acsami.8b04962


Jun 15, 2018 - Human Biomolecular Corona of Liposomal Doxorubicin: The. Overlooked Factor in Anticancer Drug Delivery. Giulio Caracciolo,*,†...
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Biological and Medical Applications of Materials and Interfaces

The human biomolecular corona of liposomal doxorubicin: The overlooked factor in anticancer drug delivery Giulio Caracciolo, Sara Palchetti, Luca Digiacomo, Riccardo Zenezini Chiozzi, Anna Laura Capriotti, Heinz Amenitsch, Paolo Maria Tentori, Valentina Palmieri, Massimiliano Papi, Francesco Cardarelli, Daniela Pozzi, and Aldo Laganà ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04962 • Publication Date (Web): 15 Jun 2018 Downloaded from http://pubs.acs.org on June 15, 2018

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The human biomolecular corona of liposomal doxorubicin: The overlooked factor in anticancer drug delivery

Giulio Caracciolo,a* Sara Palchetti,a Luca Digiacomo,a Riccardo Zenezini Chiozzi,b Anna Laura Capriotti,b Heinz Amenitsch,c Paolo Maria Tentori,d,e Valentina Palmieri,f Massimiliano Papi,f Francesco Cardarelli,g Daniela Pozzi,a Aldo Laganàb

a

Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy.

b

Department of Chemistry, Sapienza University of Rome, Rome, Italy.

c

Institute of inorganic Chemistry, Graz University of Technology, Graz, Austria.

d

Center for Nanotechnology [email protected], Istituto Italiano di Tecnologia, Pisa, Italy.

e

NEST, Scuola Normale Superiore, Pisa, Italy.

f

Istituto di Fisica, Università Cattolica del Sacro Cuore, Rome, Italy.

g

NEST - Scuola Normale Superiore, Istituto Nanoscienze - CNR (CNR-NANO), Pisa, Italy.

Keywords: biomolecular corona, bionano interactions, nanomedicine, anticancer drug delivery, liposomal doxorubicin.

*E-mail: [email protected]

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Abstract More than twenty 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, 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 (DLS), micro-electrophoresis (ME), synchrotron small angle X-ray scattering (SAXS), transmission electron microscopy (TEM), fluorescence life-time imaging microscopy (FLIM), nano-liquid chromatography tandem 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 internalization of DOX in MCF7 and MDAMB-435S human breast carcinoma. Combining FLIM and TEM experiments, we clarified that reduction in DOX intracellular content was likely due to 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.

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Introduction With 16 million new cases and 9 million deaths each year, cancer is still one of the most challenging global healthcare problems 1. It is the second cause of death in the U.S. and expected to surpass cardiovascular diseases, currently at the top of the list of most lethal disorders, by 2030 1-2

. Although there is a huge number of drugs with anticancer properties, they distribute non-

specifically through the body reaching indifferently both cancer and normal cells3. Poor concentration at the tumor site arising from dose limiting toxicity, fast removal from blood circulation and poor local accumulation has long been one of the principal reasons why in vitro promising anticancer drugs frequently failed at the level of in vivo trials4-5. The gradual maturation of nanotechnology provides an innovative perspective for cancer therapy6-7. Simultaneous encapsulation of both hydrophilic and hydrophobic drugs in nanocarriers, for instance, protects them, extend their blood circulation and promote accumulation at the target site by both passive and active targeting

6, 8-9

. Among nanodelivery systems, liposomes have received extensive

application over the past two decades for gene and drug delivery applications10-15. Doxil®, a formulation of liposomes remotely loaded with doxorubicin (DOX) and functionalized with poly(ethylene glycol) (PEG), was the first lipid nanomedicine approved by the Food and Drug Administration (FDA) in 1995 4. However, despite big efforts aimed at reducing DOX-related toxicity 16, Doxil® did not increase promises to greatly improve clinical outcome. The case of Doxil® demonstrates that progress made in exploiting nanotechnology has come to an unexpected stop in liposome research. To bring liposomes to bedside, a bottleneck must be overcome: the gap between in vitro and in vivo results

17

. Researchers believe that this gap is due several “hidden

factors” exist that have been overlooked during the last decade. The majority of such ignored factors are associated with the nanoparticle-biomolecular corona (BC)

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, a dynamic

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biomolecular layer that covers nanocarriers in vivo, changes their synthetic identity, changes in time 24 and has a deep impact on the particle fate 25-27. In liposome research the impact of BC has been long underestimated 28 because grafting PEG to liposome surface was generally assumed to inhibit protein adsorption, and consequently keep liposome functionality intact. Recently, it was clarified that PEGylation mitigates protein binding, but does not avoid BC formation

29-30

. One of

the most important implications is that clinically approved liposomal formulations could not be stealth in vivo. Since protein adsorption is not preventable on PEGylated liposomes, what the patient’s cells actually ‘see’, when coming in touch with liposome, is liposome’s BC and not the pristine vesicle31-32. The recognition of specific epitopes could have a deep impact on several biological processes, such as association with cell receptors, liposome accumulation at the target site, cell internalization, drug release and intracellular trafficking. Kostarelos et al. were the first to confirm that after administration to mice Doxil® is covered by a BC

33

. However, following

exposure of liposomes to mouse (MP) and human plasma (HP), resulting coronas were found to differ both in number and abundance of identified proteins

34

. This result indicated that

physiological outcome of liposomes in animal models such as pharmacokinetic (PK) and body distribution (BD) could be largely different from those in humans. To date, this gap makes it difficult to translate results obtained on animals into the clinic 35. Accurate understanding of the human liposome-BC could contribute to explain the limited clinical success of liposomal DOX. To test this working hypothesis, here we performed a comprehensive investigation of post-synthesis identity, biological identity and cellular response of liposomal DOX. To the authors’ knowledge, characterization of the human BC of liposomal DOX is addressed here for the first time. To this end, we employed Doxoves®, a liposomal DOX formulation with the same lipid composition and lipid/drug ratio of Doxil®. According to the manufacturer, Doxoves® exhibits comparable physical characteristics and DOX encapsulation efficiency (>98%) and is suspended in the same buffer

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(10wt% sucrose, 10mM histidine, pH 6.5). Doxoves® was incubated in HP concentrations from 5% to 50% and was thoroughly characterized by synchrotron small angle X-ray scattering (SAXS), dynamic light scattering (DLS), micro-electrophoresis (ELS), transmission electron microscopy (TEM), fluorescence-lifetime imaging microscopy (FLIM) and nano-liquid chromatography tandem mass spectrometry (nanoLC/MS-MS). Flow cytometry and fluorescence confocal microscopy were applied to investigate the biological performance of Doxoves® in MCF7 and MDA-MB-435S human breast carcinoma cells both in the absence and in the presence of BC. In vitro experiments should mimic in the best possible way what happens in vivo. As the plasma percentage is around 50% in vivo, this is the typical experimental condition used to explore the “nano-bio interactions”36. However, it should be underlined that the amount of protein “seen” by NPs in vivo is not always the same. For instance, it is well established that in interstitial fluids protein concentration can be much lower than in plasma

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. This a point of great interest in anticancer drug delivery since

particle accumulation in tumors is strictly related to passage in interstitial fluids38. Thus, investigating the evolution of the BC at plasma percentages lower than 50% could make our knowledge of the biological identity of liposomal DOX more accurate. Moreover, most studies on liposomal DOX have been performed in vitro (i.e. at low serum dilutions). The fact that the BC could evolve passing from HP percentage appropriate to in vitro cell studies to the protein regime present during in vivo studies may help to interpret experimental results and may suggest new strategies for in vitro-in vivo extrapolations. Another key factor that could affect the BC is the dynamic nature of bodily fluids30, 39-40. However, to better compare our results with findings of previous in vitro studies that were performed under static incubation, the effect of dynamic incubation was not considered here, but will be the object of future investigations. Here we show that upon interaction with HP Doxoves® is surrounded by a rich BC. Protein binding has a minor effect on Doxoves®’s size and nanostructure, while it changes appreciably liposome’s surface

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charge. Of note, Doxoves®-BC is poorly enriched of typical opsonins (i.e. complement proteins, Immunoglobulins etc.), while it is mainly composed of Apolipoproteins that can provide liposomes with a distinctive ability to target cancer cells. Despite this apparently optimal composition, Doxoves®-BC reduces internalization of DOX in MDA-MB-435S human breast carcinoma cells, while it had minor effect, if any, in MCF 7 cells. This most likely occurs due to vesicle destabilization and drug release to the plasma before cell administration. Supported by our results, we speculated that a full characterization of the in vivo liposome-BC associated to advances in prediction technologies will help to develop therapeutically efficient liposomal drugs. This knowledge is a key step to move liposome technology a step closer to clinical usage.

2. Materials and Methods 2.1 Sample preparation Doxoves® and the plain control (i.e. empty liposomes) were purchased from Formumax Scientific (Sunnyvale, CA, USA). Many factors are involved in protein adsorption to NPs. Recently, Hajipour et al.20 demonstrated that changes in the human proteome as those induced by different pathological conditions may affect the composition and structure of the BC. As a consequence, each patient may have a personalized BC. In the next future, accurate knowledge of personalized BC will be necessary to design safe and efficient nanotherapies. However, changes in the humane proteome are not only caused by the presence of a certain condition (e.g. breast cancer). Among other factors, the genetic inheritance, behavioral orientations, geographical and demographical variables affect proteomes even in healthy individuals20. This means that the BCs of two healthy subjects can differ more than those of a healthy subject and a cancer patient41. To minimize variability due to interpersonal changes in protein concentration and structure, we used commercial HP (Sigma-Aldrich, Milan, Italy). Adequate amounts of liposomes (being indifferently

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DOX-loaded or empty) were incubated for 1-hour at 37°C in plasma concentrations from 5% to 50%. BC-coated liposomes were pelletized by centrifugation at 14,000 rpm for 15 minutes at 4°C. Washing with cold PBS was used to remove the non-specific bound proteins (this procedure was replicated three times). The resulting pellet was resuspended in PBS. 2.2 Cell culture MDA-MB-435S and MCF7 cells were purchased from ATCC and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), antibiotics (1% penicillin and streptomycin) and 1% of L-Glutamine. Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere. 2.3 Size and Zeta-potential experiments Size and zeta-potential measurements were performed at room temperature using a Zetasizer Nano ZS90 (Malvern, UK). Samples were diluted 1:100. Using the same apparatus, the electrophoretic mobility, µ, was measured. From the mobility u the zeta-potential of liposomes is calculated using the Smoluchowski relation zeta-potential = uη/ε, where η and ε are the viscosity and the permittivity of the solvent phase. 2.4 Synchrotron SAXS measurements Synchrotron Small Angle X-ray Scattering (SAXS) experiments were performed at the Austrian SAXS beamline of the synchrotron light source ELETTRA (Trieste, Italy). SAXS images were recorded with a 2D pixel detector Pilatus3 1M spanning the q-range between 0.05 nm-1 and 5 nm-1 with a resolution of 5 x 10-3 nm-1 (full width at half maximum). The image conversion to 1D SAXS pattern has been perfomed with FIT2D (http://www.esrf.eu). Calibration of detectors was performed with silver behenate powder (d-spacing = 5.838 nm). Typical exposure times were 100 s. Radiation damage was not detected in the SAXS scans. Samples were held in a 1 mm glass capillary (Hilgenberg, Malsfeld, Germany). Experiments were carried put at 25 °C. ACS Paragon Plus Environment

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2.5 Transmission electron microscopy (TEM) 10 µL of sample was dropped on carbon-coated copper grids and allowed to adsorb for 1 min. The resulting film was stained with a 2% uranyl acetate solution in the dark at room temperature. A filter paper was used to adsorb excess staining solution. Afterwards, grids were air dried for 1hour before imaging with TEM SUPRA 25 (Zeiss, Germany). 2.6 Fluorescence life-time imaging microscopy (FLIM) experiments Before performing FLIM experiments, Doxoves® and Doxoves®-HP complexes were loaded into a 1.7% agarose gel in order to be immobilized. Doxoves® and Doxoves®-HP complexes were imaged by a pulsed diode laser. Operating conditions were: frequency=at 40 MHz; excitation wavelength = 470 nm; average power at the sample: 10–20 μW. Emitted radiation in the wavelength range between 500 and 650 nm was collected by a photomultiplier tube interfaced with a time correlated single photon counting card and setup (PicoHarp 300, PicoQuant, Berlin). 2.7 1D SDS-PAGE experiments For the electrophoresis experiments the pellet resulting by sample centrifugation is resuspended in 40 µL of Loading Buffer 1X (50 mM Tris-HCl, 0.01% BBP, 0.5% SDS, 10% Glycerol, 0.1M DTT) and boiled for 5 minutes at 100°C. Identical volumes (20 µL) of each sample were loaded on a gradient polyacrylamide gel (4-20% Novex TG precast gels, Thermo Fischer) and run at 100 V for approximately 150 minutes. Gels were rinsed in double-distilled water (ddH2O) and fixed for 1 hour in a fixing solution (40% EtOH and 10% CH3COOH in ddH2O) with gentle agitation at room temperature. In order to determine the differences in corona composition, we stained the proteins with Comassie for 1 hour at room temperature with gentle shaking. 2.8 Protein assay The amount of proteins adsorbed to liposomes’ surface, was quantified using the Protein Assay reagent (Pierce, Thermo Scientific, Waltham, MA, USA) following the manufacturer’s protocol. In

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Brief, Doxoves® was incubated with HP for 1-hour at 37 °C. Then, Doxoves®−HP complexes were pelleted by centrifugation (14 000 rpm, for 15 min at 4 °C). The resulting pellet was washed 3 times with PBS and resuspended in urea 8 mol/L NH4CO3 50 mmol/L. 10 µL of sample was placed into a 96-multiwell plate and then 150 μL of Protein Assay reagent was added. The measurements were carried out in triplicate. The plate was covered and shaked for 5 min. Absorbance was measured with the Glomax Discover System (Promega, Madison, WI, USA) at 660 nm. Protein amount was determined by comparing absorbance value to a standard curve. 2.9 Proteomics Experiments In solution digestion and desalting. Pellet was resuspended in 40 µl of urea 8 mol L−1, NH4HCO3 50 mmol/l (pH=7.8). Afterwards, protein solution was reduced with 2 µl of 1 DTT 200 mmol L−1, alkylated with 8 µl of IAA 200 mmol L−1 and newly added with 8 µl of DTT 200 mmol L−1. Sample solution was diluted with 50 mmol L−1 NH4HCO3 to obtain a final urea concentration of 1 mol L−1 and digested overnight with 2 µg of trypsin at 37 °C. The enzymatic reaction was stopped by adding TFA. The digested peptides were desalted using SPE C18 column, reconstituted with a suitable volume of a 0.1% formic acid solution, and stored at -80°C until analysis. Digested peptides were stored at -80° C in labeled Protein LoBind tubes for no more than one week. 2.10 NanoLC-MS/MS analysis Analysis of digested peptides was made using a Dionex Ultimate 3000 (Sunnyvale, CA, USA) nanoLC system connected to a hybrid mass spectrometer LTQ Orbitrap XL (Thermo Fisher Scientific Bremen, Germany), furnished with a nanoelectrospray ion source. Peptide mixtures were enriched by injecting 10 µL (ca. 3.3 µg) of sample 300 µm ID × 5 mm Acclaim PepMap 100 C18 precolumn, employing a premixed mobile phase H2O–ACN 95 : 5 (v/v) containing 0.1% HCOOH at 10 µL min-1 flow-rate. Peptide mixtures were separated by reversed-phase chromatography as explained elsewhere. MS spectra were collected over an m/z range of 400–1800 using a resolution ACS Paragon Plus Environment

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setting of 60,000 (full width at half-maximum at m/z 400), operating in the data-dependent mode, operating in the data dependent mode to switch automatically between Orbitrap-MS and LTQMS/MS acquisition. MS/MS spectra were collected for the five most abundant ions in the scan. All samples were measured in triplicate.

2.11 Data analysis and protein validation Raw data files, obtained from Xcalibur software, were submitted to Proteome Discover (1.2 version, Thermo Scientific) for database search using Mascot (version 2.3.2 Matrix Science). Data were searched against the SwissProt database (57.15 version, 20266 sequences). The built-in decoy search option of Mascot was used. Scaffold software (v3.6, Proteome Software, Portland, Oregon, USA). Peptide identification was validated if surpassing a 95% probability threshold set by the PeptideproPhet algorithm. Protein identification was accepted if the protein contained at least two unique peptides and the probability of correct identification was >99.0%. Unweighted spectrum counts (USC) were used to assess the consistency of biological replicates in quantitative analysis, and normalized spectrum counts (NCS) were used to retrieve protein abundance. Protein quantification was made by Scaffold that allows to normalize spectral countings (normalized spectral countings, NSCs). Finally for each protein the relative protein abundance was calculated according to the following equation 36:

(/)

RPAk = ∑

(/)

x 100

(1)

where RPAk is the percentage molecular weight normalized NSC for protein k and MW is the molecular weight in kDa for protein k. 2.12 Confocal imaging

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Cell imaging was performed by using a Leica SP5 inverted confocal microscope equipped with a 488-nm Argon laser to excite DOXO fluorescence (collection range: 500-650). A 405-nm pulsed diode laser was used to excite HOECHST fluorescence (for imaging of nuclei). During imaging, cells were maintained in thermostated chamber (37°C and 5% CO2). 2.13 FACS MCF7 and MDA- MB-435S cells were seeded in 12-well plates (150,000 cells/well) using complete medium. After 24 hours, cells were treated for 3 hours with Doxoves®-HP complexes in Optimem medium. Then cells were washed two times with cold PBS, detached with trypsin/EDTA and acquired using cytometer. Fluorescence-activated cell sorting (FACS) analysis was performed using BD LSR Fortessa. Cells were first gated using forward vs side scatter (FSC vs SSC) strategy to exclude debris and then analyzed for the specific 600 nm emission (PE channel) exploiting fluorescent proprieties of DOX42. 2.14 MTT To evaluate the cytotoxicity of Doxoves-HP complexes, cell viability of MCF7 and MDA- MB-435S cells was evaluated using 3-(4,5-dymethyl thiazol 2-yl)-2,5-diphenyl tetrazolium bromide (MTT, mitochondrial respiration analysis; Sigma-Aldrich), according to Mosmann protocol. Briefly, cells were seeded on 96-well plates (10,000 cells/well) in complete medium. After 24 hours, cells were treated with Doxoves®-HP complexes in Optimem medium for 24 hours. Then, MTT was added to each well at the final concentration of 0.5 mg/mL and plates were incubated for 3 hours at 37°C. The resulting formazan salt was dissolved with 100 μL isopropylic alcohol and the absorbance of each well was measured with Glomax Discover System (Promega, Madison, WI, USA) at 570 nm. Cell viability was calculated for each treatment as “OD of treated cells/OD of control cells” x 100. All the measures were made in triplicate. Results are given as mean ± standard deviation.

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3. Results 3.1 Size and Zeta-potential of Doxoves®-HP complexes In a recent paper of the Moghimi’s group

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, subtle physicochemical differences between four

liposomal DOX products (Doxil®, Caelyx®, DOXOrubicin® and SinaDoxosome®) with identical lipid composition and lipid/drug ratio were found. Preliminary experiments were therefore aimed at characterizing physico-chemical properties of Doxoves®. Size and zeta-potential distributions were centered at DH = 92.5 ± 1.7 nm and Zp = -34.3 ± 2.5 mV (black curves, Figure 1, panels A and B respectively). Doxoves® was incubated in plasma concentrations from 5% to 50%. Size distributions of Doxoves®-HP complexes were steady, while zeta-potential distributions of Doxoves®-HP complexes were shifted with respect to those of pristine vesicles (Figure 1, panels A and B). At low plasma concentration BC leads to a significantly larger size (DH ≈ 135 nm) and then slight modification as a function of plasma concentration was observed (Figure 1, panel C). Zetapotential values for Doxoves®-HP complexes suggest that the highly negative zeta-potential of Doxoves® is reduced (to approximately -18 mV) even at low plasma concentrations (Figure 1, panel D). Size and zeta-potential measurements suggest that BC forms even at the lowest plasma concentration (HP=5%) and that the protein layer has a main effect of particle stability in HP. Size and zeta-potential experiments were replicated on empty liposomes. Main results are shown in Figures S1 and discussed in the Supporting Information. Globally, after exposure to HP, empty liposomes exhibited larger size and higher zeta potential than those of Doxoves®. 3.2 Nanostructure of Doxoves®-HP complexes Bilayer structure of Doxoves® was investigated by synchrotron SAXS that is the method of choice for the structural characterization of materials at the nanoscale, and it is commonly used for the study of liposomes

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. SAXS patterns of Doxoves®and Doxoves®-HP complexes are depicted in

Figure 2. Of note, synchrotron SAXS scans of Doxoves®-HP complexes showed diffuse scattering

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that is characteristic of positionally not correlated bilayers 44, while diffraction Bragg peaks were not detected. This means that protein binding does not promote formation of multilamellar structure as those found in cationic liposome-HP complexes

29

. TEM images (Figure 3, panel A)

show that Doxoves® liposomes are pretty homogeneous in size. As TEM images clearly show, DOX is aggregated in the form of one-dimensional (1D) rod-like crystals. According to literature4, DOX rods can touch the lipid bilayer, inducing formation of non-spherical vesicles. Size distribution of Doxoves® as determined by TEM data analysis (Figure 3, panel B) was in good agreement with that obtained by DLS. TEM images for Doxoves® after incubation in HP are reported in Figure 3, panel C. First, no trace of multilamellar aggregates was found. This finding was consistent with synchrotron SAXS results. On the other side, TEM images showed the presence of several ruptured vesicles (Figure 3, panel D). This would suggest that, following exposure to HP, Doxoves® could not be robust enough to keep the liposome integrity. Synchrotron SAXS and TEM experiments were replicated on bare liposomes (i.e. not loaded with DOX) and results are reported in the Supporting Information (Figure S2-S3). No relevant differences between DOX-loaded and empty liposomes were found. Moreover, empty liposomes seemed to be more resistant to HP exposure. Although the exact molecular mechanisms have not been clarified so far, previous studies showed that DOX crystals touch the vesicle membrane, producing a pressure gradient between the outside and the inside of the particle (reviewed in reference 5). Such a pressure gradient is likely to break lipid vesicles leading to drug release45. Further analysis was also performed by confocal imaging and FLIM analysis on single Doxoves® and Doxoves®-HP complexes preliminarily immobilized in 1.7% agarose gel (Figure 4). Intensity analysis reveals that particles exposed to HP (50%) contain substantially less DOX as compared to the control ones (i.e. not exposed to HP). On the average the observed decrease in intensity is of a factor of about 2.5±0.4 from N=75 quantitative comparisons. This evidence supports the idea that exposure to HP and BC formation may interfere

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with Doxoves® integrity, possibly leading to DOX leakage. By the way, FLIM analysis show that, in both experiments, the Doxoves® content yields a similar lifetime, i.e. the DOX ‘aggregation’ state is analogous. In particular, the average lifetime measured within Doxoves® particles is at around 4.5 nanoseconds, a value close to the expected one for ‘conjugated’ DOX, according to the literature

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. This makes us argue that, in our experiments, such lifetime could be reasonably

assigned to the crystal form of DOX, in keeping with TEM-based observations. 3.3 The human biomolecular corona of Doxoves® Figure S4, panel A illustrates SDS-PAGE results in which Doxoves® was incubated in plasma concentrations from 5% to 50%. Band intensity was used to explore the evolution of the Doxoves®’s BC with plasma concentration.

The total amount of proteins slightly rises with

increasing percentage of plasma up to HP= 20% (i.e., higher amounts of the same plasma proteins bind at higher HP concentrations). For HP > 20%, total amount of proteins decreases. This finding indicates that low-abundant proteins compete for the surface binding sites and promote the desorption of highly-abundant plasma proteins with lower binding affinity36. Experiments were replicated on bare liposomes (Figure S4, panel B). A key step to predict the liposome’s physiological response (e.g. biodistribution) is the quantification of the corona proteins

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. 239

proteins were identified in the BCs of Doxoves® by nanoLC-MS/MS at low (HP=5%), medium (HP=20%) and high (HP=50%) plasma concentration. This finding supports the current assumption that the liposome-BC consist of a few hundred proteins 47. Figure 5 shows the Venn diagrams. A big fraction of proteins, precisely 221, were common to the three BCs; 8 proteins were “unique”, i.e. they were found only in one BC; 6 proteins were in common between two BCs. Similar findings were obtained with bare liposomes (Figure S5). As Figure 6 and Figure S6 clearly show, the BCs of both Doxoves® and bare liposomes were formed mostly by proteins with low MW and high isolectric point (pI). More in detail, MS/MS data showed that proteins < 20 kDa were the most

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abundant ones (RPA about 25%). In contrast to cationic liposomes and other classes of materials 34

, proteins with molecular weight >150 kDa were poorly abundant (RPA < 10%). Finally, plasma

proteins were grouped according to their physiological functions (Figure 7). Of note, typical “opsonins” such as immunoglobulins (RPA < 10 %) and complement proteins (RPA < 5 %) were poorly abundant in the corona of Doxoves®–HP complexes. On the other side, Doxoves® avidly bind serum clotting factors (e.g. Fibrinogen) and Apolipoproteins (15% < RPA < 30%). Comparing “coronome” of Doxoves® (Figure 7) with that of empty liposomes (Figure S7), systematic differences were found. This finding could be somewhat unexpected considering that DOX is precipitated within the interior of lipid vesicles (Figure 3) and that Doxoves®’s lipid composition is identical to that of empty liposomes. According to previous findings 48, this discrepancy in corona composition let us conclude that a certain fraction of DOX could be located at the vesicle surface, thus competing with lipids for protein binding. 3.4 Cellular uptake of Doxoves®-HP complexes in breast cancer cells Lastly, we evaluated the role played by BC on cellular uptake of Doxoves® in cancer cells. Preliminary confocal analyses were conducted on MCF7 and MDA-MB-435S human breast carcinoma cells treated with Doxoves® (HP=0%) and BC-coated Doxoves®(HP= 50%) (Figure 8). These experiments qualitatively confirm that both cell lines are interested by Doxoves® uptake. In order to get a more quantitative view on this process, we performed FACS analysis. In particular, MCF7 and MDA-MB-435S human breast carcinoma cells were treated with Doxoves® both in the absence (HP=0%) and in the presence of the BC (HP= 5%, 20% and 50%). According to literature, cellular uptake experiments were replicated at two drug concentrations (5 µg/well and 10 µg/well) and results are displayed as the percentage of fluorescent positive cells (Figure 9). BC did not significantly affect cell uptake of Doxoves® in MCF 7 cells, while it diminished uptake in MDA-

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MB-435S cells by a factor comprised between 2 (10µg/well, HP=20%) and 1.3 (5µg/well, HP=5%; 10µg/well, HP=5% and HP=50%). 3.6 Anticancer effect of Doxoves®-HP complexes in human breast cancer cells It is well recognized that encapsulated DOX can activate apoptosis in many cancer cell lines and it is the drug of choice for breast cancer treatment. To explore the role of BC on the anticancer effect of Doxoves®, MCF7 and MDA-MB-435S human breast carcinoma cells were treated with Doxoves®-HP complexes (HP= 5%, 20% and 50%). 24 hours after particle administration cell viability was evaluated by the MTT assay. Figure 10 shows that BC had a minor effect, if any, on cell viability.

Discussion Despite the progresses of liposomes in biomedical applications, few formulations have reached clinical practice

49

. Among them, liposomal DOX is FDA-approved for breast cancer and other

indications. As clarified by Barenholz 4, in liposomal DOX each single part matters and helps to optimize its performance

50

. However, despite the promises, it has been less effective than

expected and many details of its mechanism of action still remain unclear. Today, the concept is emerging that the poor clinical success of liposomal DOX could be largely due to our limited understanding of its “nano-bio-interactions” experienced in vivo 28. In biological media (e.g. blood, urine, gastric fluids etc.) NPs adsorb biomolecules to form a BC, which hinders the ability of the pristine NPs to interact with target cells 51. BC may also trigger an immune response

52

with the

result that the NP is cleared from the blood before reaching its destination. However, BC does not necessarily produce detrimental effects on NPs. Recent works have shown that BC provides NPs with a stealth effect on recognition by immune cells nanomaterials with a distinctive targeting ability

54

53

. Moreover, BC is able to provide

. Proteins of the BC may lead to specific

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biological recognition of cell receptors thus boosting cellular uptake of NPs 55-56. Such results have stimulated debate about exploitation of BC in drug delivery 35, 57-58. Cancer drug delivery strategies are currently based on the enhanced permeation and retention (EPR) effect

59-60

that passively

drives drug accumulation at the tumore site. Because of the large permeable vasculature and reduced lymphatic drainage of the tumor, particles smaller than ~ 200 nm escape from the blood vessels into the tumor. Once inside the tumor, the new “liposome interface”, i.e. the liposome-BC, could either favor or disadvantage particle association with cancer cells. If the interaction between cancer cell receptors and corona proteins is ineffective, cellular association may be poor thus leading to reduced internalization of the drug. On the opposite, if corona proteins are recognized by tumor cell receptors, liposome-cell association could be enhanced leading to optimal intracellular drug disposition. Thoroughly characterization of liposome-BC (structure, size, charge and composition) is rapidly emerging as the first step towards deciphering its role in drug delivery 35, 47, 59-60

. In this study we explored the biological identity of Doxoves®, a commercial product with

the same lipid composition, lipid/drug ratio and physical-chemical properties of Doxil®. Doxoves® was small in size and negatively charged with intraliposome precipitated DOX. Following exposure to HP, Doxoves® bound plasma proteins, but no evidence of multilamellar aggregates was found. At a first sight, one may deduce that PEGylation was the main factor to limit protein binding and, in turn, to prevent particle fusion and agglomeration. However, recent investigations 29, 61 clarified that BC can significantly affect the bilayer structure of PEGylated liposomes when they are made of cationic lipids. Moreover, some of us have shown that liposomal Irinotecan (Onivyde) a PEGylated formulation approved by the FDA for the cure of pancreatic ductal adenocarcinoma (PDAC), is covered by a rich BC whose composition resembles that of its unPEGylated counterpart 62

. Combining these evidences, we claim that the intrinsic ability of Doxoves® to limit massive

protein adsorption is mainly due to its peculiar lipid composition rather than to PEGylation (as

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commonly assumed so far). This is a crucial step in liposomal drug delivery because proteininduced membrane destabilization can result in vesicle rupture and cargo release. Size distribution is another critical issue in liposomal drug delivery. Indeed liposome degradation in blood is known to be a size-dependent process

63

: the larger the size, the faster the blood clearance. As a

consequence, size has a large impact on PK and BD of liposomal drugs, and therefore on therapeutic efficiency and nanotoxicity 64. Of note, size of Doxoves®-HP complexes was adequately small (< 130 nm) even at high plasma concentration. As above explained, the biological identity of liposomal drugs in physiological environments is emerging as a crucial factor in drug delivery. The most relevant implication is that the BC (and not the pristine lipid surface) is what is actually “seen” and “processed” by living systems in vivo. Identification and quantification of “corona proteins” is therefore a fundamental step towards understanding the physiological response of liposomal DOX. NanoLC MS/MS results in Figure 7 showed that BC Doxoves® slightly changed as a function of HP concentration. At the highest plasma concentration (HP=50%) the abundance of identified classes of proteins was in the order: Apoliproteins > Tissue Leakage proteins > HSA > Immunoglobulins > Coagulation proteins > Acute Phase proteins > Complement proteins. The relative abundance of typical opsonins (e.g. Complement proteins, Immunoglobulins and Coagulation proteins) was found to be unusually small with respect to that of other liposomal formulations 29, 62, 65. According to our current understanding 65, the reduced affinity of Doxoves® for opsonins is likely to be due to its peculiar lipid composition. This is particularly interesting because these classes of plasma proteins promote unwanted effects such as initiation of the coagulation cascade

66

, activation of complement system

67

, phagocytosis by immune cells,

accumulation in the liver and spleen and renal clearance. For instance, clotting proteins stimulate immune cells by
toll-like receptors (TLRs). On the other hand, the poor abundance of opsonins in the BC of Doxoves® could help to explain its good PK

50

. According to results of Figure 7,

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Apolipoproteins were highly enriched and constituted about 30% of the BC. Apolipoproteins are considered “dysopsonins” (i.e. prolong blood circulation) and provide liposomes with a selected targeting ability 27, 58. In fact, several apolipoproteins (e.g. APO-A1, APO-A2, APO-C2, APO-E, APO-F etc.) bind low-density lipoprotein receptor (LDLR) and scavenger receptor class B, type I (SRB1) that are overexpressed in several disease states (e.g., renal cell carcinoma, melanoma, hepatocellular carcinoma, lymphoma and atherosclerosis). As a result, liposomes decorated by apolipoproteins could be better targeting candidates than other delivery systems. If the BC Doxoves® was seemingly ideal to target breast cancer cells, entry of DOX into MCF 7 cells was as high as in the absence of BC (HP=0%), while it was largely reduced in MDA-MB-435S cells (Figure 9). About this observation, some considerations can be made. According to recent works, only a minor fraction of Apolipoproteins adsorbed to Doxoves® could have functional motifs that are correctly presented to bind cell receptors. Likewise, Apolipoproteins might be unfolded or the binding regions of the proteins may be hidden. Thus, mapping protein binding sites on the NP-BC is vital to exploit the targeting properties of BC

68-69

. Moreover, we suggest that BC induces

destabilization of some lipid vesicles (Figure 3, panel D) and that the consequent release of a fraction of the transported load (Figure 4) could explain the observed decrease in intracellular drug localization. Moreover, the anticancer ability of Doxoves® was poor both in MCF 7 and MDAMB-435S cancer cells and was poorly affected by BC (Figure 10). A previous investigation showed that Doxil® is intact intracellularly and that the free drug is more effective in killing cells in vitro as compared to its liposomal counterpart 70. This behavior could be related to the drug phase. Our TEM results of Figure 3 showed DOX was precipitated inside liposomes. A calculation shows that each vesicle encloses ~ 10,000 molecules of DOX and that the largest fraction (< 95 %) is in the crystalline phase 4. The effect of crystallization on therapeutic efficacy of DOX has not clarified so far. However, the interaction of the crystallized drug form with DNA could be disadvantaged

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compared to that of its monomeric counterpart. This key aspect is currently under investigation in our laboratories.

Conclusions In this study, we have demonstrated that, following exposure to HP, liposomal DOX does not retain its synthetic identity, but it is coated by a complex BC. These data indicate that grafting PEG to the surface of Doxoves® does not prevent protein binding (as generally assumed so far). MS/MS showed that Doxoves® preferentially bind Apolipoproteins, while opsonins were poorly enriched. Additionally, Doxoves® lipid bilayer of Doxoves® was not strong enough to keep the liposome integrity and vesicle rupture was frequently observed by TEM. FLIM demonstrated drug release from ruptured liposomes and provided explanation for the significantly reduced penetration of DOX in MCF7 and MDA-MB-435S human breast cancer cells shown by flow cytometry. Finally, cell viability studies performed 24 hours after drug administration indicated that BC does not significantly impact anticancer activity of Doxoves® when compared to pristine particles (i.e. in the absence of BC). In the next future, we expect that following exposure to physiological environments liposomal formulations will be sufficiently resistant to maintain the integrity of the lipid membrane and not release the drug outside target cells. Such optimized formulations will recruit selected plasma proteins from the environment and ‘corona proteins’ will induce receptormediated cellular association. Therefore, full characterization of the ‘bionanointerface’ will pave the way to radically improve the antitumor capabilities of liposomal drugs.

Acknowledgements

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SP is recipient of an AIRC fellowship.

Authors’ contribution. GC conceived the study, discussed data, supervised research, wrote the manuscript. SP performed 1D SDS-PAGE, FACS and MTT experiments, analyzed data, prepared figures, reviewed the manuscript. LD performed DLS, SAXS and confocal microscopy experiments, analyze data, prepared figures, reviewed the manuscript. RZC analyzed nanoLC MS/MS data. ALC conceived nanoLC MS/MS experiments, discussed data. HA supervised synchrotron SAXS experiments, discussed data, reviewed the manuscript. PMT performed FLIM experiments. VP performed TEM experiments, reviewed the manuscript. MP supervised TEM experiments, discussed data, reviewed the manuscript. FC supervised FLIM experiments, discussed data, reviewed the manuscript. DP discussed data, supervised research, reviewed the manuscript. AA supervised nanoLC MS/MS experiments, discussed data.

Supporting Information. Figure S1. Size and zeta-potential distributions of empty (i.e. not loaded with DOX) Doxoves® and empty Doxoves®-HP complexes. Figure S2. Synchrotron small-angle X-ray scattering patterns of empty Doxoves® and empty Doxoves®-HP complexes. Figure S3. TEM image of empty Doxoves®. Figure S4. Representative sodium dodecyl sulfate polyacrylamide gel electrophoresis gel image of plasma proteins obtained from both DOX-loaded and empty Doxoves® following 1-hour exposure to HP at 37 °C. Figure S5. Venn diagram reporting the number of plasma proteins adsorbed to the surface of empty Doxoves® after 1-hour incubation with HP at 37°C. Figure S6. Relative protein abundance (RPA) of corona proteins classified according to their calculated molecular weight (MW) calculated isoelectric point (pI) for empty Doxoves®-HP complexes. Figure S7. Bioinformatic classification of corona proteins found in the biomolecular corona of empty Doxoves® after 1-hour incubation with HP at 37°C as determined by nanoLC/MS-MS.

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53. Caracciolo, G.; Palchetti, S.; Colapicchioni, V.; Digiacomo, L.; Pozzi, D.; Capriotti, A. L.; La Barbera, G.; Laganà, A., Stealth Effect of Biomolecular Corona on Nanoparticle Uptake by Immune Cells. Langmuir 2015, 31 (39), 10764-10773. 54. Caracciolo, G.; Cardarelli, F.; Pozzi, D.; Salomone, F.; Maccari, G.; Bardi, G.; Capriotti, A. L.; Cavaliere, C.; Papi, M.; Laganà, A., Selective targeting capability acquired with a protein corona adsorbed on the surface of 1,2-dioleoyl-3-trimethylammonium propane/dna nanoparticles. ACS Applied Materials and Interfaces 2013, 5 (24), 13171-13179. 55. Walkey, C. D.; Olsen, J. B.; Song, F.; Liu, R.; Guo, H.; Olsen, D. W. H.; Cohen, Y.; Emili, A.; Chan, W. C., Protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles. ACS nano 2014, 8 (3), 2439-2455. 56. Palchetti, S.; Digiacomo, L.; Pozzi, D.; Peruzzi, G.; Micarelli, E.; Mahmoudi, M.; Caracciolo, G., Nanoparticles-cell association predicted by protein corona fingerprints. Nanoscale 2016, 8 (25), 12755-12763. 57. Mirshafiee, V.; Kim, R.; Park, S.; Mahmoudi, M.; Kraft, M. L., Impact of protein pre-coating on the protein corona composition and nanoparticle cellular uptake. Biomaterials 2016, 75, 295304. 58. Palchetti, S.; Pozzi, D.; Mahmoudi, M.; Caracciolo, G., Exploitation of nanoparticle-protein corona for emerging therapeutic and diagnostic applications. Journal of Materials Chemistry B 2016, 4 (25), 4376-4381. 59. Fang, J.; Nakamura, H.; Maeda, H., The EPR effect: unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effect. Advanced drug delivery reviews 2011, 63 (3), 136-151. 60. Maeda, H.; Nakamura, H.; Fang, J., The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Advanced drug delivery reviews 2013, 65 (1), 71-79. 61. Digiacomo, L.; Pozzi, D.; Amenitsch, H.; Caracciolo, G., Impact of the biomolecular corona on the structure of PEGylated liposomes. Biomaterials science 2017, 5 (9), 1884-1888. 62. Papi, M.; Caputo, D.; Palmieri, V.; Coppola, R.; Palchetti, S.; Bugli, F.; Martini, C.; Digiacomo, L.; Pozzi, D.; Caracciolo, G., Clinically approved PEGylated nanoparticles are covered by a protein corona that boosts the uptake by cancer cells. Nanoscale 2017, 9 (29), 10327-10334. 63. Harashima, H.; Hiraiwa, T.; Ochi, Y.; Kiwada, H., Size dependent liposome degradation in blood: in vivo/in vitro correlation by kinetic modeling. Journal of drug targeting 1995, 3 (4), 253261. 64. Szebeni, J.; Bedőcs, P.; Rozsnyay, Z.; Weiszhár, Z.; Urbanics, R.; Rosivall, L.; Cohen, R.; Garbuzenko, O.; Báthori, G.; Tóth, M., Liposome-induced complement activation and related cardiopulmonary distress in pigs: factors promoting reactogenicity of Doxil and AmBisome. Nanomedicine: Nanotechnology, Biology and Medicine 2012, 8 (2), 176-184. 65. Caracciolo, G.; Pozzi, D.; Capriotti, A. L.; Cavaliere, C.; Piovesana, S.; Amenitsch, H.; Laganà, A., Lipid composition: A "key factor" for the rational manipulation of the liposome-protein corona by liposome design. RSC Advances 2015, 5 (8), 5967-5975. 66. Bonté, F.; Juliano, R., Interactions of liposomes with serum proteins. Chemistry and physics of lipids 1986, 40 (2-4), 359-372. 67. Szebeni, J.; Muggia, F.; Gabizon, A.; Barenholz, Y., Activation of complement by therapeutic liposomes and other lipid excipient-based therapeutic products: prediction and prevention. Advanced drug delivery reviews 2011, 63 (12), 1020-1030. 68. Gianneli, M.; Polo, E.; Lopez, H.; Castagnola, V.; Aastrup, T.; Dawson, K., Label-free in-flow detection of receptor recognition motifs on the biomolecular corona of nanoparticles. Nanoscale 2018. ACS Paragon Plus Environment

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69. Lara, S.; Alnasser, F.; Polo, E.; Garry, D.; Lo Giudice, M. C.; Hristov, D. R.; Rocks, L.; Salvati, A.; Yan, Y.; Dawson, K. A., Identification of receptor binding to the biomolecular corona of nanoparticles. ACS nano 2017, 11 (2), 1884-1893. 70. Seynhaeve, A. L.; Dicheva, B. M.; Hoving, S.; Koning, G. A.; ten Hagen, T. L., Intact Doxil is taken up intracellularly and released doxorubicin sequesters in the lysosome: evaluated by in vitro/in vivo live cell imaging. Journal of controlled release 2013, 172 (1), 330-340.

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Figure 1. (A) Intensity dynamic light scattering (DLS) distributions of Doxoves® (black curve) and Doxoves®-HP complexes: HP= 5% (dark blue curve); HP= 10% (light blue curve); HP= 20% (dark green curve); HP= 30% (light green curve); HP= 40% (orange curve); HP= 50% (red curve). (B) Zetapotential distributions of Doxoves® (black curve) and Doxoves®-HP complexes. (C) Hydrodynamic diameter of Doxoves® (black circle) and of Doxoves®-HP complexes . (D) Zeta-potential of Doxoves® (black circle) and of Doxoves®-HP complexes. In panels B-D, the same color code than panel A was used.

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Figure 2. Synchrotron small-angle X-ray scattering (SAXS) patterns of Doxoves® (black points) and Doxoves®-HP complexes: HP= 5% (dark blue points); HP= 10% (light blue points); HP= 20% (dark green points); HP= 40% (orange points); HP= 50% (red points). Dashed lines are the best fits to the data using weighted linear combinations of the SAXS patterns of Doxoves® and HP.

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Figure 3. (A) Transmission electron microscopy (TEM) image of Doxoves®. Scale bar: 1 micrometer. long and fiber-like crystals of doxorubicin are clearly shown in the inset. (B) Size distribution of Doxoves®. Solid and dashed lines indicate median and average of size distribution respectively (C) Representative TEM images of Doxoves®-HP complexes (HP=50%). Scale bar: 100 nm (D) TEM images of broken Doxoves®. Scale bar: 200 nm.

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Figure 4. Representative FLIM images showing Doxoves® (left panel) and Doxoves®-HP complexes (HP=50%, right panel) immobilized in a 1.7% agarose gel. The FLIM image denotes the fluorescence lifetimes measured at each pixel and displayed as color contrast image. The corresponding false-color lookup table represents the lifetime distribution. Scale bars: 1 micrometer.

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Figure 5. Venn diagram reporting the number of plasma proteins adsorbed to the surface of Doxoves® after 1 h incubation with human plasma (HP) at 37°C: HP=5% (blue), HP=20% (green), and HP =50% (red).

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Figure 6. (A) Relative protein abundance (RPA) of corona proteins classified according to their calculated molecular weight (MW) for Doxoves®-HP complexes. (B) RPA of identified plasma proteins classified according to their calculated isoelectric point (pI) for Doxoves®-HP complexes. Color code: HP=5% (blue histograms), HP=20% (green histograms), and HP =50% (red histograms).

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Figure 7. Bioinformatic classification of corona proteins found in the biomolecular corona of Doxoves® after 1 h incubation with human plasma (HP) at 37°C as determined by nanoLC/MS-MS. Proteins were grouped according to their physiological functions at different HP concentration: (A) pie charts, (B) histograms. (C) Scatter plot depicting the measured relative protein abundances under different conditions of plasma concentrations (HP=5%, HP=20%, HP=50%). The diagonal represents an ideal reference distribution of data points with equal protein abundances under the investigated conditions. (D) Distance\Delta between each of the experimental data points and the reference diagonal. Results are sorted in descending order to rank the identified corona proteins according to their sensitivity to the plasma concentration.

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Figure 8. Live-cell confocal imaging of Doxoves® uptake in MCF-7 (upper panels) and MDA-MB 435S (lower panels) cells, in absence of the BC (left panels) or in presence of BC (upon exposure of liposomes to 50% HP, right panels). Red signal derives from DOX, blue signal derives from HOECHST and localizes the nuclei (see Materials and Methods for further experimental details). Scale bar, as indicated: 10 micrometers.

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Figure 9. Cell uptake liposomal doxorubicin given as the percentage of fluorescent positive MCF 7 (panel A) and MDA-MB-435S (panel B) cancer cells in the absence (HP=0%) and in the presence of biomolecular corona bound to Doxoves® after 1 h incubation with human plasma (HP): HP=5% (blue histograms), HP=20% (green histograms), and HP =50% (red histograms).

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Figure 10. Cell viability of Doxoves®-HP complexes after at 24 h after administration in MCF 7 and MDA-MB-435S cancer cells (panels A and B). According to literature, all the experiments were replicated at two DOX concentrations. Dashed lines indicate cell viability of untreated cells.

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