Brain targeting by liposome-biomolecular corona boosts anti-cancer

Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

pubs.acs.org/chemneuro

Brain Targeting by Liposome−Biomolecular Corona Boosts Anticancer 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*,‡ †

Istituto Neurologico Mediterraneo Neuromed, Via dell’Elettronica 86077 Pozzilli (IS), Italy Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy § Department of Chemistry, Sapienza University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy ∥ Department of Oncohematology, Ospedale Pediatrico Bambino Gesu’, Viale San Paolo 15, 00146 Rome, Italy ⊥ Department of Anesthesiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States

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‡

S Supporting Information *

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

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INTRODUCTION

central obstacle to the management of malignant glioma is the inability to effectively deliver a therapeutic agent to the tumor. A significant challenge in treating GBM is the ability of a drug to cross the blood−brain barrier (BBB), the brain’s own defense system, which actively blocks or expels curative drugs from entering the brain. Several approaches have been explored to overcome this issue. Nanoparticles (NPs) showed promising capacity as carriers to increase the bioavailability of traditional chemotherapeutic drugs for brain tumors, such as TMZ, doxorubicin hydrochloride, irinotecan hydrochloride, and vincristine sulfate.7−9 Among different types of NPs, liposomes have received

Glioblastoma multiforme (GBM) is the most common brain tumor, with an annual incidence of 3.19 per 100 000 in the United States.1,2 GBMs are histologically and heterogeneous tumors and have historically been classified by clinical presentation as either primary or secondary depending on evidence of a pre-existing lower-grade glioma.3 The current standard of care combines maximal surgical resection, followed by radiotherapy with concomitant and adjuvant Temozolomide (TMZ). Despite this multimodal approach, median survival is limited to 16−19 months, with approximately 25−30% of the patients alive at 2 y after diagnosis.4 Patients whose tumors display epigenetic silencing of the DNA repair enzyme Omethyl-guanine-methyltransferase experience better outcomes.4,5 Given the poor survival with currently approved treatments, new therapeutic options for GBM are needed.6 A © XXXX American Chemical Society

Received: July 9, 2018 Accepted: July 17, 2018 Published: July 17, 2018 A

DOI: 10.1021/acschemneuro.8b00339 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience

Figure 1. Exploitation of the bionano interactions for brain delivery. Following administration in vivo, Temozolomide-loaded cationic liposomes (CLs) get covered by a biomolecular corona (BC) that can mask targeting surface ligands thus resulting in unpredictable off-target interactions. On the other side, proteins forming the CL−BC have their own biological purpose including specific interactions related to their function and can therefore promote favorable interactions with specific receptors of target cells. To exploit BC in targeted brain drug delivery designed liposomes should recruit plasma proteins with the highest affinity for cellular receptors overexpressed at the blood−brain barrier. (e.g., scavenger receptor class B, type I, and lowdensity lipoprotein receptor).

nervous system (CNS) related diseases (Figure 1). NPs bind to receptors located at the BBB29 and are internalized by a process referred to as adsorptive mediated transcytosis.30 Efficient transcytosis across the BBB is an important strategy for accessing drug targets within the CNS. Despite extensive research the number of studies reporting successful delivery of macromolecules or macromolecular complexes to the CNS has remained very low so far. Evidently, to gain maximal benefit from these novel developments and to enable improved delivery of drugs to the brain, we need to explore whether BC facilitates liposomes interactions with BBB and consequently affects efficacy of drugs for brain tumors. We have recently investigated BC-mediated liposome−cancer cell interactions using a library of liposomes of various size (e.g., 100 nm vs 250 nm) and surface chemistry (e.g., cationic vs anionic liposomes).31,32 For the entire library, a total of 436 distinct plasma proteins were detected. It was noteworthy that, of the entire pool of possible descriptors, a very small set of BC fingerprints (BCFs) demonstrated the greatest impact on cell association. Notably, the same proteins were also identified in previous works as being highly relevant to correlating NP-cell association.33 To exploit the targeting nature of BC, here we encapsulated TMZ in four binary CL formulations made of the widely used cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3([N-(N′,N′-dimethylaminoethane)-carbamoyl]-cholesterol (DC-Chol) and neutral lipids dioleoylphosphatidylethanolamine (DOPE) and cholesterol.34 The choice of synthesizing binary formulations depended on the fact that multicomponent liposomal formulations (e.g., ternary, quaternary lipidic mixtures, etc.) present numerous defects in the lipid bilayer due to the nonideal miscibility of the lipid species. As previously demonstrated,35−37 such defects makes multicomponent formulations less stable under interaction with biomembranes and plasma proteins. This aspect is unfavorable in view of the

widespread application as drug and gene delivery vectors with a unique history of successful clinical translation.10−14 Current targeting strategies for brain drug delivery are based on the functionalization of liposomes through appropriate ligands that could be recognized by receptors at the BBB.15,16 For instance, a recent study showed that encapsulation of TMZ in a tumortargeting cationic liposome (CL) enhances anticancer efficacy in a mouse model of GBM.17 However, despite promising results, no targeted liposomal therapeutics have been clinically approved for brain drug delivery. One possible reason could be the existence of “hidden factors” at the nanobio interface, which created a vast gap between bench discoveries and clinical translation of nanotechnologies.9,18−20 It is now widely accepted that when NPs are injected into biological fluids, such as the blood, biomolecules form a complex layer around them, referred to as “biomolecular corona” (BC).21,22 Following introduction in the bloodstream, liposomes are surrounded by high concentrations of plasma proteins that bind to the lipid surface leading to formation of liposome−BC. Main factors shaping liposome−BC are (i) the physicochemical properties of lipid surface;23 (ii) the protein source;24 and (iii) the physiological environment.24,25 Being the biological interface seen by cells, BC is thought to give a new “biological identity” to the liposomes by encrypting information that controls their bioactivity (e.g., cellular association and intracellular fate).26 At present, researchers believe that understanding and controlling the bionano interactions of liposomes with biological media (e.g., blood, lympha, and interstitial fluids) is central for their clinic translation.27,28 Liposome−BC would include proteins engaged from the blood that could lead the vesicle to interact with specific receptors expressed on the plasma membrane of target cells. Currently, the concept is emerging that BC-based nanomedicine could generate innovative treatments for the central B


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ACS Chemical Neuroscience possible in vivo application of CL formulations. As cationic lipid species we employed DOTAP and DC-Chol that have been extensively used both in vitro38 and in vivo.39,40 Two binary CL formulations were synthesized mixing DOTAP and DC-Chol with DOPE. Due to its “cone-like” molecular structure, DOPE promotes the formation of highly fusogenic inverse phases upon interaction with cellular membranes resulting in the efficient release of transported load.41 In addition, when mixed with either DOTAP and DC-Chol, DOPE promotes adsorption of apolipoproteins that could provide CLs with distinctive ability to target cancer cells.42 A second couple of binary CLs were prepared by mixing DOTAP and DC-Chol with cholesterol. Previous investigations clarified that liposomes rich in cholesterol bind less protein and are more resistant in vivo than cholesterol-free liposomes.43 However, recent studies have shown that, in combination with either DOTAP and DC-Chol, cholesterol promotes the adverse adsorption of opsonins (e.g., immunoglobulins, complement proteins, etc.).42 This has been an overlooked factor in lipid-mediated drug delivery and deserves further investigation. In the following text, CL formulations will be synthetically indicated as follows: CL1 (DOTAP/cholesterol), CL2 (DOTAP/DOPE), CL3 (DCChol/DOPE), and CL4 (DC-Chol/cholesterol). TMZ-loaded liposome−BC complexes were thoroughly characterized by dynamic light scattering (DLS), electrophoretic light scattering (ELS), and nanoliquid chromatography tandem mass spectrometry (nano-LC MS/MS). Following 1-h exposure to human plasma (HP), the surface of TMZ-loaded CLs was covered by complex and heterogeneous BCs. CL-BC complexes were used to deliver TMZ to human umbilical vein endothelial cells (HUVECs) and U-87 MG cells. While HUVECs represent one of the most common in vitro models of the BBB, U-87 MG is a human GBM cell line derived from malignant glioma. We found that the CL formulation exhibiting the highest levels of targeting BCFs, had the major uptake resulting in large growth inhibition of HUVECs. Furthermore, TMZ-loaded CL2−BC complexes were much more effective in killing U-87 cells than TMZ and TMZ-loaded CL2. Thus, the selected formulation can both target the endothelial cells to cross the BBB and efficiently kill tumor cells once in the brain. The enhanced efficacy of BCdecorated CLs suggests that exploitation of BC holds great promise as a more efficient nanomedicine for GBM.

Figure 2. Size (A) and zeta-potential (B) of CLs (diagonal patterned histograms), Temozolomide (TMZ)-loaded CLs (vertical patterned histograms), and TMZ-loaded CL−biomolecular corona complexes (full histograms): CL1 (DOTAP/cholesterol), CL2 (DOTAP/ DOPE), CL3 (DC-Chol/DOPE), CL4 (DC-Chol/cholesterol).

in size (hydrodynamic diameter, DH ≈ 100−150 nm). In line with previous findings,17 TMZ-loaded CLs were roughly twice bigger in size than bare CLs and maintained positive surface charge (Figure 2). Following 1-h exposure to HP, TMZ-loaded CL−BC complexes were bigger in size than TMZ-loaded CLs, with the increase in size ranging between 20 and 40 nm (Figure 2). According to the previous reports,21,26 such enlargement is compatible with formation of a thick BC at the vesicle surface. Particle size distributions were further characterized by the polydispersity index (PdI) that is reported in Table 2 for the

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RESULTS AND DISCUSSION Encapsulation efficiency (EE) and drug loading content (DLC) were determined by UV−vis experiments44 (Figure S1), and results are summarized in Table 1. As evident all CLs exhibited EE and DLC in agreement with previous results.17 Then, size and zeta-potential of CLs were analyzed (Figure 2). Bare CLs were positively charged (zeta-potential ≈ 40−55 mV) and small

Table 2. Polydispersity Index (PdI) of Bare Cationic Liposomes (CLs), TMZ-Loaded CLs (TMZ/CL), and TMZLoaded CL−BC Complexes (TMZ/CL/BC)a CL1 CL2 CL3 CL4

Table 1. Encapsulation Efficiency (EE) and Drug Loading Content (DLC) of TMZ in Cationic Lipid (CL) Formulationsa CL1 CL2 CL3 CL4

EE (%)

DLC (%)

54.9 ± 8.5 41.0 ± 7.1 49.6 ± 3.0 48.0 ± 2.8

22.0 ± 3.4 16.6 ± 2.8 19.8 ± 1.2 19.2 ± 1.1

CL

TMZ/CL

TMZ/CL/BC

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

0.23 ± 0.01 0.06 ± 0.05 0.08 ± 0.02 0.19 ± 0.05

0.49 ± 0.10 0.42 ± 0.08 0.35 ± 0.05 0.26 ± 0.03

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

three experimental conditions (i.e., pristine CLs, TMZ-loaded CLs, and TMZ-loaded CL−BC complexes). PdI, which is commonly used to indicate the degree of uniformity of a size distribution of particles, shows that CLs and TMZ-loaded CLs were homogeneous in size, while TMZ-loaded CL−BC complexes exhibited a broader size distribution. Due to

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

C


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ACS Chemical Neuroscience preferential absorption of negatively charged plasma proteins, zeta-potential of TMZ-loaded CL−BC complexes was found to be negative. This result is fully in agreement with the previous reports showing that BC tends to give a nanomaterial a zeta potential between −10 and −30 mV irrespective of nanomaterial physical−chemical properties.21,32,45−47 Such so-called “normalization” of zeta potentials is due to the fact that most plasma proteins carry a net negative charge at physiological pH.21 As discussed above, a transport technology enabling liposomal drugs to overcome the BBB and enter GBM is still not available. Indeed, despite the increasing ability to engineering liposomes with a precise control of the surface functionalization,14 the targeting capacity is lost when liposomes are embedded in complex biological media, where issues associated with the complexity of the surrounding environment highly increase. Liposomes can be rapidly covered by BC and this biomolecular layer is the ultimate vesicle surface “seen” by living systems. Since formation of BC is unavoidable even for stealth liposomes,48−50 researchers are trying to exploit the tumortargeting nature of BC as it was as an “endogenous trigger” capable to promote favorable interactions with receptors overexpressed in target cells.51 In addition, the formation of BC can protect drug release/burst-effects in body by adding more drug protective layers at the surface of nanocarriers.52 The latest research has explored the correlation between BC composition and association with cancer cells.33,53 In the first step, BC proteins are identified and quantified by tandem mass spectrometry (MS/MS).54 Next, BC composition is correlated with cellular uptake by computational methods such as quantitative structure−activity relationships (QSARs). This approach has resulted in the identification of BCFs that are likely to promote NP uptake in target cells. For instance, Walkey et al.33 used NP properties and BCFs to predict the associations between a library of 105 gold NPs with A549 human lung epithelial carcinoma cells. More recently, we have shown that a small portion of the liposome−BC, which comprises 8 BCFs (Vitronectin, APOA1, APOA2, APOB, APOC2, Ig heavy chain V−III region BRO, vitamin K-dependent protein, and Integrin beta3), controls cell association of liposomes with cancer cells.32,55 More specifically, it was shown that the higher the abundance of the 8 BCFs, the higher the cell association with human cervical cancer cell line (HeLa) and human prostate cancer cell line (PC3). Thus, in the present investigation, we first characterized BCs by liquid-chromatography MS/MS (LC-MS/ MS). We identified 219, 192, 135, and 190 plasma proteins in the BCs of CL1, CL2, CL3, and CL4, respectively. The full list of proteins identified by nanoLC-MS/MS is given in Tables S1−S4 in the Supporting Information (SI). Venn diagrams of Figure 3 show that, a large number of proteins, exactly 91, were found to be in common among the four BCs. This significant overlapping confirms that surface charge is among the main not specific factors shaping BC.21,56,57 In addition, a lower but relevant number of unique proteins was found (36 for CL1, 28 for CL2, 2 for CL3, and 40 for CL4, respectively). In addition, overlapping of the 20 most abundant common proteins was far from being 100% (Table S5 in the ESI). These observations support the idea that lipid composition is a key specific factor in shaping liposome− BC.42 To exploit the targeting-nature of liposome−BC, first we evaluated the total abundance of BCFs. According to Figure 4 panel A, CL2 exhibited the highest abundance of BCFs. However, plasma proteins other than BCFs could promote favorable interactions with target cells. Among identified

Figure 3. (A) Venn diagrams of proteins identified in the biomolecular coronas (BCs) of CLs: CL1 (DOTAP/Cholesterol), CL2 (DOTAP/ DOPE), CL3 (DC-Chol/DOPE), CL4 (DC-Chol/Cholesterol). (B) Venn diagrams of panel A with surface proportional to the number of identified proteins. Points inside each element represent proteins of that set. (C) Circular plot depicting the protein composition of the investigated coronas. Each quadrant represents a lipid formulation: proteins in common among the four BCs are represented by gray curve histograms. Colored circumference arcs have lengths proportional to the fraction of proteins belonging to one or more formulations, as indicated by the corresponding links in the inner circle. Thus, for each quadrant, the set of circumference arcs defines a curvilinear histogram that describes the protein composition of a formulation, in terms of unique and common elements with respect to the other ones.

fingerprints, Vitronectin is recognized by αVβ3 integrins, also known as the Vitronectin receptor, which are overexpressed on many solid tumors and in tumor neovasculature. In a previous work, the Vitronectin-enriched BC of cationic liposomes was exploited to target highly metastatic ductal carcinoma cells overexpressing αvβ3 integrins.58 This is a point of great general interest since tumor cells overexpress integrin αvβ3 in various states of activation. For instance, the high level of αvβ3 in metastatic cancer cells circulating in the bloodstream contribute to cancer spreading.59 Apolipoproteins bind specific lipoprotein receptors, including scavenger receptor class B, type I (SR-BI) and low-density lipoprotein receptor (LDLR) that are overexpressed in a huge number of pathological conditions (e.g., renal cell carcinoma, melanoma, hepatocellular carcinoma, lymphoma, and atherosclerosis). SR-BI is a high-density lipoprotein (HDL) receptor that facilitates the uptake of cholesterol esters from circulating lipoproteins, while LDLR mediates the endocytosis of cholesterol-rich LDL. Notably, brain microvascular endothelial cells found in the BBB are enriched of SR-BI and LDLR, which are essential to the endocytosis/transcytosis of cargos through brain microvascular endothelial cells D


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Figure 4. Relative protein abundance of biomolecular corona fingerprints (Vitronectin, APOA1, APOA2, APOB, APOC2, Ig heavy chain V−III region BRO, vitamin K-dependent protein, and Integrin beta3) and Apolipoproteins in the biomolecular corona of CL1−CL4 formulations. BCFs were chosen according to refs 33 and 35.

Figure 5. (A) Cellular uptake of CL−BC complexes 10 min after administration to HUVECs cells. Statistical significance was evaluated by Student’s t test: * P < 0.05, ** P < 0.001, *** not significant. (B) Cell viability of HUVECs cells following 10 min incubation with CL−BC complexes. Statistical significance was evaluated by Student’s t test with respect to control (i.e., not treated cells): * P < 0.05, ** P < 0.001, *** not significant. Where not displayed, cell viability was not significantly different from that of control.

(BMEC).60 This binding ability makes NPs coated with an Apolipoprotein-enriched BC superior candidates for brain drug delivery when compared with other nanoparticle systems.51 Of note, Figure 4 panel B shows that the abundance of Apolipoproteins in the BC of CLs was in the order: CL2 > CL3 > CL1 > CL4. Given their peculiar enrichment in BCFs and Apolipoproteins, CL2 and CL3 were therefore identified as the most promising formulations to deliver TMZ across the BBB. Among the well characterized in vitro BBB models, most of them are developed using animal cells isolated from brain microvessels. Moreover, the majority of human in vitro models, commonly found in cancer literature, uses HUVECs.61 We therefore treated HUVECs with CL−BC complexes and evaluated cellular uptake after 10 min incubation (Figure 5). According to literature experiments were performed at three TMZ doses: 5, 50, and 100 μM.17 FACS results of Figure 5, panel A, showed that, at the highest drug concentration, BC of CL2 exhibited the highest percentage of fluorescent-positive cells, i.e. the highest targeting ability. This result was in line with our predictions of targeting ability of CL−BC complexes based on the enrichment of BCFs (Figure 4). FACS experiments also showed that 10 min incubation had a minor effect on cell viability (Figure 5, panel B). Next, we treated HUVECs with CLs both in the presence and in the absence of BC and evaluated cell viability after and 1-h incubation. Indeed, at longer incubation times, high uptake rates are linked to greater inhibition of cell growth. In the absence of BC (Figure 6, panel A) cell viability decreased with increasing TMZ dose, but no clear correlation with liposomal formulation was found. On

the other side, dose-dependent cell survival of HUVECs was markedly affected by BC. Figure 6, panel B shows that growth inhibition ability of CL−BC complexes was in the order: CL2 > CL4 > CL1 > CL3. As above-discussed, BC of CL2 exhibited the highest targeting ability, while that of CL3 did not confirm our predictions. According to recent literature, protein abundance is not the only factor controlling protein cell interactions. In principle, Apolipoproteins adsorbed to CL3 could have functional motifs buried inside or not correctly presented at the liposome surface. Mapping protein binding sites on the liposome−BC is an urgent task for future research.62−65 In summary, combined MS/MS findings and cellular studies let us identify CL2 as the most promising formulation to deliver TMZ across the BBB. Finally, we evaluated the antitumor activity of TMZ in U-87 MG human glioblastoma cell line. Preliminary experiments showed that the free drug produced a time- and dose-dependent growth inhibition (Figure 7). According to previous findings by Kim et al.,17 the effect of TMZ was significant at 50−100 μM and the time of exposure was also critical for inhibiting cell proliferation. When U-87 cells were treated with TMZ-loaded CL2 at the highest TMZ concentration (100 μM), inhibition of cellular growth was slightly higher than that achieved with free TMZ (Figure 7). On the other side, BC boosted inhibitory effect of TMZ by factor ∼5 E


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by BC on the interaction between CLs and the machinery of U87 cells will be the object of future investigations. However, the improved cancer killing ability of TMZ-loaded CL−BC complexes, as those forming in vivo,46,66 compared to free TMZ and TMZ-loaded CLs lets us suggest that BC-based nanomedicine may hold great promise as a more effective therapy for GBM.

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CONCLUSIONS We have synthesized a set of four TMZ-encapsulating liposome formulations that recruited plasma proteins from HP. Most of identified proteins were previously categorized as BC fingerprints and promoted favorable NP−cancer cell association. We found that the CL formulation exhibiting the highest levels of targeting fingerprints, had also the major impact on HUVECs that are an established model of in vitro BBB.63−65 When administered to U-87 cells, designer formulation enhanced antitumor efficacy compared by a factor 5 compared to corona free CLs. Our results indicate that exploitation of BC could be a valuable means to develop targeted nanomedicine with superior ability to cross the BBB and enhanced anticancer efficacy. This study was performed on a simplified in vitro model of the BBB whose behavior not necessarily reflects the in vivo situation. Future work will lead to a fundamental understanding of transport mechanisms across more realistic BBB models, which is key for further improvements of treatments for brain diseases and other CNS related diseases. Although recent studies have demonstrated that predicting liposome−cell association by BC fingerprints provides valuable new insights, this approach requires the mapping of plasma proteins on the liposome surface. Deciphering the recognition between corona proteins and cell receptors63,64,67 could help us toward understanding exactly how BC-decorated lipid vesicles interact with cells and biological barriers, potentially activating different biological pathways. To this end, reproducible methods must be clearly established that comprise the recovery of liposomes after dynamic incubation68 and/or systemic administration in vivo,46,69,70 thorough characterization of the BC, and evaluation of corresponding biological interactions. We foresee that these future advances will yield new opportunities for accelerating the clinical translation of liposomal drugs for brain delivery.

Figure 6. Cell growth inhibition of HUVECs cells following 1-h incubation with CLs both in the absence (A) and presence (B) of biomolecular corona at three drug concentrations (5, 50, and 100 μM): CL1 (DOTAP/cholesterol), CL2 (DOTAP/DOPE), CL3 (DC-Chol/ DOPE), CL4 (DC-Chol/cholesterol). Statistical significance was evaluated with respect to control (i.e., not treated cells, CT): * P < 0.05, ** P < 0.001, *** not significant.

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METHODS

Preparation of CLs. DOTAP, DC-Chol, DOPC, and DOPE were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and used without further purification. Texas-Red DOPE was purchased from Thermo Fisher Scientific. CLs were prepared according to standard procedures. In brief, appropriate amounts of lipids were dissolved at neutral/total lipid (mol/mol) = 0.5. Lipid films were hydrated (final lipid concentration is 1 mg mL−1) with ultrapure water for size, zetapotential, laser scanning confocal microscopy (LSCM), and flow cytometry experiments. For proteomics experiments lipid films were hydrated with a dissolving 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. Preparation of TMZ-Loaded CLs. Incorporation of TMZ into cationic liposome was performed using the dehydratation-rehydratation method.1 Liposomal formulations were made combining 1 mg of TMZ with lipids in ratio 1:1 (molar ratio), this mixture was dissolved in 1 mL of chloroform and 0.2 mL of methanol and placed on a rotary evaporator set at 65 °C for 4 h to produce the film layer. The resulting film was rehydrated with 2.5 mL of PBS and then extruded 20 times through a 0.1 μm polycarbonate carbonate filter by the Avanti MiniExtruder (Avanti Polar Lipids, Alabaster, AL). Following 1-h exposure to HP, TMZ-loaded CL−BC complexes were formed. Fluorescently labeled CLs were prepared using DOPE-Texas red (Thermo Fisher

Figure 7. Enhanced efficacy of TMZ-loaded CL2−BC complexes (green diamonds) compared to free TMZ (gray circles) and TMZloaded CLs (green circles). Statistical significance was evaluated with respect to control (i.e., not treated cells, CT): * P < 0.05, ** P < 0.001.

with respect to free drug. This finding indicates that BC can trigger specific NP−cell interactions (e.g., cellular uptake, endosomal escape, lysosomal degradation and nuclear translocation). For example, Digiacomo et al.51 showed that liposome−BC promotes a switch in the uptake mechanism of multicomponent CLs from micropinocytosis to clathrindependent endocytosis. Accurate clarification of the role played F


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Cell Viability Assays. A portion of 1 ×104 cells was seeded in 96well plates and allowed to adhere for 12 h. The cultures were exposed to different concentrations of TMZ alone 5, 50, 100 μM or TMZ encapsulated within CLS for 24, 48 and 72 h, followed by MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma−Aldrich, assay). Briefly, 5 mg/mL MTT in 100 μL of DMEM without phenol red was added to the cultured cells for 2 h. Cells were washed by phosphate buffered saline (PBS) and lysed by 100 mL of DMSO. The concentrations of MTT were examined colorimetrically; absorbance was determined at 570 nm. All measurements were carried out by triplicate in three different replicates. The data results of the growth curve were normalized at logarithmic scale. The results were analyzed based on variance analysis (ANOVA).

Scientific). To evaluate TMZ encapsulation efficiency, free TMZ was separated from the TMZ encapsulated-liposomes using Vivavspin 500 (5 kDa MWCO, GE Healthcare), then the UV−vis absorption spectra of both free and encapsulated TMZ was measured by a Jasco V-630 spectrophotometer. Concentrations of free and encapsulated TMZ were evaluated by applying the Lambert−Beer law to the 330 nm absorption peak (Figure S1). Absolute amounts of free and encapsulated TMZ were determined by relating the measured concentrations to the sample volumes. Finally, EE and DLC were measured as

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

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(1)

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

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.8b00339.

Size and Zeta-Potential Experiments. All size and zeta-potential measurements were made on a Zetasizer Nano ZS90 (Malvern, U.K.) at 25 °C. CLs, TMZ-loaded CLs, and TMZ-loaded CL−BC complexes were diluted 1:100 with distilled water and size and zeta-potential results are given as mean ± standard deviation of five replicates. Proteomics Experiments. For proteomics experiments lipid films were hydrated with a dissolving 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. The obtained solutions were extruded 20 times through a 0.1 μm polycarbonate carbonate filter with the Avanti Mini-Extruder (Avanti Polar Lipids, Alabaster, AL). CLs were incubated with HP (1:1 v/v) and then incubated at 37 °C for 60 min. This volume ratio was chosen because it is mimetic of in vivo conditions. After incubation, the samples were centrifuged 15 min at 14 000 rpm followed by pellet resuspension; this procedure was repeated three times to wash the sample and remove loosely bound proteins. NanoLC−MS/MS analysis, data analysis, and protein validation were performed as reported elsewhere.48 Cell Cultures. Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Allendale, NJ, USA) and were maintained in EGM-2 BulletKit medium (Lonza). Cells were used until passage number 4. After mechanical dissociation, U-87 MG (ATCC) single cells were resuspended in F10 medium and centrifuged at 1000 g for 5 min. The pellet was resuspended in the F10 growth medium supplemented with 10% fetal calf serum (FCS, Life Technologies Ltd., Milano, Italy) and cells were plated in Petri plates (Falcon Primaria, Lincoln Park, NJ, USA). The medium was then changed every 3 days. After 14−15 days, cells were trypsinized, replated into 24-well plates at a density of 25 × 103 cells/well and shifted into D-MEM Glutamax without serum (Life Technologies Ltd., Milano, Italy). USA). The medium was then changed every 3 days. After 14−15 days, cells were trypsinized, replated into 24-well plates at a density of 25 × 103 cells/well, and shifted into D-MEM Glutamax without serum (Life Technologies Ltd., Milano, Italy). FACS. HUVECs cells were seeded in 12-well plates (150 000 cells/ well) using complete medium. After 24 h, cells were treated for 3 h with fluorescently labeled TMZ-loaded CLs in Optimem medium. Then cells were washed two times with cold PBS, detached with trypsin/ EDTA, and acquired using a cytometer. Fluorescence-activated cell sorting (FACS) analysis was performed using BD LSR Fortessa (BD Bioscience). TMZ Treatment of U-87 MG Cell Lines. In vitro response to treatment with TMZ was evaluated under three conditions: (i) free TMZ; (ii) TMZ encapsulated in CLS before incubation in HP (i.e., in the absence of BC, in the text defined “TMZ-loaded CLs”); (iii) TMZ encapsulated in CLS after incubation in HP (i.e., in the presence of BC, in the text defined “TMZ-loaded CL−BC complexes”). To this end, we plated U-87 MG cells in 48-well plates at 1 × 104 cells per well in DMEM supplemented with 10% FBS and incubated them at 37 °C in an atmosphere containing 5% CO2. The following day the cells were treated for 24, 48, and 72 h at the following concentrations: 5, 50, and 100 μM. After treatment, cell count was made using a Bürker chamber; before the count, the cells were colored with trypan blue dye (Sigma St. Louis, MO, USA) to discriminate live cells from dead.

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Figure S1. Absorption spectra of TMZ as a function of drug concentration. Tables S1−S4. Full list of corona proteins identified onto the surface of CL1-CL4 liposomes following 1 h incubation with human plasma. Table S5. Top 20 most abundant proteins identified in protein corona of CL1-CL4 liposomes following 1 h incubation with human plasma (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Luca Digiacomo: 0000-0003-3362-7160 Anna Laura Capriotti: 0000-0003-1017-9625 Morteza Mahmoudi: 0000-0002-2575-9684 Giulio Caracciolo: 0000-0002-8636-4475 Author Contributions

A.A. performed cell viability experiments, conceived the study, discussed data, supervised research, and reviewed the manuscript; S.P. performed FACS and cell viability experiments, analyzed data, prepared figures, and reviewed the manuscript; L.D. performed DLS and UV−vis absorption experiments, analyzed data, prepared figures, and reviewed the manuscript; D.P. conceived the study, discussed data, and wrote the manuscript; A.L.C. conceived nanoLC MS/MS experiments, analyzed nanoLC MS/MS data, and reviewed the manuscript; L.F. conceived the study, discussed data, supervised research, and reviewed the manuscript; M.A.O. performed cell viability experiments, discussed data, and reviewed the manuscript; G.T. discussed the data and reviewed the manuscript; R.R. provided HUVECS cells, discussed the data, and reviewed the manuscript; I.S. conceived the study, discussed data, and reviewed the manuscript; M.M. conceived the study, discussed data, and wrote the manuscript; G.C. conceived the study, discussed data, supervised research, and wrote the manuscript. Author Contributions #

A.A. and S.P. contributed equally.

Notes

The authors declare no competing financial interest. G


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ACKNOWLEDGMENTS GC, DP, and LD gratefully acknowledge support by Sapienza University of Rome (Projects H2020; protocol number PH11715C7916B7A6).

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