decoupling TE and cytotoxicity allowed us to combine them in a unique coherent vision. While bulk self-assembly produces highly efficient and highly toxic MC lipoplexes, microfluidics manufacture leads to less efficient, but less cytotoxic complexes. This discrepancy is ascribed to two main factors controlling lipid-mediated cell transfection, i.e. the lipoplex concentration at the cell surface and the lipoplex arrangement at the nanoscale. Further research is required to optimize microfluidic manufacturing of lipoplexes to obtain highly efficient and not cytotoxic gene delivery systems.

The protein corona of circulating PEGylated liposomes.

Following systemic administration, liposomes are covered by a 'corona' of proteins, and preserving the surface functionality is challenging. Coating the liposome surface with polyethylene glycol (PEG) is the most widely used anti-opsonization strategy, but it cannot fully preclude protein adsorption. To date, protein binding has been studied following in vitro incubation to predict the fate of liposomes in vivo, while dynamic incubation mimicking in vivo conditions remains largely unexplored. The main aim of this investigation was to determine whether shear stress, produced by physiologically relevant dynamic flow, could influence the liposome-protein corona. The corona of circulating PEGylated liposome was thoroughly compared with that formed by incubation in vitro. Systematic comparison in terms of size, surface charge and quantitative composition was made by dynamic light scattering, microelectrophoresis and nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). Size of coronas formed under static vs. dynamic incubation did not appreciably differ from each other. On the other side, the corona of circulating liposomes was more negatively charged than its static counterpart. Of note, the variety of protein species in the corona formed in a dynamic flow was significantly wider. Collectively, these results demonstrated that the corona of circulating PEGylated liposomes can be considerably different from that formed in a static fluid. This seems to be a key factor to predict the biological activity of a liposomal formulation in a physiological environment.

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.

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.

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.

Time evolution of nanoparticle-protein corona in human plasma: relevance for targeted drug delivery.

When nanoparticles (NPs) enter a biological fluid (e.g., human plasma (HP)), proteins and other biomolecules adsorb on the surface leading to formation of a rich protein shell, referred to as "protein corona". This corona is dynamic in nature and its composition varies over time due to continuous protein association and dissociation events. Understanding the time evolution of the protein corona on the time-scales of a particle's lifetime in blood is fundamental to predict its fate in vivo. In this study, we used lipid NPs, the cationic lipid 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl] (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE), that are among the most promising nanocarriers both in vitro and in vivo. Here, we investigated the time evolution of DC-Chol-DOPE NPs upon exposure to HP. On time scales between 1 and 60 minutes, nanoliquid tandem mass spectrometry revealed that the protein corona of DC-Chol-DOPE NPs is mainly constituted of apolipoproteins (Apo A-I, Apo C-II, Apo D, and Apo E are the most enriched). Since the total apolipoprotein content is relevant, we exploited the protein corona to target PC3 prostate carcinoma cell line that expresses high levels of scavenger receptor class B type 1 receptor, which mediates the bidirectional lipid transfer between low-density lipoproteins, high-density lipoproteins, and cells. Combining laser scanning confocal microscopy experiments with flow cytometry we demonstrated that DC-Chol-DOPE/HP complexes enter PC3 cells by a receptor-mediated endocytosis mechanism.

Artificial Protein Coronas Enable Controlled Interaction with Corneal Epithelial Cells: New Opportunities for Ocular Drug Delivery.

Topical administration is the most convenient route for ocular drug delivery, but only a minor fraction is retained in the precorneal pocket. To overcome this limitation, numerous drug delivery systems (DDS) have been developed. The protein corona (PC) is the layer of biomolecules (e.g., proteins, sugars, lipids, etc.) that forms around DDS in physiological environments by non-covalent interaction. The PC changes the DDS physical-chemical properties, providing them with a completely novel biological identity. The specific involvement of PC in ocular drug delivery has not been addressed so far. To fulfill this gap, here we explored the interaction between a library of four cationic liposome-DNA complexes (lipoplexes) and mucin (MUC), one of the main components of the tear film. We demonstrate that MUC binds to the lipoplex surface shifting both their size and surface charge and reducing their absorption by primary corneal epithelial cells. To surpass such restrictions, we coated lipoplexes with two different artificial PCs made of Fibronectin (FBN) and Val-Gly-Asp (VGA) tripeptide that are recognized by receptors expressed on the ocular surface. Both these functionalizations remarkedly boosted internalization in corneal epithelial cells with respect to pristine (i.e., uncoated) lipoplexes. This opens the gateway for the exploitation of artificial protein corona in targeted ocular delivery, which will significantly influence the development of novel nanomaterials.

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.

Impact of the protein corona on nanomaterial immune response and targeting ability.

Over the last decade nanomaterials have had a major impact on human health for the early detection and treatment of many diseases. The future success of clinically translatable nanomaterials lies in the combination of several functionalities to realize a personalized medical experience for patients. To maintain promises, concerns arising from toxic potential and off-target accumulation of nanomaterials must be addressed first. Upon introduction to a complex biological system (e.g., following systemic administration), nanomaterials interact with all the encountered biomolecules and form the protein corona, a complex coating of plasma proteins that provides them with a totally new biological identity. As the protein corona controls the nanomaterial behavior in vivo, a precise knowledge of the relationship between biological identity and physiological response is needed but not yet achieved. Based on impressive progress made thus far, this review critically discusses how the protein corona activates immune response and influences the targeted delivery of nanomaterials. Furthermore, we comment on emerging strategies to manipulate protein binding in order to promote formation of designer artificial coronas and achieve a desired therapeutic outcome. We conclude by debating challenges that must be overcome to obtain widespread clinical adoption of nanomaterials. This article is categorized under: Nanotechnology Approaches to Biology > Cells at the Nanoscale Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Therapeutic Approaches and Drug Discovery > Emerging Technologies.

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.

A mechanistic explanation of the inhibitory role of the protein corona on liposomal gene expression.

The past three decades have witnessed fast advances in the use of cationic liposome-DNA complexes (lipoplexes) for gene delivery applications. However, no lipoplex formulation has reached into the clinical practice so far. The primary drawback limiting clinical use of lipoplexes is the lack of mechanistic understanding of their low transfection efficiency (TE) in vivo. In physiological environments, lipoplexes are coated by a protein corona (PC) that mediates the interactions with the cell machinery. Here we show that the formation of PC can change the interactions of multicomponent (MC) lipoplexes with our cell model (i.e., HeLa). At the highest lipoplex concentration, the formation of PC can reduce the TE of MC lipoplexes from 60% to <5%. Combining dynamic light scattering and synchrotron small-angle X-ray scattering (SAXS), we clarify that the formation of PC modifies physical-chemical properties of MC lipoplexes so as to affect their TE. Moreover, we examined single transfection barriers by a combination of fluorescence-activated cell sorting, single-cell real-time fluorescence confocal microscopy, and synchrotron SAXS. We demonstrate that PC formation has the ability to modify the relative contribution of caveolae-mediated endocytosis and macropinocytosis in lipoplexes uptake, in favor of the latter, increasing accumulation of PC-decorated lipoplexes into degradative lysosomal compartments. Finally, we report evidences that PC reduces the structural stability of lipoplexes against solubilization by cellular lipids, likely favoring premature DNA release and cytosolic digestion by DNAase. These combined effects revealed here offer a comprehensive mechanistic explanation on the reason behind reduction in gene expression of MC lipoplexes.

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.

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.

Author Correction: Interplay of protein corona and immune cells controls blood residency of liposomes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

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.

The biomolecular corona of gold nanoparticles in a controlled microfluidic environment.

Nanoparticles (NPs) exposed to biological media are coated by proteins and other biomolecules forming a biomolecular corona (BC) on the particle surface. Recent studies have shown that shear stress as that created by laminar fluid flow generates more complex coronas with systematic changes in composition with respect to counterparts formed under static incubation. However, in most studies reported so far, dynamic environments have been produced by peristaltic pumps and comparing experimental results appears challenging. On the other side, generating shear stress by microfluidic devices could help to remove user variability and ensure better reproducibility of experimental data. This study was therefore aimed at exploring formation of NP-BC in a microfluidic environment. To this end, 100 nm gold nanoparticles and human plasma (HP) were used as models for nano-formulation and biological medium. We injected gold nanoparticles and HP in each of the islets of a remote-controlled microfluidic cartridge. Static incubation was used as a reference. BC-decorated NPs were thoroughly characterized by dynamic light scattering (DLS), micro-electrophoresis (ME), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and nano-liquid chromatography tandem mass spectrometry (nano-LC MS/MS). By varying the incubation time from 30 s to 2.5 min we demonstrate that BC is already determined by the earliest exposure time point and does not appreciably evolve in time. DLS and ME results demonstrate that the BC formed in a microfluidic chip is thicker and more negatively charged than its counterpart formed under static incubation. SDS-PAGE and nano-LC MS/MS revealed that the incubation procedure had a major effect on BC composition. As an example, immunoglobulins are the most abundant plasma proteins of the BC generated in a microfluidic environment (relative protein abundance ∼30%), while tissue leakage proteins (relative protein abundance ∼26%) are the most enriched proteins when the BC is formed upon static incubation. Potential implications in emerging biomedical research arenas are discussed.

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.