Brain Targeting by LiposomeâBiomolecular Corona Boosts Anticancer

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Brain targeting by liposome-biomolecular corona boosts anti-cancer efficacy of Temozolomide in glioblastoma cells Antonietta Arcella, Sara Palchetti, Luca Digiacomo, Daniela Pozzi, Anna Laura Capriotti, Luigi Frati, Maria Antonietta Oliva, Georgia Tsaouli, Rossella Rota, Isabella Screpanti, Morteza Mahmoudi, and Giulio Caracciolo ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00339 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 18, 2018

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ACS Chemical Neuroscience

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Brain targeting by liposome-biomolecular corona boosts anti-cancer efficacy of Temozolomide in

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glioblastoma cells

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Antonietta Arcella,a# Sara Palchetti,b# Luca Digiacomo,b Daniela Pozzi,b Anna Laura Capriotti,c Luigi

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Frati,a Maria Antonietta Oliva,a Georgia Tsaouli,b Rossella Rota,d Isabella Screpanti,b Morteza

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Mahmoudi,e Giulio Caracciolob*

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a.

Istituto Neurologico Mediterraneo Neuromed, Via dell’Elettronica 86077 Pozzilli (IS), Italy

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b.

Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291,

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00161 Rome, Italy

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c.

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Italy

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d. Dept. of Oncohematology | Ospedale Pediatrico Bambino Gesu', Viale San Paolo 15, 00146

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Roma - Italy

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e.

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Boston, MA 02115, USA

Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome,

Department of Anesthesiology, Brigham and Women’s Hospital, Harvard Medical School,

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*E-mail: [email protected]

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#equal contribution

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ACS Paragon Plus Environment

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Temozolomide (TMZ) is the current first-line chemotherapy for treatment of glioblastoma

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multiforme (GBM). However, similar to other brain therapeutic compounds, access of TMZ to

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brain tumors is impaired by the blood–brain barrier (BBB) leading to poor response for GBM

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patients. To overcome this major hurdle, we have synthesized a set of TMZ-encapsulating

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nanomedicines made of four cationic liposome (CL) formulations with systematic changes in lipid

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composition and physical-chemical properties. The targeting nature of this nanomedicine is

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provided by the recruitment of proteins, with natural targeting capacity, in the biomolecular

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corona (BC) layer that forms around CLs after exposure to human plasma (HP). TMZ-loaded CL-BC

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complexes were thoroughly characterized by dynamic light scattering (DLS), electrophoretic light

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scattering (ELS) and nano-liquid chromatography tandem mass spectrometry (nano-LC MS/MS).

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BCs were found to be enriched of typical BC fingerprints (BCFs) (e.g. Apolipoproteins, Vitronectin,

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and Vitamin K-dependent protein), which have a substantial capacity in binding to receptors that

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are overexpressed at the BBB (e.g. scavenger receptor class B, type I and low-density lipoprotein

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receptor). We found that the CL formulation exhibiting the highest levels of targeting BCFs, had

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larger uptake in human umbilical vein endothelial cells (HUVECs) that are commonly used as in

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vitro model of the BBB. This formulation could also deliver TMZ to human glioblastoma U-87 MG

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cell line and thus substantially enhance their anti-tumor efficacy compared to corona free CLs.

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Thus, we propose that the BC-based nanomedicines may pave a more effective way for efficient

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treatment of GBM.

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Keywords: biomolecular corona, nanobio interface, nanomedicine, drug delivery, Temozolomide,

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Glioblastoma.

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Introduction

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Glioblastoma multiforme (GBM) is the most common brain tumor, with an annual incidence of

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3.19 per 100 000 in the United States (1, 2). GBMs are histologically and heterogeneous tumors

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and have historically been classified by clinical presentation as either primary or secondary

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depending on evidence of a pre-existing lower-grade glioma (3). The current standard of care

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combines maximal surgical resection, followed by radiotherapy with concomitant and adjuvant

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temozolomide (TMZ). Despite this multimodal approach, median survival is limited to 16 to 19

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months, with approximately 25% to 30% of the patients alive at 2 years after diagnosis (4).

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Patients whose tumors display epigenetic silencing of the DNA repair enzyme O-methyl-guanine-

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methyltransferase experience better outcomes (4, 5). Given the poor survival with currently

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approved treatments, new therapeutic options for GBM are needed (6). A central obstacle to the

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management of malignant glioma is the inability to effectively deliver therapeutic agent to the

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tumor. A significant challenge in treating GBM is the ability of a drug to cross the blood-brain

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barrier (BBB), the brain's own defense system, which actively blocks or expels curative drugs from

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entering the brain. Several approaches have been explored to overcome this issue.

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Nanoparticles (NPs) showed promising capacity as carriers to increase the bioavailability of

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traditional chemotherapeutic drugs for brain tumors, such as TMZ, doxorubicin hydrochloride,

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irinotecan hydrochloride, and vincristine sulfate (7-9). Among different types of NPs, liposomes

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have received widespread application as drug and gene delivery vectors with the unique history of

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successful clinical translation (10-14). Current targeting strategies for brain drug delivery are based

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on the functionalization of liposomes through appropriate ligands that could be recognized by

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receptors at the BBB (15, 16). For instance, a recent study showed that encapsulation of TMZ in a

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tumor-targeting cationic liposome (CL) enhances anti-cancer efficacy in a mouse model of GBM

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(17). However, despite promising results, no targeted liposomal therapeutics has been clinically


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approved for brain drug delivery. One possible reason could be the existence of “hidden factors”

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at the nano-bio interface, which created a vast gap between bench discoveries and clinical

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translation of nanotechnologies (9, 18-20).

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It is now widely accepted that when NPs are injected into biological fluids, such as the blood,

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biomolecules form a complex layer around them, referred to as “biomolecular corona” (BC) (21,

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22). Following introduction in the bloodstream, liposomes are surrounded by high concentrations

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of plasma proteins that bind to the lipid surface leading to formation of liposome-BC. Main factors

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shaping liposome-BC are: i) the physicochemical properties of lipid surface (23); ii) the protein

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source (24) and iii) the physiological environment (24, 25). Being the biological interface seen by

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cells , BC is thought to give a new “biological identity” to the liposomes by encrypting information

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that controls their bioactivity (e.g. cellular association and intracellular fate) (26). At present,

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researchers believe that understanding and controlling the bio-nano-interactions of liposomes

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with biological media (e.g. blood, lympha, and interstitial fluids) is central for their clinic

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translation (27, 28). Liposome-BC would include proteins engaged from the blood that could lead

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the vesicle to interact with specific receptors expressed on the plasma membrane of target cells.

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Currently, the concept is emerging that BC-based nanomedicine could generate innovative

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nanomedicine for the treatment of central nervous system (CNS) related diseases (Figure 1). NPs

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bind to receptors located at the BBB (29) and are internalized by a process referred to as

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adsorptive mediated transcytosis (30). Efficient transcytosis across the BBB is an important

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strategy for accessing drug targets within the CNS. Despite extensive research the number of

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studies reporting successful delivery of macromolecules or macromolecular complexes to the CNS

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has remained very low so far. Evidently, to gain maximal benefit from these novel developments

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and to enable improved delivery of drugs to the brain, we need to explore whether BC facilitates

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liposomes interactions with BBB and consequently affect efficacy of drugs for brain tumors. We


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have recently investigated BC-mediated liposome-cancer cell interactions using a library of

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liposomes of various size (e.g. 100 nm vs. 250 nm) and surface chemistry (e.g. cationic vs. anionic

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liposomes) (31, 32). For the entire library, a total of 436 distinct plasma proteins were detected. It

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was noteworthy that, of the entire pool of possible descriptors, a very small set of BC fingerprints

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(BCFs) demonstrated the greatest impact on cell association. Notably, the same proteins were also

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identified in previous works as being highly relevant to correlating NP-cell association (33). To

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exploit the targeting nature of BC, here we encapsulated TMZ in four binary CL formulations made

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of the widely used cationic lipids 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP) and 3(-[N-

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(N’,N’-dimethylaminoethane)-carbamoyl]-cholesterol

(DC-Chol)

and

neutral

lipids

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dioleoylphosphatidylethanolamine (DOPE) and Cholesterol (34). The choice of synthesizing binary

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formulations depended on the fact that multi-component liposomal formulations (e.g. ternary,

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quaternary lipidic mixtures, etc.) present numerous defects in the lipid bilayer due to the non-ideal

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miscibility of the lipid species. As previously demonstrated (35-37), such defects makes

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multicomponent formulations less stable under interaction with bio-membranes and plasma

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proteins. This aspect is unfavorable in view of the possible in vivo application of CL formulations.

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As cationic lipid species we employed DOTAP and DC-Chol that have been extensively used both in

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vitro (38) and in vivo (39, 40). Two binary CL formulations were synthesized mixing DOTAP and DC-

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Chol with DOPE. Due to its "cone-like" molecular structure DOPE promotes the formation of highly

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fusogenic inverse phases upon interaction with cellular membranes resulting in the efficient

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release of transported load (41). In addition, when mixed with either DOTAP and DC-Chol, DOPE

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promotes adsorption of apolipoproteins that could provide CLs with distinctive ability to target

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cancer cells (42). A second couple of binary CLs were prepared by mixing DOTAP and DC-Chol with

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Cholesterol. Previous investigations clarified that liposomes rich in cholesterol bind less protein

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and are more resistant in vivo than cholesterol-free liposomes (43). However, recent studies have


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shown that, in combination with either DOTAP and DC-Chol, cholesterol promotes the adverse

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adsorption of opsonins (e.g. immunoglobulins, complement proteins etc.) (42). This has been an

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overlooked factor in lipid-mediated drug delivery and deserves further investigation. In the

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following text, CL formulations will be synthetically indicated as follows: CL1 (DOTAP/Cholesterol),

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CL2 (DOTAP/DOPE), CL3 (DC-Chol/DOPE), and CL4 (DC-Chol/Cholesterol). TMZ-loaded liposome-BC

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complexes were thoroughly characterized by dynamic light scattering (DLS), electrophoretic light

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scattering (ELS), and nano-liquid chromatography tandem mass spectrometry (nano-LC MS/MS).

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Following 1-h exposure to human plasma (HP), the surface of TMZ-loaded CLs was covered by

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complex and heterogeneous BCs. As CL2-BC complexes exhibited the highest levels of targeting

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BCFs, they were used to deliver TMZ to human umbilical vein endothelial cells (HUVECs) and U-87

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MG cells. While HUVECs represent one of the most common in vitro models of the BBB, U-87 MG

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is a human GBM cell line derived from malignant glioma. We found that the CL formulation

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exhibiting the highest levels of targeting BCFs, had the major uptake resulting in large growth

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inhibition of HUVECs. Furthermore, TMZ-loaded CL2-BC complexes were much more effective in

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killing U-87 cells than TMZ and TMZ-loaded CL2. Thus, the selected formulation can both target

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the endothelial cells to cross the BBB and efficiently kill tumor cells once in the brain. The

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enhanced efficacy of BC-decorated CLs suggests that exploitation of BC holds great promise as a

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more efficient nanomedicine for GBM.

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Results and Discussion

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Encapsulation efficiency (EE) and drug loading content (DLC) were determined by UV-VIS

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experiments (44) (Figure S1) and results are summarized in Table 1. As evident all CLs exhibited EE

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and DLC in line with previous findings (17). Then, size and zeta-potential of CLs were analyzed

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(Figure 2). Bare CLs were positively charged (zeta-potential ≈ 40-55 mV) and small in size

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(hydrodynamic diameter, DH ≈ 100-150 nm). In line with previous findings (17), TMZ-loaded CLs

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were roughly twice bigger in size than bare CLs and maintained positive surface charge (Figure 2).

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Following 1-h exposure to HP, TMZ-loaded CL-BC complexes were bigger in size than TMZ-loaded

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CLs, with increase in size ranging between 20 and 40 nm (Figure 2). According to the previous

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reports (21, 26), such enlargement is compatible with formation of a thick BC at the vesicle

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surface. Particle size distributions were further characterized by the polydispersity index (PdI) that

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is reported in Table 2 for the three experimental conditions (i.e. pristine CLs, TMZ-loaded CLs and

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TMZ-loaded CL-BC complexes). PdI, which is commonly used to indicate the degree of uniformity

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of a size distribution of particles, confirm that all kinds of vesicles were acceptably homogeneous

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in size. More in detail, CLs and TMZ-loaded CLs were homogeneous in size, while TMZ-loaded CL-

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BC complexes exhibited a broader size distribution. Due to preferential absorption of negatively

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charged plasma proteins, zeta-potential of TMZ-loaded CL-BC complexes was found to be

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negative. This result is fully in agreement with the previous reports showing that BC tends to give a

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nanomaterial a zeta potential between - 10 mV and - 30 mV irrespective of nanomaterial physical-

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chemical properties (21, 32, 45-47). Such so-called ‘normalization’ of zeta potentials is due to the

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fact that most plasma proteins carry a net negative charge at physiological pH (21).

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As discussed above, a transport technology enabling liposomal drugs to overcome the BBB and

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enter GBM is still not available. Indeed, despite the increasing ability to engineering liposomes

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with a precise control of the surface functionalization (14), the targeting capacity is lost when ACS Paragon Plus Environment

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liposomes are embedded in complex biological media, where issues associated with the

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complexity of the surrounding environment highly increase. Liposomes can be rapidly covered by

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BC and this biomolecular layer is the ultimate vesicle surface “seen” by living systems. Since

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formation of BC is unavoidable even for stealth liposomes (48-50), researchers are trying to exploit

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the tumor-targeting nature of BC as it was as an “endogenous trigger” capable to promote

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favorable interactions with receptors overexpressed in target cells (51). In addition, the formation

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of BC can protect drug release/burst-effects in body by adding more drug protective layers at the

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surface of nanocarriers (52).

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Latest research has explored the correlation between BC composition and association with cancer

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cells (33, 53). In the first step, BC proteins are identified and quantified by tandem mass

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spectrometry (MS/MS) (54). Next, BC composition is correlated with cellular uptake by

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computational methods such as quantitative structure-activity relation (QSAR). This approach has

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resulted in the identification of BCFs that are likely to promote NP uptake in target cells. For

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instance, Walkey et al. (33) used NP properties and BCFs to predict the associations between a

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library of 105 gold NPs with A549 human lung epithelial carcinoma cells. More recently, we have

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shown that a small portion of the liposome-BC, which comprises 8 BCFs (Vitronectin, APOA1,

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APOA2, APOB, APOC2, Ig heavy chain V–III region BRO, Vitamin K-dependent protein, and Integrin

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beta3), controls cell association of liposomes with cancer cells (32, 55). More specifically, it was

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shown that the higher the abundance of the 8 BCFs, the higher the cell association with human

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cervical cancer cell line (HeLa) and human prostate cancer cell line (PC3). Thus, in the present

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investigation, we first characterized BCs by liquid-chromatography MS/MS (LC-MS/MS). We

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identified 219, 192, 135 and 190 plasma proteins in the BCs of CL1, CL2, CL3 and CL4 respectively.

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The full list of proteins identified by nanoLC-MS/MS is given in Tables S1-S4 in the ESI†.


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Venn diagrams of Figure 3 show that, a large number of proteins, exactly 91, were found to be in

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common amongst the four BCs. This significant overlapping confirms that surface charge is among

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the main not specific factors shaping BC (21, 56, 57). In addition, a lower but relevant number of

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unique proteins was found (36 for CL1, 28 for CL2, 2 for CL3 and 40 for CL4 respectively). In

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addition, overlapping of the twenty most abundant common proteins was far from being 100%

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(Table S5 in the ESI). These observations support the idea that lipid composition is a key specific

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factor in shaping liposome-BC (42). To exploit the targeting-nature of liposome-BC, first we

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evaluated the total abundance of BCFs. According to Figure 4 panel A, CL2 exhibited the highest

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abundance of BCFs. However, plasma proteins other than BCFs could promote favorable

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interactions with target cells. Among identified fingerprints, Vitronectin is recognized by αVβ3

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integrins, also known as the vitronectin receptor, which are overexpressed on many solid tumors

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and in tumor neovasculature. In a previous work, the vitronectin-enriched BC of cationic

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liposomes was exploited to target highly metastatic ductal carcinoma cells over-expressing αvβ3

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integrins (58). This is a point of great general interest since tumor cells over-express integrin αvβ3

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in various states of activation. For instance, the high level of αvβ3 in metastatic cancer cells

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circulating in the blood stream contribute to cancer spreading (59). Apolipoproteins bind specific

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lipoprotein receptors, including scavenger receptor class B, type I (SR-BI) and low-density

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lipoprotein receptor (LDLR) that are overexpressed in a huge number of pathological conditions

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(e.g., renal cell carcinoma, melanoma, hepatocellular carcinoma, lymphoma, and atherosclerosis).

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SR-BI is a high-density lipoprotein (HDL) receptor that facilitates the uptake of cholesterol esters

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from circulating lipoproteins, while LDLR mediates the endocytosis of cholesterol-rich LDL.

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Notably, brain microvascular endothelial cells found in the BBB are enriched of SR-BI and LDLR,

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which are essential to the endocytosis/transcytosis of cargos through brain microvascular

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endothelial cells (BMEC) (60). This binding ability makes NPs coated with an Apolipoprotein-


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enriched BC superior candidates for brain drug delivery when compared with other nanoparticle

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systems (51). Of note, Figure 4 panel B shows that the abundance of Apolipoproteins in the BC of

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CLs was in the order: CL2>CL3>CL1>CL4. Given their peculiar enrichment in BCFs and

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Apolipoproteins, CL2 and CL3 were therefore identified as the most promising formulations to

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deliver TMZ across the BBB. Among the well characterized in vitro BBB models, most of them are

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developed using animal cells isolated from brain microvessels. Moreover, the majority of human in

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vitro models, commonly found in cancer literature, uses HUVECs (61). We therefore treated

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HUVECs with CL-BC complexes and evaluated cellular uptake after 10-min incubation (Figure 5).

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According to literature experiments were performed at three TMZ doses: 5 µM, 50 µM and 100

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µM (17). FACS results of Figure 5, panel A showed that, at the highest drug concentration, BC of

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CL2 exhibited the highest percentage of fluorescent-positive cells, i.e. the highest targeting ability.

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This result was in line with our predictions of targeting ability of CL-BC complexes based on the

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enrichment of BCFs (Figure 4). FACS experiments also showed that 10-min incubation had a minor

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effect on cell viability (Figure 5, panel B). Next, we treated HUVECs with CLs both in the presence

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and in the absence of BC and evaluated cell viability after and 1-h incubation. Indeed, at longer

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incubation times, high uptake rates are linked to greater inhibition of cell growth. In the absence

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of BC (Figure 6, panel A) cell viability decreased with increasing TMZ dose, but no clear correlation

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with liposomal formulation was found. On the other side, dose-dependent cell survival of HUVECs

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was markedly affected by BC. Figure 6, panel B shows that growth inhibition ability of CL-BC

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complexes was in the order: CL2>CL4>CL1>CL3. As above discussed, BC of CL2 exhibited the

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highest targeting ability, while that of CL3 did not confirm our predictions. According to recent

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literature, protein abundance is not the only factor controlling protein cell interactions. In

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principle, Apolipoproteins adsorbed to CL3 could have functional motifs buried inside or not

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correctly presented at the liposome surface. Mapping protein binding sites on the liposome-BC is


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an urgent task for future research (62-65). In summary, combined MS/MS findings and cellular

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studies let us identify CL2 as the most promising formulation to delivers TMZ across the BBB.

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Finally, we evaluated the antitumor activity of TMZ in U-87 MG human glioblastoma cell line.

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Preliminary experiments showed that the free drug produced a time- and dose-dependent growth

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inhibition (Figure 6, panels A-D). According to previous findings by Kim et al. (17), the effect of

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TMZ was significant at 50-100 µM and the time of exposure was also critical for inhibiting cell

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proliferation. When U-87 cells were treated with TMZ-loaded CL2 at the highest TMZ

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concentration (100 µM), inhibition of cellular growth was slightly higher than that achieved with

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free TMZ (Figure 6, panel E). On the other side, BC boosted inhibitory effect of TMZ by factor ∼ 5

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with respect to free drug. This finding indicates that BC can trigger specific NP-cell interactions

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(e.g. cellular uptake, endosomal escape, lysosomal degradation and nuclear translocation). For

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example, Digiacomo et al. (51) showed that liposome-BC promotes a switch in the uptake

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mechanism of multicomponent CLs from micropinocytosis to clathrin-dependent endocytosis.

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Accurate clarification of the role played by BC on the interaction between CLs and the machinery

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of U-87 cells will be the object of future investigations. However, the improved cancer killing

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ability of TMZ-loaded CL-BC complexes, as those forming in vivo (46, 66), compared to free TMZ

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and TMZ-loaded CLs let us suggest that BC-based nanomedicine may hold great promise as a more

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effective therapy for GBM.

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Conclusions

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We have synthesized a set of four TMZ-encapsulating liposome formulations that recruited plasma

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proteins from HP. Most of identified proteins were previously categorized as BC fingerprints and

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promoted favorable NP- cancer cell association. We found that the CL formulation exhibiting the


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highest levels of targeting fingerprints, had also the major impact on HUVECs that are an

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established model of in vitro BBB (63-65). When administered to U-87 cells, designer formulation

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enhanced anti-tumor efficacy compared by a factor 5 compared to corona free CLs. Our results

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indicate that exploitation of BC could be a valuable means to develop targeted nanomedicine with

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superior ability to cross the BBB and enhanced anticancer efficacy. This study was performed on a

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simplified in vitro model of the BBB whose behavior not necessarily reflects the in vivo situation.

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Future work will lead to a fundamental understanding of transport mechanisms across more

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realistic BBB models, which is key for further improvements of treatments for brain diseases and

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other CNS related diseases. Although recent studies have demonstrated that predicting liposome-

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cell association by BC fingerprints provides valuable new insights, this approach requires the

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mapping of plasma proteins on the liposome surface. Deciphering the recognition between corona

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proteins and cell receptors (63, 64, 67) could help us towards understanding exactly how BC-

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decorated lipid vesicles interact with cells and biological barriers, potentially activating different

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biological pathways. To this end, reproducible methods must be clearly established that comprise

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the recovery of liposomes after dynamic incubation (68) and/or systemic administration in vivo

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(46, 69, 70), thorough characterization of the BC, and evaluation of corresponding biological

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interactions. We foresee that these future advances will yield new opportunities for accelerating

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the clinical translation of liposomal drugs for brain delivery.

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Material and methods

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Preparation of CLs. DOTAP, DC-Chol, DOPC and DOPE were purchased from Avanti Polar Lipids

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(Alabaster, AL, USA) and used without further purification. Texas-Red DOPE was purchased from

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Thermo Fisher Scientific. CLs were prepared according to standard procedures. In brief,


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appropriate amounts of lipids were dissolved at neutral/total lipid (mol/mol)=0.5. Lipid films were

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hydrated (final lipid concentration is 1 mg mL-1) with ultrapure water for size, zeta-potential, laser

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scanning confocal microscopy (LSCM) and flow cytometry experiments. For proteomics

4

experiments lipid films were hydrated with a dissolving buffer (Tris–HCl, pH 7.4, 10 mmol L-1; NaCl,

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150 mmol L-1; EDTA, 1 mmol L -1) and stored at 4 °C.

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Preparation of TMZ-loaded CLs. Incorporation of TMZ into cationic liposome was performed using

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the dehydratation-rehydratation method (1). Liposomal formulations were made combining 1 mg

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of TMZ with lipids in ratio 1:1 (molar ratio), this mixture was dissolved in 1 ml of chloroform and

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0.2 ml of methanol and placed on a rotary evaporator set at 65°C for 4 hours to produce the film

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layer. The resulting film was rehydrated with 2.5 ml of PBS and then extruded 20 times through a

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0.1 μm polycarbonate carbonate filter by the Avanti Mini-Extruder (Avanti Polar Lipids, Alabaster,

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AL). Following 1-h exposure to HP, TMZ-loaded CL-BC complexes were formed. Fluorescently

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labelled CLs were prepared using DOPE-Texas red (Thermo Fisher Scientific). To evaluate TMZ

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encapsulation efficiency, free TMZ was separated from the TMZ encapsulated-liposomes using

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Vivavspin 500 (5kDa MWCO, GE Healthcare), then the UV-Vis absorption spectra of both free and

16

encapsulated TMZ was measured by a Jasco V-630 spectrophotometer. Concentrations of free and

17

encapsulated TMZ were evaluated by applying the Lambert-Beer law to the 330 nm absorption

18

peak (Figure S1). Absolute amounts of free and encapsulated TMZ were determined by relating

19

the measured concentrations to the sample volumes. Finally, EE and DLC were measured as:

20

EE=100 x (mass of the drug in liposome)/(initial mass of the drug used) (1)

21

DLC=100 x (mass of encapsulated drug)/(mass of liposome) (2).

22


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Page 14 of 31

1

Size and Zeta-potential experiments. All size and zeta-potential measurements were made on a

2

Zetasizer Nano ZS90 (Malvern, U.K.) at 25 °C. CLs, TMZ-loaded CLs and TMZ-loaded CL-BC

3

complexes were diluted 1:100 with distilled water and size and zeta-potential results are given as

4

mean ± standard deviation of five replicates.

5

Proteomics experiments. For proteomics experiments lipid films were hydrated with a dissolving

6

buffer (Tris-HCl, pH 7.4, 10 mmol L−1; NaCl, 150 mmol L−1; EDTA, 1 mmol L−1) and stored at 4 °C.

7

The obtained solutions were extruded 20 times through a 0.1 μm polycarbonate carbonate filter

8

with the Avanti Mini-Extruder (Avanti Polar Lipids, Alabaster, AL). CLs were incubated with HP (1:1

9

v/v) and then incubated at 37 °C for 60 min. This volume ratio was chosen because it is mimetic of

10

in vivo conditions. After incubation, the samples were centrifuged 15 min at 14,000 rpm followed

11

by pellet resuspension; this procedure was repeated three times to wash the sample and remove

12

loosely bound proteins. NanoLC–MS/MS analysis, data analysis and protein validation were

13

performed as elsewhere reported (48).

14

Cell cultures. Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Lonza

15

(Allendale, NJ, USA) and were maintained in EGM-2 BulletKit medium (Lonza). Cells were used

16

until passage number 4. After mechanical dissociation, U-87 MG (ATCC®) single cells were

17

resuspended in F10 medium and centrifuged at 1000 g for 5 min. The pellet was resuspended in

18

the F10 growth medium supplemented with 10% fetal calf serum (FCS, Life Technologies Ltd,

19

Milano, Italy) and cells were plated in Petri plates (Falcon Primaria, Lincoln Park, NJ, USA). The

20

medium was then changed every 3 days. After 14– 15 days, cells were trypsinized, re-plated into

21

24-well plates at a density of 25 · 103 cells/well and shifted into D-MEM Glutamax without serum

22

(Life Technologies Ltd, Milano, Italy). USA). The medium was then changed every 3 days. After 14–

23

15 days, cells were trypsinized, re-plated into 24-well plates at a density of 25 · 103 cells/well and

24

shifted into D-MEM Glutamax without serum (Life Technologies Ltd, Milano, Italy). ACS Paragon Plus Environment

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1

FACS. HUVECs cells were seeded in 12-well plates (150,000 cells/well) using complete medium.

2

After 24 hours, cells were treated for 3 hours with fluorescently labelled TMZ-loaded CLs in

3

Optimem medium. Then cells were washed two times with cold PBS, detached with trypsin/EDTA

4

and acquired using cytometer. Fluorescence-activated cell sorting (FACS) analysis was performed

5

using BD LSR FortessaTM (BD Bioscience).

6

TMZ treatment of U-87 MG cell lines. In vitro response to treatment with TMZ was evaluated

7

under three conditions: i) free TMZ; ii) TMZ encapsulated in CLS before incubation in HP (i.e. in the

8

absence of BC, in the text defined “TMZ-loaded CLs”); iii) TMZ encapsulated in CLS after incubation

9

in HP (i.e. in the presence of BC, in the text defined “TMZ-loaded CL-BC complexes”). To this end,

10

we plated U-87 MG cells in 48-well plates at 1 × 104 cells per well in DMEM supplemented with

11

10% FBS and incubated them at 37°C in an atmosphere containing 5% CO2. The following day the

12

cells were treated for 24, 48 and 72h at the following concentrations: 5, 50 and 100 µM. After

13

treatment, cell count was made using a Bürker chamber; before the count the cells were colored

14

with trypan blue dye (Sigma St. Louis, MO, USA) to discriminate live cells from dead.

15

Cell viability assays. 1 x 104 cells were seeded in 96-well plates and allowed to adhere for 12h. The

16

cultures were exposed to different concentrations of TMZ alone 5, 50, 100 µM or TMZ

17

encapsulated within CLS for 24, 48 and 72h, followed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-

18

diphenyltetrazolium bromide] (Sigma–Aldrich, assay). Briefly, 5mg/ml MTT in 100 µl of DMEM

19

without phenol red was added to the cultured cells for 2 h. Cells were washed by phosphate

20

buffered saline (PBS) and lysed by 100 ml of DMSO. The concentrations of MTT were examined

21

colorimetrically; absorbance was determined at 570 nm. All measurements were carried out by

22

triplicate in three different replicates. The data results of the growth curve were normalized at

23

logarithmic scale. The results were analyzed based on variance analysis (ANOVA).

24


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Page 16 of 31

1

Acknowledgements

2

GC, DP and LD gratefully acknowledge support by Sapienza University of Rome (Projects H2020;

3

protocol number: PH11715C7916B7A6).

4

5

6

Supporting Information. Figure S1. Absorption spectra of TMZ as a function of drug concentration. Tables

7

S1-S4. Full list of corona proteins identified onto the surface of CL1-CL4 liposomes following 1 hour

8

incubation with human plasma. Table S5. Top 20 most abundant proteins identified in protein corona of

9

CL1-CL4 liposomes following 1 hour incubation with human plasma.

10 11

Authors’ contribution. AA performed cell viability experiments, conceived the study, discussed data,

12

supervised research, reviewed the manuscript; SP performed FACS and cell viability experiments, analyzed

13

data, prepared figures, reviewed the manuscript; LD performed DLS and UV-VIS absorption experiments,

14

analyzed data, prepared figures, reviewed the manuscript; DP conceived the study, discussed data, wrote

15

the manuscript; ALC conceived nanoLC MS/MS experiments, analyzed nanoLC MS/MS data, reviewed the

16

manuscript; LF conceived the study, discussed data, supervised research, reviewed the manuscript; MAO,

17

performed cell viability experiments, discussed data, reviewed the manuscript; GT discussed the data,

18

reviewed the manuscript; RR provided HUVECS cells, discussed the data, reviewed the manuscript; IS

19

conceived the study, discussed data, reviewed the manuscript; MM conceived the study, discussed data,

20

wrote the manuscript; GC conceived the study, discussed data, supervised research, wrote the manuscript.

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References 1. McFaline-Figueroa JR, Lee EQ. Brain Tumors. The American journal of medicine. 2018. 2. Groneberg DA, Addicks A-M, Bendels MH, Quarcoo D, Jaque J, Brüggmann D. Glioblastoma research: US and international networking achievements. Oncotarget. 2017;8(70):115730-5. 3. Omuro A, DeAngelis LM. Glioblastoma and other malignant gliomas: a clinical review. Jama. 2013;310(17):1842-50. 4. Gilbert MR, Wang M, Aldape KD, Stupp R, Hegi ME, Jaeckle KA, et al. Dose-dense temozolomide for newly diagnosed glioblastoma: a randomized phase III clinical trial. Journal of clinical oncology. 2013;31(32):4085-91. 5. Hegi ME, Diserens A-C, Gorlia T, Hamou M-F, de Tribolet N, Weller M, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. New England Journal of Medicine. 2005;352(10):997-1003. 6. Anjum K, Shagufta BI, Abbas SQ, Patel S, Khan I, Shah SAA, et al. Current status and future therapeutic perspectives of glioblastoma multiforme (GBM) therapy: A review. Biomedicine & Pharmacotherapy. 2017;92:681-9. 7. Bernal GM, LaRiviere MJ, Mansour N, Pytel P, Cahill KE, Voce DJ, et al. Convection-enhanced delivery and in vivo imaging of polymeric nanoparticles for the treatment of malignant glioma. Nanomedicine: Nanotechnology, Biology and Medicine. 2014;10(1):149-57. 8. Xi G, Robinson E, Mania-Farnell B, Vanin EF, Shim K-W, Takao T, et al. Convection-enhanced delivery of nanodiamond drug delivery platforms for intracranial tumor treatment. Nanomedicine: Nanotechnology, Biology and Medicine. 2014;10(2):381-91. 9. Krol S, Macrez R, Docagne F, Defer G, Laurent S, Rahman M, et al. Therapeutic benefits from nanoparticles: the potential significance of nanoscience in diseases with compromise to the blood brain barrier. Chemical reviews. 2012;113(3):1877-903. 10. Allen TM, Cullis PR. Liposomal drug delivery systems: from concept to clinical applications. Advanced drug delivery reviews. 2013;65(1):36-48. 11. Barenholz YC. Doxil®—The first fda-approved nano-drug: Lessons learned. Journal of controlled release. 2012;160(2):117-34. 12. Pozzi D, Marchini C, Cardarelli F, Rossetta A, Colapicchioni V, Amici A, et al. Mechanistic understanding of gene delivery mediated by highly efficient multicomponent envelope-type nanoparticle systems. Molecular pharmaceutics. 2013;10(12):4654-65. 13. Palchetti S, Pozzi D, Marchini C, Amici A, Andreani C, Bartolacci C, et al. Manipulation of lipoplex concentration at the cell surface boosts transfection efficiency in hard-to-transfect cells. Nanomedicine: Nanotechnology, Biology, and Medicine. 2017;13(2):681-91. 14. Gao W, Hu C-MJ, Fang RH, Zhang L. Liposome-like nanostructures for drug delivery. Journal of Materials Chemistry B. 2013;1(48):6569-85. 15. Belhadj Z, Zhan C, Ying M, Wei X, Xie C, Yan Z, et al. Multifunctional targeted liposomal drug delivery for efficient glioblastoma treatment. Oncotarget. 2017;8(40):66889. 16. Zhao G, Huang Q, Wang F, Zhang X, Hu J, Tan Y, et al. Targeted shRNA-loaded liposome complex combined with focused ultrasound for blood brain barrier disruption and suppressing glioma growth. Cancer letters. 2018. 17. Kim S-S, Rait A, Kim E, DeMarco J, Pirollo KF, Chang EH. Encapsulation of temozolomide in a tumortargeting nanocomplex enhances anti-cancer efficacy and reduces toxicity in a mouse model of glioblastoma. Cancer letters. 2015;369(1):250-8. 18. Mahmoudi M. Protein corona: The golden gate to clinical applications of nanoparticles. The International Journal of Biochemistry & Cell Biology. 2016. 19. Mahmoudi M, Lynch I, Ejtehadi MR, Monopoli MP, Bombelli FB, Laurent S. Protein− nanoparƟcle interactions: opportunities and challenges. Chemical reviews. 2011;111(9):5610-37. 20. Zanganeh S, Spitler R, Erfanzadeh M, Alkilany AM, Mahmoudi M. Protein corona: Opportunities and challenges. The international journal of biochemistry & cell biology. 2016;75:143-7. 21. Walkey CD, Chan WCW. Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment. Chemical Society Reviews. 2012;41(7):2780-99. ACS Paragon Plus Environment

17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

Page 18 of 31

22. Mahmoudi M, Bertrand N, Zope H, Farokhzad OC. Emerging understanding of the protein corona at the nano-bio interfaces. Nano Today. 2016. 23. Capriotti AL, Caracciolo G, Cavaliere C, Foglia P, Pozzi D, Samperi R, et al. Do plasma proteins distinguish between liposomes of varying charge density? Journal of Proteomics. 2012;75(6):1924-32. 24. Caracciolo G, Pozzi D, Capriotti AL, Cavaliere C, Piovesana S, La Barbera G, et al. The liposomeprotein corona in mice and humans and its implications for in vivo delivery. Journal of Materials Chemistry B. 2014;2(42):7419-28. 25. Colapicchioni V, Tilio M, Digiacomo L, Gambini V, Palchetti S, Marchini C, et al. Personalized liposome–protein corona in the blood of breast, gastric and pancreatic cancer patients. The International Journal of Biochemistry & Cell Biology. 2016;75:180-7. 26. Caracciolo G. Liposome–protein corona in a physiological environment: Challenges and opportunities for targeted delivery of nanomedicines. Nanomedicine: Nanotechnology, Biology and Medicine. 2015;11(3):543-57. 27. Caracciolo G, Farokhzad OC, Mahmoudi M. Biological Identity of Nanoparticles In Vivo: Clinical Implications of the Protein Corona. Trends in Biotechnology. 2017;35(3):257-64. 28. Mahmoudi M, Yu M, Serpooshan V, Wu JC, Langer R, Lee RT, et al. Multiscale technologies for treatment of ischemic cardiomyopathy. Nature nanotechnology. 2017;12(9):845. 29. Gaillard PJ, Appeldoorn CC, Dorland R, van Kregten J, Manca F, Vugts DJ, et al. Pharmacokinetics, brain delivery, and efficacy in brain tumor-bearing mice of glutathione pegylated liposomal doxorubicin (2B3-101). PloS one. 2014;9(1):e82331. 30. Bramini M, Ye D, Hallerbach A, Nic Raghnaill M, Salvati A, Åberg C, et al. Imaging approach to mechanistic study of nanoparticle interactions with the blood–brain barrier. ACS nano. 2014;8(5):4304-12. 31. Bigdeli A, Palchetti S, Pozzi D, Hormozi-Nezhad MR, Baldelli Bombelli F, Caracciolo G, et al. Exploring Cellular Interactions of Liposomes Using Protein Corona Fingerprints and Physicochemical Properties. ACS nano. 2016. 32. Palchetti S, Digiacomo L, Pozzi D, Peruzzi G, Micarelli E, Mahmoudi M, et al. Nanoparticles-cell association predicted by protein corona fingerprints. Nanoscale. 2016;8(25):12755-63. 33. Walkey CD, Olsen JB, Song F, Liu R, Guo H, Olsen DWH, et al. Protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles. ACS nano. 2014;8(3):2439-55. 34. Caracciolo G, Amenitsch H. Cationic liposome/DNA complexes: from structure to interactions with cellular membranes. European Biophysics Journal. 2012;41(10):815-29. 35. Caracciolo G, Pozzi D, Amenitsch H, Caminiti R. Multicomponent cationic lipid-DNA complex formation: Role of lipid mixing. Langmuir. 2005;21(25):11582-7. 36. Caracciolo G, Pozzi D, Caminiti R, Amenitsch H. Two-dimensional lipid mixing entropy regulates the formation of multicomponent lipoplexes. Journal of Physical Chemistry B. 2006;110(42):20829-35. 37. Caracciolo G, Pozzi D, Caminiti R, Marchini C, Montani M, Amici A, et al. Enhanced transfection efficiency of multicomponent lipoplexes in the regime of optimal membrane charge density. Journal of Physical Chemistry B. 2008;112(36):11298-304. 38. Pozzi D, Marchini C, Cardarelli F, Rossetta A, Colapicchioni V, Amici A, et al. Mechanistic understanding of gene delivery mediated by highly efficient multicomponent envelope-type nanoparticle systems. Molecular Pharmaceutics. 2013;10(12):4654-65. 39. Simberg D, Weisman S, Talmon Y, Faerman A, Shoshani T, Barenholz Y. The role of organ vascularization and lipoplex-serum initial contact in intravenous murine lipofection. Journal of Biological Chemistry. 2003;278(41):39858-65. 40. Gjetting T, Arildsen NS, Christensen CL, Poulsen TT, Roth JA, Handlos VN, et al. In vitro and in vivo effects of polyethylene glycol (PEG)-modified lipid in DOTAP/cholesterol-mediated gene transfection. International journal of nanomedicine. 2010;5:371. 41. Pozzi D, Caracciolo G, Caminiti R, De Sanctis SC, Amenitsch H, Marchini C, et al. Toward the Rational Design of Lipid Gene Vectors: Shape Coupling between Lipoplex and Anionic Cellular Lipids Controls the Phase Evolution of Lipoplexes and the Efficiency of DNA Release. ACS Applied Materials & Interfaces. 2009;1(10):2237-49.


18

Page 19 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52


42. Caracciolo G, Pozzi D, Capriotti AL, Cavaliere C, Piovesana S, Amenitsch H, et al. Lipid composition: a “key factor” for the rational manipulation of the liposome–protein corona by liposome design. RSC Advances. 2015;5(8):5967-75. 43. Moghimi SM, Patel HM. Tissue specific opsonins for phagocytic cells and their different affinity for cholesterol-rich liposomes. FEBS letters. 1988;233(1):143-7. 44. Verma J, Van Veen HA, Lal S, Van Noorden CJ. Delivery and cytotoxicity of doxorubicin and temozolomide to primary glioblastoma cells using gold nanospheres and gold nanorods. European Journal of Nanomedicine. 2016;8(1):49-60. 45. Palchetti S, Colapicchioni V, Digiacomo L, Caracciolo G, Pozzi D, Capriotti AL, et al. The protein corona of circulating PEGylated liposomes. Biochimica et Biophysica Acta - Biomembranes. 2016;1858(2):189-96. 46. Amici A, Caracciolo G, Digiacomo L, Gambini V, Marchini C, Tilio M, et al. In vivo protein corona patterns of lipid nanoparticles. RSC Advances. 2017;7(2):1137-45. 47. Papi M, Caputo D, Palmieri V, Coppola R, Palchetti S, Bugli F, et al. Clinically approved PEGylated nanoparticles are covered by a protein corona that boosts the uptake by cancer cells. Nanoscale. 2017;9(29):10327-34. 48. Pozzi D, Colapicchioni V, Caracciolo G, Piovesana S, Capriotti AL, Palchetti S, et al. Effect of polyethyleneglycol (PEG) chain length on the bio-nano-interactions between PEGylated lipid nanoparticles and biological fluids: from nanostructure to uptake in cancer cells. Nanoscale. 2014;6(5):2782-92. 49. Digiacomo L, Pozzi D, Amenitsch H, Caracciolo G. Impact of the biomolecular corona on the structure of PEGylated liposomes. Biomaterials science. 2017;5(9):1884-8. 50. Caracciolo G. Clinically approved liposomal nanomedicines: lessons learned from the biomolecular corona. Nanoscale. 2018. 51. Digiacomo L, Cardarelli F, Pozzi D, Palchetti S, Digman M, Gratton E, et al. An apolipoproteinenriched biomolecular corona switches the cellular uptake mechanism and trafficking pathway of lipid nanoparticles. Nanoscale. 2017;9(44):17254-62. 52. Behzadi S, Serpooshan V, Sakhtianchi R, Müller B, Landfester K, Crespy D, et al. Protein corona change the drug release profile of nanocarriers: the “overlooked” factor at the nanobio interface. Colloids and Surfaces B: Biointerfaces. 2014;123:143-9. 53. Liu R, Jiang W, Walkey CD, Chan WC, Cohen Y. Prediction of nanoparticles-cell association based on corona proteins and physicochemical properties. Nanoscale. 2015;7(21):9664-75. 54. Lai ZW, Yan Y, Caruso F, Nice EC. Emerging Techniques in Proteomics for Probing Nano–Bio Interactions. ACS nano. 2012;6(12):10438-48. 55. Bigdeli A, Palchetti S, Pozzi D, Hormozi-Nezhad MR, Baldelli Bombelli F, Caracciolo G, et al. Exploring Cellular Interactions of Liposomes Using Protein Corona Fingerprints and Physicochemical Properties. ACS Nano. 2016;10(3):3723-37. 56. Caracciolo G, Pozzi D, Candeloro De Sanctis S, Laura Capriotti A, Caruso G, Samperi R, et al. Effect of membrane charge density on the protein corona of cationic liposomes: Interplay between cationic charge and surface area. Applied Physics Letters. 2011;99(3). 57. Walkey CD, Olsen JB, Guo H, Emili A, Chan WC. Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake. Journal of the American Chemical Society. 2012;134(4):2139-47. 58. Caracciolo G, Cardarelli F, Pozzi D, Salomone F, Maccari G, Bardi G, et al. 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):13171-9. 59. Ganguly KK, Pal S, Moulik S, Chatterjee A. Integrins and metastasis. Cell adhesion & migration. 2013;7(3):251-61. 60. Mooberry LK, Sabnis NA, Panchoo M, Nagarajan B, Lacko AG. Targeting the SR-B1 receptor as a gateway for cancer therapy and imaging. Frontiers in pharmacology. 2016;7:466. 61. Drolez A, Vandenhaute E, Julien S, Gosselet F, Burchell J, Cecchelli R, et al. Selection of a relevant In vitro blood-brain barrier model to investigate pro-metastatic features of human breast cancer cell lines. PloS one. 2016;11(3):e0151155.


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Page 20 of 31

62. Kelly PM, Åberg C, Polo E, O'Connell A, Cookman J, Fallon J, et al. Mapping protein binding sites on the biomolecular corona of nanoparticles. Nature nanotechnology. 2015;10(5):472-9. 63. Lara S, Alnasser F, Polo E, Garry D, Lo Giudice MC, Hristov DR, et al. Identification of receptor binding to the biomolecular corona of nanoparticles. ACS nano. 2017;11(2):1884-93. 64. Polo E, Collado M, Pelaz B, del Pino P. Advances toward More Efficient Targeted Delivery of Nanoparticles in Vivo: Understanding Interactions between Nanoparticles and Cells. ACS nano. 2017;11(3):2397-402. 65. 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. 66. Hadjidemetriou M, Al-Ahmady Z, Mazza M, Collins RF, Dawson K, Kostarelos K. In vivo biomolecule corona around blood-circulating, clinically used and antibody-targeted lipid bilayer nanoscale vesicles. ACS nano. 2015;9(8):8142-56. 67. Kelly PM, Åberg C, Polo E, O'Connell A, Cookman J, Fallon J, et al. Mapping protein binding sites on the biomolecular corona of nanoparticles. Nature nanotechnology. 2015. 68. Pozzi D, Caracciolo G, Digiacomo L, Colapicchioni V, Palchetti S, Capriotti AL, et al. The biomolecular corona of nanoparticles in circulating biological media. Nanoscale. 2015;7(33):13958-66. 69. Hadjidemetriou M, Al-Ahmady Z, Kostarelos K. Time-evolution of in vivo protein corona onto bloodcirculating PEGylated liposomal doxorubicin (DOXIL) nanoparticles. Nanoscale. 2016;8(13):6948-57. 70. Corbo C, Molinaro R, Taraballi F, Toledano Furman NE, Hartman KA, Sherman MB, et al. Unveiling the in Vivo Protein Corona of Circulating Leukocyte-like Carriers. ACS nano. 2017;11(3):3262-73.

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1

Captions to Figures

2

Figure 1. Exploitation of the bionano interactions for brain delivery. Following administration in

3

vivo, temozolomide-loaded cationic liposomes (CLs) get covered by a biomolecular corona (BC)

4

that can mask targeting surface ligands thus resulting in unpredictable off-target interactions. On

5

the other side, proteins forming the CL-BC have their own biological purpose including specific

6

interactions related to their function and can therefore promote favorable interactions with

7

specific receptors of target cells. To exploit BC in targeted brain drug delivery designed liposomes

8

should recruit plasma proteins with the highest affinity for cellular receptors over-expressed at the

9

blood-brain barrier. (e.g. scavenger receptor class B, type I and low-density lipoprotein receptor).

10

Figure 2. Size (A) and zeta-potential (B) of CLs (diagonal patterned histograms), temozolomide

11

(TMZ)-loaded CLs (vertical patterned histograms) and TMZ-loaded CL-biomolecular corona

12

complexes (full histograms). CL1 (DOTAP/Cholesterol), CL2 (DOTAP/DOPE), CL3 (DC-Chol/DOPE),

13

CL4 (DC-Chol/ Cholesterol).

14

Figure 3. (A) Venn diagrams of proteins identified in the biomolecular coronas (BCs) of CLs: CL1

15

(DOTAP/Cholesterol), CL2 (DOTAP/DOPE), CL3 (DC-Chol/DOPE), CL4 (DC-Chol/ Cholesterol). (B)

16

Venn diagrams of panel A with surface proportional to the number of identified proteins. Points

17

inside each element represent proteins of that set. (C) Circular plot depicting the protein

18

composition of the investigated coronas. Each quadrant represents a lipid formulation: proteins in

19

common amongst the four BCs are represented by gray curve histograms. Colored circumference

20

arcs have lengths proportional to the fraction of proteins belonging to one or more formulations,

21

as indicated by the corresponding links in the inner circle. Thus, for each quadrant, the set of

22

circumference arcs defines a curvilinear histogram that describes the protein composition of a

23

formulation, in terms of unique and common elements with respect to the other ones.


21


Page 22 of 31

1

Figure 4. Relative protein abundance of biomolecular corona fingerprints (Vitronectin, APOA1,

2

APOA2, APOB, APOC2, Ig heavy chain V–III region BRO, Vitamin K-dependent protein, and Integrin

3

beta3) and Apolipoproteins in the biomolecular corona of CL1-CL4 formulations. BCFs were chosen

4

according to references 33 and 35.

5

Figure 5. (A) Cellular uptake of CL-BC complexes 10-min after administration to HUVECs cells.

6

Statistical significance was evaluated by Student’s t-test: *P < 0.05, **P < 0.001, *** not

7

significant. (B) Cell viability of HUVECs cells following 10-min incubation with CL-BC complexes.

8

Statistical significance was evaluated by Student’s t-test with respect to control (i.e. not treated

9

cells): *P < 0.05, **P < 0.001, *** not significant. Where not displayed, cell viability was not

10

significantly different from that of control.

11

Figure 6. Cell growth inhibition of HUVECs cells following 1-h incubation with CLs both in the

12

absence (A) and in the presence (B) of biomolecular corona at three drug concentrations (5 µM, 50

13

µM and 100 µM). CL1 (DOTAP/Cholesterol), CL2 (DOTAP/DOPE), CL3 (DC-Chol/DOPE), CL4 (DC-

14

Chol/ Cholesterol). Statistical significance was evaluated with respect to control (i.e. not treated

15

cells, CT): *P < 0.05, **P < 0.001, *** not significant.

16

Figure 7. Enhanced efficacy of TMZ-loaded CL2-BC complexes (green diamonds) compared to free

17

TMZ (gray circles) and TMZ-loaded CLs (green circles). Statistical significance was evaluated with

18

respect to control (i.e. not treated cells, CT): *P < 0.05, **P < 0.001.

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1

2

3

Table 1. Encapsulation efficiency (EE) and drug loading content (DLC) of TMZ in cationic lipid (CL)

4

formulations. Results are given as average of three independent measurements ± standard

5

deviation.

CL1 CL2 CL3 CL4

EE (%) 54.9 ± 8.5 41.0 ± 7.1 49.6 ± 3.0 48.0 ± 2.8

DLC (%) 22.0 ± 3.4 16.6 ± 2.8 19.8 ± 1.2 19.2 ± 1.1

6

7

Table 2. Polydispersity index (PdI) of bare cationic liposomes (CLs), TMZ-loaded CLs (TMZ/CL) and

8

TMZ-loaded CL-BC complexes (TMZ/CL/BC). Results are given as average of three independent

9

measurements ± standard deviation.

CL1 CL2 CL3 CL4

CL 0.25 ± 0.09 0.15 ± 0.02 0.16 ± 0.05 0.19 ± 0.1

TMZ/CL 0.23 ± 0.01 0.06 ± 0.05 0.08 ± 0.02 0.19 ± 0.05

TMZ/CL/BC 0.49 ± 0.10 0.42 ± 0.08 0.35 ± 0.05 0.26 ± 0.03

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Graphical Table of Contents

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The liposome-biomolecular corona that forms in vivo can be exploited to cross the blood-brain

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barrier and enhance the antitumor efficacy of free drugs.

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Brain Targeting by LiposomeâBiomolecular Corona Boosts Anticancer

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