Fate of PLA and PCL-Based Polymeric ... - ACS Publications

Jan 20, 2016 - Animal Models of Triple-Negative Breast Cancer ... (2) PLA8 NPs were not internalized in the human TNBC cell line (MDA-MB-231); (3) PCL...
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Fate of PLA and PCL-Based Polymeric Nanocarriers in Cellular and Animal Models of Triple-Negative Breast Cancer Leopoldo Sitia,† Raffaele Ferrari,‡ Martina B. Violatto,† Laura Talamini,† Luca Dragoni,§ Claudio Colombo,‡ Laura Colombo,† Monica Lupi,† Paolo Ubezio,† Maurizio D’Incalci,† Massimo Morbidelli,‡ Mario Salmona,† Davide Moscatelli,§ and Paolo Bigini*,† †

IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, 20156 Milano, Italia Institute for Chemical and Bioengineering, ETH Zurich, CH-8093 Zurich, Switzerland § Dipartimento di Chimica Materiali e Ingegneria Chimica “G. Natta”, Politecnico di Milano, 20133 Milano, Italia ‡

S Supporting Information *

ABSTRACT: An integrated platform to assess the interaction between nanocarriers and biological matrices has been developed by our group using poly methyl-methacrylate nanoparticles. In this study, we exploited this platform to evaluate the behavior of two biodegradable formulations, polyε-caprolactone (PCL3) and poly lactic-acid (PLA8), respectively, in cellular and animal models of triple-negative breast cancer (TNBC). Both NPs shared the main physicochemical parameters (size, shape, ζ-potential) and exclusively differentiated on the material on which they are composed. Our results showed that (1) PLA8 NPs, systemically injected in mice, underwent rapid degradation without penetration into tumors; (2) PLA8 NPs were not internalized in the human TNBC cell line (MDA-MB-231); (3) PCL3 NPs had a longer bioavailability, reached the tumor parenchyma, and efficiently penetrated in MDA-MB-231 cells. Our data highlight the relevance of the material selection to both improve bioavailability and target tropism, and make PCL3 NPs an interesting tool for the development of nanodrugs against TNBC.

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the biological matrices (e.g., biological fluids, cells, organs).13 In this context, it is widely known that size, shape, and surface charge may greatly modify the biodistribution of nanocarriers, their interaction with biological barriers, 14 and cellular internalization.15 The standardization of physicochemical features of NPs is required to determine their correlation with biological targets in many therapeutic fields. In oncology, one of the main goals of nanotherapy is both to improve the bioavalilability in tumor mass and to penetrate in cancer cells for a targeted release. The ability to penetrate cells and release therapeutic compounds in the cytoplasm becomes even more relevant for cancer cells lacking efficient systems of particular receptors on the cell surface. Triple-negative breast cancer (TNBC) belongs to this class of cells. This is characterized by concomitant absence of the three main receptors (estrogen, progesterone, and HER2) commonly expressed in many breast tumors. The poor prognosis and a lack of efficient pharmacological treatments need to be addressed by alternative strategies, such as the development of efficient nanodrugs. In this context, the direct delivery to intracellular region of therapeutic compounds could

INTRODUCTION Over the last decades, an increasing number of nanometric systems for biomedical uses has been proposed.1 The latest advancements in material science and nanotechnology were applied in pharmacology to produce emulsions, nanocrystals, liposomes, viral vectors, to be tested as drug-delivery agents in several fields, including cancer therapy.2−5 In this context, polymeric nanoparticles (NPs) have been exploited for their stability, handiness, tunable properties, and biocompatibility.6−8 These features confer to polymeric NPs many advantages such as (i) the loading of high quantities of hydrophobic agents; (ii) the high variability in terms of size modulation and surface functionalization (to improve the tropism to a target organ and/or increase the half-life in the bloodstream); (iii) the possibility to reduce the systemic toxicity and to optimize the drug release through the modulation of NP biodegradability. The use of polymeric NPs for both diagnostics and therapeutic purposes has been widely described.9−11 However, in spite of a large evidence of preclinical studies, their clinical exploitation is still very limited. Nowadays, only few formulations based on polymeric NPs are commercially available, and just a few more are under clinical investigation.12 The translational path needs to be a strict series of checkpoints that should start from the understanding of how the different parameters of neosynthesized nanovectors may influence their interaction with © XXXX American Chemical Society

Received: October 22, 2015 Revised: January 18, 2016

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Biomacromolecules represent a novel approach,16 and some recent studies associated with the use of NPs have shown promising results.17,18 On the basis of our recent experience, where an integrated system of in vivo and ex vivo analyses was optimized using non degradable poly(methyl methacrylate) nanoparticles (PMMA NPs) to understand the behavior of polymeric NPs in cells and bearing-cancer mice,19 we evaluated the biodistribution and the target penetration at both tissue and cellular level. The process of synthesis was aimed to produce NPs similar in size, shape, external surface, amount of fluorescent dye, and process of polymerization but differing on the polymeric compound. Starting from our recent analyses, we selected a polymer made of a poly hydroxyethyl methacrylate (HEMA) backbone grafted with a well-controlled number of polyester units; 3 and 8 for poly(ε-caprolactone) (PCL3) and poly(lactic acid) (PLA)8-based NPs, respectively.19,20 Both the materials and the length of the grafted chains led to different degradation time of the NPs. In detail, 2 and 5 days for PLA8 and PCL3 NPs, respectively, are required to reach the full degradation in cell medium, a relevant fluid in which is it possible to mimic the NP behavior in vivo.21 We carefully selected those two materials for their well-known biocompatibility and usage in biological applications,22 while we set up these formulation in order to ensure a complete degradation and to limit issues as polymer accumulation into cells. As exhaustively described by our group,9,19 the fluorescent dye Rhodamine-B (RhB) was linked to the polymeric matrix of NPs through a covalent bond in order to allow their traceability in vitro and in vivo. The process of RhB bounding before the polymerization of the material represents our warranty to avoid the risk of biological dye elution that is one of the main problems occurring in studies of nanotracking when the tracers is physically adsorbed to the shell of the NP. Before comparing the behavior of the two different materials in tissues and cells, an exhaustive evaluation of their stability in biological fluids was performed. Finally, all the results obtained at the different steps of biological complexity were integrated to define an overall comparison of the potential and drawbacks of each material. The purpose of the work is therefore 2-fold: to understand how the level of biodegradability can influence the fate of NPs and, at the same time, to provide a method easily reproducible with other materials and other therapeutic targets.

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Nanoparticle Synthesis and Characterization. RhB was linked to the HEMA molecule through an esterification performed in the presence of DCC as transesterification agent and DMAP as catalyst as already published by our group and described in detail in the Supporting Information (SI) section.9 Through this procedure, a fluorescent macromonomer (HEMA-RhB) has been obtained and copolymerized with the different monomers in order to produce nonbiodegradable and biodegradable NPs. Macromonomers based of ε-caprolactone and lactide were synthesized through ROP using a procedure reported in the literature.23,24 Briefly, the reaction was carried out in bulk conditions, without solvent. Monomer (caprolactone or actide) was first heated up in a stirred flask at 130 ± 1 °C with temperature controlled by an external oil bath. A mixture of Sn(Oct)2 and HEMA at a given molar ratio (1/200) was prepared and stirred until complete dissolution of Sn(Oct)2. HEMA solution was then added at the desired molar ratio with respect to the monomer to initiate the reaction which was carried out for 2 h. Final macromonomers with 3 CL units (HEMA-CL3) and 8 lactoyl repeating units (HEMA-LA8) have been produced and used for further emulsion polymerization step. Detailed macromonomer characterization is reported in the SI section. Polymeric NPs were produced via emulsion copolymerization of the proper monomer (MMA, HEMA-CL3, or HEMA-LA8) and a small amount of HEMA-RhB (0.01% w/w in respect to the monomer) as described elsewhere.25 Monomer-starved semibatch emulsion polymerization of the produced macromonomers was carried out in a 50 mL three-necked glass flask. This procedure consists in loading initially all of the hydrophilic macromonomer (HEMA-Ch, 15% w/w and HEMA-PEG, 11% w/w) with deionized water (45 mL) in the reactor as in a normal batch polymerization. After purging and the addition of the initiator (dissolved in 2.5 mL of deionized water), the more hydrophobic monomer (MMA, HEMA-CL3, or HEMA-LA8) was fed into the reactor with a rate of 2 mL/h using a syringe pump (Model NE-300, New Era Pump System, U.S.A.); the reaction was run at 80 °C for 2 h. The final solid content is 2.5 g (5% w/w). CTMAB was removed after synthesis through dialysis (Slide-ALyzer Dialysis Cassette 3,5 KD 0,5−3 mL, Thermo Scientific) against water for 24 h. NPs have been characterized in terms of average diameter and polydispersitiy index (PDI) by dynamic light scattering (DLS, Malvern, Zeta nano ZS) using the cumulant method as defined by ISO (standard document 13321:1996E) and in terms of fluorescence properties with a spectrofluorimeter (Infinite 200 PRO series, Tecan Trading AG, Switzerland). All the reported data are the average of three independent measurements of the same sample. The ζ-potential was also evaluated through DLS measurements. The detailed synthesis and characterization are reported in the SI section. Measurement of Degradation in Biological Fluids. In order to characterize the interaction of NPs with proteins and the influence on NP colloidal stability, PMMA, PCL3 and PLA8 NPs have been incubated for 72 h at 37 °C with distilled water and/or human serum (10% of human serum in distilled water). Atomic force microscopy (AFM) experiments were carried out on a Multimode AFM with a Nanoscope V system operating in tapping mode using standard phosphorus-doped silicon probes (thickness range, 3.5−4.5 μm; length, 115−135 μm; width, 30−40 μm; spring constant, 20−80 N/ m, Veeco/Digital Instruments) with a scan rate in the 0.5−1.2 Hz range, proportional to the area scanned. Freshly cleaved muscovite mica discs (Veeco/Digital Instruments) were used for deposition of the two different NP-serum solutions that were added to freshly cleaved mica at room temperature. After 1 min, the samples were washed and dried under gentle nitrogen flow. AFM images of NP distribution were analyzed for diameter and height with the Scanning probe image processor (SPIP) Version 5.1.6 data analysis package to describe NP structures. To exclude the interference of possible artifacts, extra control samples, such as freshly cleaved mica and freshly cleaved mica soaked with ultrapure water or serum alone, were also used. All the topographic patterns and SPIP characterization described in the text were confirmed by additional measurements in a minimum of 10 different, well-separated areas.

EXPERIMENTAL SECTION

Materials. Rhodamine B (RhB, Sb sensitivity 99% purity, SigmaAldrich), 4-(dimethylamino)-pyridine (DMAP; Sigma-Aldrich), acetonitrile (99% purity, Sigma-Aldrich), trifluoroacetic acid (SigmaAldrich), methyl methacrylate (MMA, 99% purity, Sigma-Aldrich), azobis(2-methylpropionamidine) dihydrochloride (>99% purity, Fluka), ε-caprolactone (CL, 99%, Sigma-Aldrich), L,L-lactide (LT, PURAC, 99% purity), 2-ethylhexanoic acid tin(II) salt (Sn(Oct)2,∼95%), poly(ethylene glycol) methyl ether methacrylate (HEMA-PEG9, Molecular weight: ca. 475 Da, Sigma-Aldrich), ethanol (EtOH, ≥99.7%, Sigma-Aldrich), cetyltrimetylamonium bromide (CTMAB, Sigma-Aldrich, >99% purity), and [2-(methacryloyloxy)ethyl] trimethylammonium chloride, 80% weight, solution in water (HEMA-Ch, Sigma-Aldrich) were all used without further treatment. Dulbecco’s Modified Eagle’s Medium-high glucose, L-glutamine, fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were purchased from Biowest. Hoechst 33258 (Hoechst 33258, Pentahydrate (bis-Benzimide), 10 mg/mL solution in water, Molecular ProbesTM) and Fluormount (fluormount mounting medium, 25 mL, Bio-Optica) were used. B

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(antirabbit α-tubulin, cell signaling 1:100), the early endosomes (EEA1), and the mature lysosomes (LAMP 2), as previously described by Esposito et al.26 Cells were visualized 6 or 24 h after NP incubation, respectively. Animals. Female athymic Foxn1 nu/nu mice, 6−8 weeks old, were obtained from Harlan Laboratories (Bresso, Italy). Mice were maintained under specific pathogen-free condition in the Institute’s Animal Care Facilities and regularly checked by a certified veterinarian who is responsible for health monitoring, animal welfare supervision, and experimental protocol revision. Procedures involving animals and their care were conducted in conformity with the institutional guidelines at the IRCCS - Institute for Pharmacological Research “Mario Negri” in compliance with national (Decreto Legge nr 116/92, Gazzetta Ufficiale, supplement 40, February 18, 1992; Circolare nr 8, Gazzetta Ufficiale, July 14, 1994) and international laws and policies (EEC Council Directive 86/609, OJL 358, 1, Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, eighth edition, 2011). This project of research has been reviewed by IRCCS-IRFMN Animal Care and Use Committee (IACUC) and approved by the Italian “Istituto Superiore di Sanità” (code: 17/01 D Appl 3). MDA-MB-231 cells (1 × 107) were subcutaneously injected in the right flank of the mice. The growing tumor masses were measured with the aid of a Vernier caliper, and the tumor weights were calculated (weight = 1/2 length × width)2. When tumor load reached a weight of about 300−500 mg, mice were randomized in the experimental groups. Biodistribution of PLA8 and PCL3 NPs was studied in tumor-bearing mice intravenously treated with 200 μL of NPs (1 × 1013 NPs/mL). Three animals were treated with a physiological solution and kept as control. To study NP biodistribution, histological studies were performed as previously described.19 Then at 1, 4, 24, and 120 h after administration, animals were deeply anesthetized with an overdose of ketamine (75 mg/kg) metodimine (1 mg/kg) before sacrifice (n = 5 per experimental group). Liver, kidneys, spleen, and tumor were removed, immediately frozen in liquid nitrogen, and stored at −80 °C until use. Serial sections (30 μm) of each organ were collected and stained with Hoechst 33258 (2 μg/mL in PBS, 40 min, RT) for nuclei visualization. The sections were imaged with Nanozoomer HT 2.0 (Hamamatsu Photonics, Hamamatsu City, Japan) instrument to obtain rapid fluorescent scans of the entire surface of organ sections, with high anatomical resolution. For a limited number of liver and tumor sections, immunohistochemistry of the endothelial walls and two markers of macrophages were performed. Serial sections were collected and kept under vacuum in a cold dark room overnight. In all the conditions, a blocking solution (PBS 0.1 M pH 7.4 + FBS 5% + Triton X-100 0.1%, 30′) was added before washing with PBS (3 × 5 min). Primary antibodies were employed as follows: Endothelial vessels: rat monoclonal antibody anti-CD 31 (1:400) + FBS 10% in PBS 0.1 M pH 7.4 without Ca2+/ Mg2+, 2 h RT, O/N at 4 °C; Resident macrophages: rat α-CD 68 (1:200) + Triton 0.1% + NGS 3% in PBS 0.1 M pH 7.4 without Ca2+/Mg2+, 2 h RT, O/N at 4 °C; Activated macrophages: rat α-CD 11b (1:800) + Triton 0.1% + NGS 1% in PBS 0.1 M pH 7.4 without Ca2+/Mg2+, 2 h RT, O/N at 4 °C. After primary antibody staining, sections were washed with PBS (3 × 5 min) and antirat Alexa488 IIa antibody was used (diluted 1:1000) and incubated for 60 min at RT. At the end of secondary antibodies incubation, sections were washed with PBS (3 × 5 min), and nuclei were stained with Hoechst 33258. Statistical Analysis. All data were expressed as mean ± SD. Student’s t tests were used, and p values ≤0.05 were considered significant. All statistical analyses were done using the Graph Pad Prism version 6.00 for Windows (Graph-Pad Software, San Diego, CA, U.S.A.).

Cellular Internalization. For in vitro studies, MDA-MB-231 cells were cultured in DMEM-high glucose containing 10% FBS and 1% Lglutamine and maintained at 37 °C in a humidified atmosphere at 5% CO2 in T25 or T75 cm2 flasks (Iwaky Bibby Sterilin, Staffordshire, U.K.). For the evaluation of NP cellular internalization, MDA-MB-231 cells were seeded on round glass slides in 24-well Multiwell plates (Iwaky Bibby Sterilin) at concentration of 10 000 cells/mL and left in adhesion for 24 h. Culture medium was then removed and cells were incubated with PMMA, PCL3 and PLA8 NP solutions at equivalent concentration of 2.5 × 1010 NPs/mL (#NPs/v). The internalization study was performed at three different time points of incubation with NPs 6, 24, and 72 h, respectively. At the end of incubation, cells were washed three times in PBS, fixed with 4% paraformaldehyde in PBS for 40 min, and nuclei were stained with Hoechst 33258 (2 μg/mL in PBS, incubation for 40 min). Glass slides were mounted on cover glass slides with 2−3 drops of Fluormont and stored at 4 °C until acquisition with confocal microscopy, as described in the following sections. Three replicated samples for each condition have been analyzed. For colocalization experiments, cells were cultured as described for internalization assay, and NPs were incubated for 24 h at the concentration of 2.5 × 1010 NPs/mL. At the end of incubation, cells were washed three times in PBS, fixed with 4% paraformaldehyde in PBS for 40 min. A blocking solution (PBS-FBS 5%) was added before washing again with PBS (3 times, 5′). For subcellular localization studies, the antibody anti-GM130 (specific for the Golgi Apparatus) and two different antibodies directed against early or late lysosomes (anti-EAA1 and anti-LAMP2 antibodies respectively) were employed as follows: Golgi Apparatus: Primary monoclonal mouse antibody antiGM130 (1:400) (BD Transduction Laboratories) + Triton X-100 0.05% + FBS 10% in PBS 0.1 M pH 7.4 without Ca2+/Mg2+, O/N at 4 °C. Early Lysosomes: Primary EEA1 (DB bioscience) (1:400) + Triton X-100 0.05% + FBS 10% in PBS 0.1 M pH 7.4 without Ca2+/ Mg2+, O/N at 4 °C; Mature Lysosomes: Polyclonal rat antibody (GeneTex, Inc.) anti-LAMP2 (1:400) + Triton X-100 0.05% + FBS 10% in PBS 0.1 M pH 7.4 without Ca2+/Mg2+, O/N at 4 °C. Secondary FITC conjugated antibody (Invitrogen) (1:1000) was incubated for 1 h at RT in a PBS-FBS 0.1% solution. At the end of incubation, wells were washed with PBS (3 times, 5′), and nuclei were stained with Hoechst 33258 (2 μg/mL in PBS, incubation for 40 min). Glass slides were taken from the wells, mounted on cover glass slides with 2−3 drops of Fluormont, and stored at 4 °C until acquisition. Confocal Acquisition and Fluorescence Quantification. All samples were then analyzed with an Olympus Fluoview microscope BX61 with confocal system FV500, equipped with specific lasers λexc = 405 nm, λexc = 488 nm and λexc = 546 nm to visualize Hoechst 33258, FITC (only for subcellular localization studies) and RhB, respectively. Images were pseudocolored (blue for Hoechst 33258, green for FITC and AF 488, red for RhB) and the signal obtained from the three channels was automatically merged by Olympus Fluoview software. 3D reconstruction was performed using Imaris 5.0 (Bitplane) software. Semiquantitative measurements were carried out by setting the laser potency at a threshold value as low as to maximally reduce the tissue autofluorescence. Once the parameters of acquisition were established, all fields of view (9 for each experimental group) were acquired by keeping them constant in order to standardize the quantification. The images were then processed by the cell segmentation software Tissue Quest (TissueGnosticsGmbH, Austria) as previously described9 and widely reported (seeSI). Briefly, the following parameters were investigated: (1) distribution of NPs (average and total surface of the events associated with red NP signal); (2) intensity of the signal associated with RhB and NPs (intensity/pixels); (3) the spread and the intensity of immunofluorescent markers (EEA1, LAMP2 and GM130). Using the nuclei counting function provided by the software, the mean number of cells (Hoechst 33258 positive spots) for each experimental group was calculated. This enabled us to calculate area and intensity of RhB staining per single cell. To better define the interaction between PCL3 NPs and subcellular organelles, Super Resolution Microscopy (N-SIM Nikon) experiments were carried out using antibodies directed against the cytoskeleton

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RESULTS AND DISCUSSION Synthesis and Characterization: The Influence of the Material on Biological Fluids Stability. The first requirement to compare data obtained from NPs made of different C

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unveiled the formation of aggregates even after 72 h of incubation in distilled water (Figure 1 C, green circle). The high degree of degradability of this material was further highlighted after incubation in 10% serum (Figure 1C’), in which they completely lost their monodispersity and were degraded after incubation in protein-rich fluids (green circle). The data obtained with AFM confirm that serum has a strong influence on NP stability and degradation time. As expected, this is even more evident as NP biodegradability increases. It is widely known that more degradable particles have a different behavior depending upon the fluid in which they interact. They can modify their distribution, localization, and metabolism and therefore affect their potential in vivo efficacy.28,29 As previously reported for PMMA NPs,9 fluorimetric analysis of PCL3 and PLA8 NPs revealed that the conjugation of RhB with each kind of monomer did not interfere with the amplitude of fluorescence or the peaks of emission−excitation did not vary with respect to free RhB (Figure S5). This is an important characterization to follow the fate of NPs in both cells and tissues. In Vivo Studies: The Influence of the Material on NP Tumor Penetration. In recent years, a growing number of studies has focused the attention on developing carriers to internalize therapeutic compounds directly inside cancer cells.30 Nanopharmacology plays a pivotal role in this scenario, and the selection of the best material(s) of nanocarriers may greatly influence their efficacy. This investigation requires a tight integration from in vitro and in vivo results, and this is the most direct way to establish the actual potential of a candidate nanocarrier for translational purposes.31 Parameters such as the stability in the bloodstream, the avidity of filter organs, and the ability to pass through biological barriers should be carefully elucidated before studying the efficiency of loading and/or release for each kind of nanodrug. It is in fact widely recognized that the main NP properties (e.g., size, shape, and surface charge) that influence their overall behavior in terms of organ targeting, bloodstream half-life, and cellular internalization are their physico-chemial properties and surface properties (e.g., size, shape, surface charge, surface functionalization, and aspect ratio).27,32 Other important properties that regulate NP biological fate both in vitro and in vivo are more correlated with the intrinsic properties of the NP core material, such as rigidity,33,34 flexibility,35 elasticity,36 and biodegradability.37 Due to the complexity of this scenario, understanding the behavior of polymeric NPs after systemic administration in mouse models of human disorders is a fundamental starting point to select the most promising nanomaterials for theranostics. To this aim, our study was directly carried out on an athymic mouse bearing a human TNBC. In recent years, some perplexities have emerged on the use of immunodeficient mice bearing human tumors due to the increased half-life in the bloodstream and the increased EPR effect.38 Conversely, there is an emerging body of evidence arguing that the mechanisms of cellular uptake, tumor growth, and response to anticancer drug in murine cancer models can be misleading and somehow confounding to design a therapy for humans. Both approaches have points of strength and weakness. The decision to transplant human TNBC in immunodepressed mice merely stems from our intention to integrate the in vivo analysis with a careful investigation at the single-cell level (for this reason, it seemed more appropriate to focus our attention on human cells).

materials is to guarantee the quality of their synthesis through the evaluation of their main physicochemical parameters. Through DLS, we measured size, polydispersity index (PDI), and surface charge of the three types of NPs used in the present study. Data for PMMA, PCL3, and PLA8 NPs are listed in Table 1, which confirms the quality of the three products. Table 1. Physico-Chemical Properties of Biodegradable PCL3 and PLA8 NPs and Nondegradable PMMA NPs Obtained with DLS sample

size [nm]

PDI [−]

ζ potential [mV]

PMMA NPs PCL3 NPs PLA8 NPs

32.5 36.6 38.8

0.102 0.096 0.152

+54.4 +51.5 +47.2

All the NPs have similar average size (Figure S4), narrow size distribution with low PDI values, and similar values of positive ζ-potential. Consequently, we can compare the three different formulations in terms of interactions with biological matrices, excluding any influence of size. The low PDI indicates the presence of homogeneously distributed particles, without the presence of large aggregates in the system, whereas the positive ζ-potential is another important parameter that confers high colloidal stability to the suspension. All of these features influence the potential of biodegradable polymeric NPs as drug delivery agents. In fact, it is widely known that the early interactions of administered particles with biological fluids strongly influence NP biodistribution and the ability to pass biological barriers and arrive at their target.27 To fully assess NP colloidal stability, a separate analysis of NP dispersion in deionized water and in biological fluids was conducted by AFM analysis (Figure 1). No NP aggregation was

Figure 1. Representative AFM analysis of the distribution of PMMA (A), PCL3 (B), and PLA8 (C) NPs after 72 h of incubation in distilled water and in human serum.

previously observed when incubating PMMA NPs in water for at least 72 h.21 No alteration in terms of stability and monodispersity was observed with AFM for PMMA NPs 72 h after either water (Figure 1A) or 10% serum (Figure 1A’) incubation. Similarly to PMMA, PCL3 NPs showed a high stability in water (Figure 1 B), whereas they seem to slightly aggregate during the incubation in 10% of serum (Figure 1B’). In the panel, the presence of still monodisperse NPs and of clusters of condensed NPs enclosed in the yellow rings demonstrate a progressive loss of colloidal integrity and the aggregation of the material. Very interestingly, PLA8 NPs D

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Figure 2. Biodistribution of PCL3 and PLA8 NPs observed with fluorescence microscopy. Histological sections of liver at different magnification levels are reported. The image of PBS-treated control mouse (lower left section) is compared with images of NP-treated animals sacrified at 1, 4, 24, and 120 h after intravenous injection. NPs are visualized in red (RhB signal) and cells in blue (Hoechst 33258 staining). In the lower right panel, histograms showing the percentage area occupied by PCL3 and PLA8 NPs in liver, spleen, and kidney are reported at different time points after NP administration. All data are represented as mean ± SD, and statistical analysis was performed by Student’s t test. Degree (significant increase of PCL3 NP signal compared to PLA8 NPs; asterisk (significant increase of PLA8 NP signal compared to the PCL3 NPs).

disappearance from the bloodstream.20 In the parenchyma, heterogeneous red staining can be observed. This pattern of distribution strongly suggests a selective NP internalization in specific cells. 24 h after administration (first row, third column) no PCL3 NPs were found in the bloodstream (yellow arrows), while the parenchyma is rich of NPs. The image at higher magnification (second row, third column) further confirmed this indication. We also found a dramatic decrease of signal in vessels and endothelial cells. After 120 h from administration (first row, fourth column), PCL3 NPs almost completely disappeared from liver sections. This reduction is even more appreciable in the image at a higher magnification (second row, fourth column). The kinetics of distribution, accumulation, and elimination of PCL3 NPs in the liver is not surprising. There is an extensive literature that reports an effective and selective tropism of many nanomaterials at the hepatic level.39 The simultaneous observation of parenchyma and vessels allowed us to evaluate relevant pharmacokinetics steps, such as bloodstream permanence, biological barriers passage, and tissue accumulation. It is likely that PCL3 NP accumulation was related to their uptake by resident macrophage (Kupfer cells). Immunohistochemical images with a specific marker of liver macrophages (CD 68, green) show a high colocalization with NPs (Figure S9). These

Representative liver sections from TNBC mice treated with PCL3 or PLA8 NPs are reported in Figure 2. The immediate immersion in liquid nitrogen after tissue withdrawal enables us to “freeze” NPs in both vessels and parenchyma. In PCL3 NPtreated mice, a progressive increase of red fluorescence associated with NPs has been observed in the liver parenchyma (blue) up to 24 h, followed by a drastic reduction of the signal at the last observation time (120 h). A more accurate analysis of the liver parenchyma shows a progressive change in the localization of the signal at different times. One hour after injection (first row, first column), PCL3 NPs were mainly concentrated in specific areas (see yellow arrows). The image at higher magnification (second line, first column) showed that PCL3 NPs were mainly associated with vessels. This signal did not overlap the endothelial surfaces of the vessel. Four hours after administration, a stronger signal related to PCL3 NPs was found in the parenchyma, while a marked reduction was observed inside vascular structures (yellow arrows). Such behavior suggests a migration from the bloodstream to the liver parenchyma. The image at higher magnification (second row, second column) shows the presence of NPs on the border of the vessels (linked with the endothelium). Immunohistochemical analysis with a specific marker for endothelium, CD 31, (Figure S8) allowed us to confirm the reliability of this study. No NPs were found inside the vessels, confirming their E

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Biomacromolecules observational data confirm the hypothesis of a hepatic clearance mediated by an immunocompetent cell.27 The bottom panel (third and fourth rows) shows representative images of liver sections of TNBC mice treated with the same doses of PLA8 NPs sacrificed at the same times after administration. One hour after treatment, the signal associated with PLA8 NPs appears to be relatively low at the parenchyma level and, very interestingly, also in correspondence to the vessels (green arrows, third row, first column). The image at higher magnification confirms this global observation. It is interesting to note that the signal associated with PLA8 NPs is concentrated in the tissue and absent in the vessels; this distribution considerably differs from PCL3 NPs. These data seem to suggest a decisive influence on the stability of the material in circulation, starting from the first hours after systemic administration. Four hours after administration, PLA8 NPs were still observed in the liver parenchyma (third row, second column), and no NPs were found in vessels (green arrows). The image at higher magnification (fourth row, second column) shows a marked presence of PLA8 NPs within the parenchyma and around the vessels. No difference in PLA8 NPs distribution was observed between 4 and 24 h after administration. Similarly to PCL3 NPs, the liver of TNBC mice treated with PLA8 NPs shows a strong clearance 120 h after administration. Quite interestingly, a weak but detectable staining associated with NPs was observed around blood vessels at this time point for PLA8 NPs exclusively (green arrows, fourth row, fourth column). The lower right panel in Figure 2 shows histograms related to the quantification of NP accumulation in liver (left), spleen (middle), and kidney sections (right). As expected from observational data, the percentage of the surface associated with the signal of PCL3 NPs (light gray bars) in the liver is significantly higher compared to PLA8 NPs (dark gray bars). This significant difference was also found analyzing spleen sections. In contrast, in the kidneys, PLA8 NPs signal was significantly higher than PCL3 NPs: a possible explanation for this behavior may be related to different degradability of the two materials. It is in fact known that smaller NPs preferentially undergo to a renal accumulation and excretion.40 It is worthwhile to notice that the final NP degradation product is a poly HEMA chain (with RhB attached) which, being hydrosoluble, can be mainly excreted through the kidneys. This observation may be considered as a further proof of the PLA8 degradation. In addition, the AFM analysis (Figure 1C) clearly showed that the PLA8 NPs undergo to a marked fragmentation 72 h after incubation in serum. The evaluation of the efficacy of uptake and release from filter organs is a crucial point to define the potential risks in terms of NP bioaccumulation in TNBC models. However, a detailed analysis of the interaction between NPs and target organs is necessary to determine their therapeutic potential. Representative images regarding distribution of PCL3 NPs and PLA8 NPs in the tumor parenchyma are shown in Figure 3A,B, 1 h (left) and 24 h (right) after NP administration. The presence of signal associated with PCL3 NPs 1 h after treatment is limited to the outer surfaces, the lamina of the tumor (higher magnification image, bottom right), and the vascular structures (bottom left). At 24 h, PCL3 NPs have left the vessel (bottom left) and invaded the tumor parenchyma (bottom right). Quantitative data confirm this morphological observation (Figure 3C). A progressive increase of the signal associated with PCL3 NPs (light gray bars) was observed in the first 24 h

Figure 3. Biodistribution of PCL3 (A) and PLA8 (B) NPs observed in tumor sections 1 h (left column) and 24 h (right column) after NP administration. C: histogram showing the percentage area occupied by PCL3 and PLA8 NPs in tumor is reported at different time points after NP administration. All data are represented as mean ± SD, and statistical analysis was performed by Student’s t test. Degree (significant increase of PCL3 NP signal compared to PLA8 NPs).

after NP administration. The integration between quantitative and histological analyses clearly suggests that these NPs are able to efficiently cross the biological barrier of the tumor and migrate into the parenchyma. The AFM image (Figure 1B) shows that 24 h after serum incubation, PCL3 NPs are still monodispersed and maintained their original shape. In addition, we found that the stability was greatly reduced from 24 to 72 h. This profile of degradation perfectly fits the kinetics of tumor accumulation. This overall result makes this material extremely interesting to release the cargo in this temporal window. A strong segregation of NPs in CD 68 positive cells was observed in the liver (Figure S7). The presence of infiltration by immune cells and murine macrophage in tumor may somehow limit the interaction between PCL3 NPs and cancer cells, potentially reducing the effect of targeted delivery. However, the lack of colocalization between PCL3 NPs (red) and CD 11b (green) seems to exclude this hypothesis (Figure S10). This is a crucial point to select a material for NPdependent drug delivery. Similar to filter organs, a dramatic reduction of PCL3 NPs was observed at the fifth day after the treatment. Figure 3B shows a very limited presence of PLA8 NPs in tumor parenchyma. Some NPs can be displayed in highmagnification images (lower panels) 24 h after administration. This evidence is confirmed by the quantitative data (dark gray F

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Figure 4. (A) Representative confocal microscopy images showing the accumulation of red signal (associated with NPs) in MDA-MB-231 cells (identified by the presence of nuclei in blue). All the images were obtained exciting the samples with standardized acquisition parameters, in order to enable objective quantitative comparisons. The merge between the two signals is reported in the images. (B) Quantitative evaluation of the average fluorescent area per cell positive to the presence of NPs. Data are represented as mean ± SD, and statistical analysis was performed by Student’s t test. (C) Relative high-magnification pictures showing the different localization of NPs. PMMA NP incubation led to a massive internalization of NP (see green arrows) with a strong perinuclear localization. A homogeneous distribution of red spots was observed also in cells exposed to PCL3 NPs (green arrow). On the contrary, PLA8 NPs mainly remained confined on the external cell surface, interacting with the membrane (yellow arrow). The cartoons in panel D mimics this pattern of staining for the three types of NPs.

4B) confirmed these observational data. For each kind of NPs, the signal associated with RhB progressively increased with time. However, neither the efficiency of uptake (measured as quantification for each single histogram) nor the timedependent increase was similar for the three materials. PCL3 NPs showed a faster internalization. In fact, 24 h after incubation, they significantly differed from the other two materials. However, the rate of PLA8 NP internalization was drastically less efficient compared to the two other materials. The different kinetics among PCL3 and PLA8 NPs was furthermore confirmed by high-magnification pictures (Figure 4C) where representative images of the red fluorescence in a single cell are reported 24 h after incubation with NPs. Both PMMA (upper panel), PCL3 (lower panel) NPs were uniformly distributed inside the cytoplasm, mainly as “spot-like” structures of different sizes with a tendency to accumulate within the perinuclear region of the cells (green arrows). This peculiar pattern of staining is likely related to the active transport of NPs in endosomal vesicles, commonly known to mediate their cellular internalization and processing.41 It has been already reported that the stiffness of the material may influence the interaction between NPs and cell surface and, consequently, the vesicle formation.30 This assumption well fits with the pattern of staining associated with PLA8 NPs (middle panel) and also with their high level of degradation 72 h after

bars) in which the amount of NPs is extremely low without increasing in the first day after treatment (Figure 3C). Our data seem to indicate that the PCL3 NPs may have a much greater potential than PLA8 NPs in this type of tumor. However, a more detailed analysis at the single cell level is strongly required to define their real impact in this kind of breast cancer. In Vitro Studies: The Influence of the Material on TNBC Penetration, Subcellular Localization, and Cellular Homeostasis. In order to establish if and how the material may influence the interaction between NPs and cancer cells, the behavior of either PCL3 or PLA8 NPs was tested in MDA-MB231 cells. In this final part of the study the experiments with PMMA NPs were included,19 although they were not repeated in mice for obvious ethical reasons. Representative images of NP−cell interaction are depicted in Figure 4A. In lower-magnification pictures, the pattern of NP distribution for PMMA (upper panels), PLA8 (middle panels), and PCL3 (lower panels) is shown at 6, 24, and 72 h after incubation. A fast accumulation and a progressive increase of NPs (red signal) can be observed in both PMMA and PCL3 NPs (upper and lower panel). On the contrary, cells incubated with PLA8 NPs (middle panel) showed a lesser extent of staining, and in general, the red signal did not coordinate with the nuclear staining. The quantification of fluorescence (Figure G

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Figure 5. (A) Representative pictures of triple staining for PMMA, PLA8 and PCL3 NPs (red), Hoechst 33258 (blue), EEA1 (green, first column), GM130 (green, second column), and LAMP2 (green, third column) after 24 h of incubation with NPs. The merge between Nomarski and fluorescent acquisitions is reported. Yellow arrows show areas of colocalization between NPs and immunocytochemical markers. (B) Endosomal (EEA1) and lysosomal (LAMP2) immunoreactivity levels (expressed as the integration of the fluorescence intensity with the spread of signal per cell). Data are reported as mean ± S.E.M, (n = 10). Student’s t test was carried out to analyze statistical difference between control conditions and to evaluate possible time-dependent difference for each single material used.

Figure 5A. The visualization of the cell shape (obtained superimposing dark-fields with Nomarski) confirmed the different subcellular localization between stable (PMMA and PCL3) and more degradable materials (PLA8). An intense signal for EEA1, the marker of early endosomes (left column), is detectable in cells exposed to both PMMA NPs (upper panel) and PCL3 NPs (middle panel). In contrast, the green signal associated with early endosomes in cells exposed to PLA8 NPs (lower panel) was very low. In agreement with our previous study, the increase of the fluorescence level for EEA1 (Figure 5B, upper panel) was strongly dependent on PMMA NP internalization. PCL3 NPs had a similar trend, whereas PLA8 NPs did not lead to an increase of lysosomal signal. This result suggests that NP internalization influences the activation of the metabolic machinery. The confocal analysis (Figure 5A, left column) also showed that the green signal (EEA1) and the red signal (NPs) are often segregated in the same region, suggesting a tight correlation among the two components. This series of experiments at subcellular levels was very important for pharmacological purposes. Shifting from pharmacology to

serum incubation (Figure 1C). In contrast to the two other materials, the signal associated with PLA8 NPs was almost exclusively confined to the peripheral region of the cell (yellow arrow). They are not able to be internalized, as clearly shown by the low intensity of the intracellular signal and the slow increase of fluorescence from the 6th to the 72nd hour of incubation. The merge between red signal and the shape of cells (Figure 5A, lower panel) confirmed the low ability of PLA8 NPs to penetrate inside MDA-MB-231 cells. This difference on cell internalization among the three materials may greatly influence their therapeutic potential. In fact, for a targeted delivery, the passage of biological barriers and the localization in pathological areas is necessary but, very often, not sufficient if NPs are not able to efficiently interact with molecular targets of cancer cell.41,42 A deep understanding of the processes regulating NP internalization and subcellular localization is therefore crucial to develop finely tuned NPs with enhanced therapeutic potential.43 The interactions between the three different NPs and markers related to the cellular trafficking (such as endosomes, early and late lysosomes) and a marker of golgi apparatus to reveal the perinuclear region are shown in H

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Figure 6. (A) Representative super-resolution microscopy pictures showing, in the first row, the signal of tubulin filaments (α-tubulin, left column), early endosomes (EEA1, middle column), and lysosomes (LAMP 2, right column) 6 h after incubation with PCL3 NPs (second row). The third and the fourth row of panel A show the merge between the two signals at lower and higher degree of magnification. (B) Super-resolution microscopy showing the merge between LAMP 2 (green) and PCL3 NPs (red) after 24h of NP incubation. High magnifications of the areas are contoured by the white box.

The “perinuclear area” is not the final destination for biodegradable and biocompatible nanomaterials; the two possible scenarios are the following (1) NPs are stable and require a prolonged activation of the lysosomal machinery; (2) the stability in cells is weak, and the lysosomal activation is fast but less prolonged. This last hypothesis may give a great advantage in terms of drug release and therapeutic efficacy. The quantification of the marker associated with mature lysosomes, LAMP2, (Figure 5B, lower panel), enabled us to better understand if the three different materials may exert specific lysosomal response. The relationship between the stability of the material and the duration of the lysosomal activation is particularly evident in PMMA NPs, confirming the hypothesis of an active system of clearance involving these very stable particles (left histogram). Interestingly, lysosome fluorescence

nanopharmacology, is crucial to include a further checkpoint represented by the so-called “intracellular pharmacokinetics”. Thinking about potential delivery of SiRNAs and/or MiRNAs though nanocarriers, the fate of NPs after cell entry should be considered. NP metabolism or segregation in off-target districts may greatly influence the pharmacological response and therefore the prognosis for patients. For this reason, this study carefully investigated the interaction between NPs and subcellular targets at different time points. The segregation of PCL3 NPs in endosomes suggests that a retrieval transport from the periphery to the perinuclear area is occurring.44 The colocalization between NPs (red) and the golgi marker GM130 (green, Figure 5A, middle column) confirms this hypothesis. As expected, no overlapping between the two colors was found in cells incubated with PLA8 NPs (middle column, lower panel). I

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interestingly, the lower magnified pictures revealed that, in spite of a very high content of green-light rings (the external membrane of these lysosomes), the colocalization with the clusters of NPs (red signal) is quite modest. Another interesting feature of this image is that the few red spots colocalize with the external border of lysosomes and do not seem enclosed in these structures. This particular localization is even clearer in the higher magnified picture (white arrow, lower panel), suggesting that a first penetration of NPs is just starting to occur in mature lysosomes. Figure 6B shows the interaction between organelles and PCL3 NPs after 24 h of incubation in cells. At this time point, we found a large amount of clusters of NPs enwrapped in the endosomes (lower magnified picture, left panel). Higher magnified picture confirms this efficient system of clearance played by lysosome 1 day after NP-internalization (right panel). This difference between the localization of PCL3 NPs and mature lysosomes from the sixth to the 24th hour of incubation is extremely important to propose a therapeutic window in which the cargo should be released thus escaping to the naturally occurring degradation of the nanovector. Overall, these results indicate that, due to the migration in tumor parenchyma in TNBC mice and the efficient internalization and permanence inside cell cytoplasm, PCL3 NPs can be considered as an extremely interesting tool to be developed for intracellular drug-delivery purposes.

in cells incubated with PCL3 NPs drastically reduced after 72 h of NP incubation (right histogram). This observation might suggest that when PCL3 NPs start to degrade, they evade from these vesicles and might remain free in the cytoplasm. Moreover, due to their biodegradability, they can be processed by the cells more rapidly than nondegradable PMMA NPs. As expected, the levels of lysosome-related fluorescence in cells incubated with PLA8 NPs (middle histogram) were almost similar to those observed in untreated cells. A little and transient increase of signal was observed in cells incubated for 24 h but completely disappeared at the latter time point. The combination of the results obtained from confocal microscopy (Figure 5A) and the quantification of the signal (Figure 5B) are extremely important to confirm the tight association between the penetration of PCL3 NPs in the target cells and the fast increase of endosomal and lysosomal organelles. Super resolution microscopy (Figure 6) was carried out to carefully evaluate the spatial interaction between PCL3 NPs and specific structures of the cell at two key time points of our study, 6 and 24 h, respectively. Representative images of the subcellular localization of PCL3 NPs are depicted in Figure 6A. In the upper panels, three different markers have been visualized by immunofluorescence in light green (cytoskeleton−left; early endosomes−middle; mature lysosomes−right), and in the middle panels, the same field of view is instead excited with the laser associated with RhB. This technique of acquisition enabled us to resolve several single vesicles and clusters of NPs. The merge of the two signals reported in the third row and the relative higher magnified pictures in the lower panels of Figure 6A, gave us important indications on the mechanism of intracellular penetration of PCL3 NPs. The images in the left columns of the Figure 6A clearly show that clusters of PCL3 NPS are both included in the network of αtubulin filaments (white arrows) and penetrated into the core of the cytoplasm (red spots in the lower magnified picture) already after 6 h of incubation. This result confirms the efficient NP penetration previously shown in Figure 5 at a lower level of resolution. Figure 5 also revealed that 6 h after PCL3 NPs incubation, the signal of RhB and the marker of early endosomes EEA1 are somehow overlapping, even though no increase of EEA1 expression was observed. Super-resolution microscopy helped us to emphasize this colocalization. In Figure 6A (middle columns), it is clearly demonstrated that a relevant amount of clusters of NPs overlapped with the green spots corresponding endosomal vesicles (lower magnified picture). This colocalization is further emphasized by the higher magnified picture (bottom panel), in which it is possible to see groups of NPs (red-yellow color) enclosed in light green bodies. The white arrows depicted clearly revealed the presence of two NPs in the same endosome. This observational result strongly supports the hypothesis that an efficient internalization of PCL3 NPs in endosomes rapidly occurs in these kinds of cells. In contrast with what we observed for EEA1, a marked increase of the signal associated with LAMP2 was observed after 6 h of NP incubation (see Figure 5A, right histograms). Understanding whether this quick increase corresponds to a fast internalization of NPs in these low-pH bodies is a crucial point to establish the potential activity of NPs as cargo transporter in TNBC cells. In the merged pictures of the right column of Figure 6A, high-resolution pictures of the interaction between PCL3 NPs and mature lysosomes are reported. Very

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CONCLUSIONS In the present study, we developed an imaging-based approach to evaluate the different interactions of polymeric NPs with tumor cells and their different biodistribution in tumor-bearing animals, depending on their different stability. It was shown that administered NPs diffuse in the bloodstream long enough to reach the tumor sight. Once there, the interaction with endothelial walls of tumor vessels favors NP extravasation and accumulation in the tumor mass. The main outcomes achieved by our comparative analysis are reported in Table 2. Table 2. Summary of the Main Features of PCL3 and PLA8 NPs, Respectively, in Our Experimental Model stability in serum bloodstream permanence liver and spleen migration kidney migration tumor penetration bioaccumulation MDA-MB-231 internalization intracellular localization lysosomal activation

PCL3 NPs

PLA8 NPs

high high

low low

high

medium

low high low very high

high very low very low very low

cytoplasmic, perinuclear low

peripheral (outer surface of cell membrane) absent

These results clearly revealed the potential of PCL3 NPs for future development as nanodrugs in TNBC. On the contrary, the PLA8 formulation did not seem reliable for this kind of application due to the fast degradation profile. Our results represent a first pass toward a robust process of nanocarrier selection for the delivery of drugs in human TNBC. J

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(7) Kamaly, N.; Xiao, Z.; Valencia, P. M.; Radovic-Moreno, A. F.; Farokhzad, O. C. Targeted polymeric therapeutic nanoparticles: design, development and clinical translation. Chem. Soc. Rev. 2012, 41 (7), 2971−3010. (8) Tosi, G.; Bortot, B.; Ruozi, B.; Dolcetta, D.; Vandelli, M. A.; Forni, F.; Severini, G. M. Potential use of polymeric nanoparticles for drug delivery across the blood-brain barrier. Curr. Med. Chem. 2013, 20 (17), 2212−25. (9) Cova, L.; Bigini, P.; Diana, V.; Sitia, L.; Ferrari, R.; Pesce, R. M.; Khalaf, R.; Bossolasco, P.; Ubezio, P.; Lupi, M.; Tortarolo, M.; Colombo, L.; Giardino, D.; Silani, V.; Morbidelli, M.; Salmona, M.; Moscatelli, D. Biocompatible fluorescent nanoparticles for in vivo stem cell tracking. Nanotechnology 2013, 24 (24), 245603. (10) Janowski, M.; Bulte, J. W.; Walczak, P. Personalized nanomedicine advancements for stem cell tracking. Adv. Drug Delivery Rev. 2012, 64 (13), 1488−507. (11) Mura, S.; Couvreur, P. Nanotheranostics for personalized medicine. Adv. Drug Delivery Rev. 2012, 64 (13), 1394−416. (12) Etheridge, M. L.; Campbell, S. A.; Erdman, A. G.; Haynes, C. L.; Wolf, S. M.; McCullough, J. The big picture on nanomedicine: the state of investigational and approved nanomedicine products. Nanomedicine 2013, 9 (1), 1−14. (13) Dawidczyk, C. M.; Russell, L. M.; Searson, P. C. Recommendations for Benchmarking Preclinical Studies of Nanomedicines. Cancer Res. 2015, 75 (19), 4016−20. (14) Voigt, J.; Christensen, J.; Shastri, V. P. Differential uptake of nanoparticles by endothelial cells through polyelectrolytes with affinity for caveolae. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (8), 2942−7. (15) Harush-Frenkel, O.; Debotton, N.; Benita, S.; Altschuler, Y. Targeting of nanoparticles to the clathrin-mediated endocytic pathway. Biochem. Biophys. Res. Commun. 2007, 353 (1), 26−32. (16) de Ruijter, T. C.; Veeck, J.; de Hoon, J. P.; van Engeland, M.; Tjan-Heijnen, V. C. Characteristics of triple-negative breast cancer. J. Cancer Res. Clin. Oncol. 2011, 137 (2), 183−92. (17) Deng, X.; Cao, M.; Zhang, J.; Hu, K.; Yin, Z.; Zhou, Z.; Xiao, X.; Yang, Y.; Sheng, W.; Wu, Y.; Zeng, Y. Hyaluronic acid-chitosan nanoparticles for co-delivery of MiR-34a and doxorubicin in therapy against triple negative breast cancer. Biomaterials 2014, 35 (14), 4333−44. (18) Inoue, S.; Patil, R.; Portilla-Arias, J.; Ding, H.; Konda, B.; Espinoza, A.; Mongayt, D.; Markman, J. L.; Elramsisy, A.; Phillips, H. W.; Black, K. L.; Holler, E.; Ljubimova, J. Y. Nanobiopolymer for direct targeting and inhibition of EGFR expression in triple negative breast cancer. PLoS One 2012, 7 (2), e31070. (19) Sitia, L.; Paolella, K.; Romano, M.; Violatto, M.; Ferrari, R.; Fumagalli, S.; Colombo, L.; Bello, E.; De Simoni, M.; D’Incalci, M.; Morbidelli, M.; Erba, E.; Salmona, M.; Moscatelli, D.; Bigini, P. An integrated approach for the systematic evaluation of polymeric nanoparticles in healthy and diseased organisms. J. Nanopart. Res. 2014, 16 (7), 1−16. (20) Lupi, M.; Colombo, C.; Frapolli, R.; Ferrari, R.; Sitia, L.; Dragoni, L.; Bello, E.; Licandro, S. A.; Falcetta, F.; Ubezio, P.; Bigini, P.; Salmona, M.; D’Incalci, M.; Morbidelli, M.; Moscatelli, D. A biodistribution study of PEGylated PCL-based nanoparticles in C57BL/6 mice bearing B16/F10 melanoma. Nanotechnology 2014, 25 (33), 335706. (21) Colombo, C.; Dragoni, L.; Gatti, S.; Pesce, R. M.; Rooney, T. R.; Mavroudakis, E.; Ferrari, R.; Moscatelli, D. Tunable Degradation Behavior of PEGylated Polyester-Based Nanoparticles Obtained Through Emulsion Free Radical Polymerization. Ind. Eng. Chem. Res. 2014, 53 (22), 9128−9135. (22) Soppimath, K. S.; Aminabhavi, T. M.; Kulkarni, A. R.; Rudzinski, W. E. Biodegradable polymeric nanoparticles as drug delivery devices. J. Controlled Release 2001, 70 (1−2), 1−20. (23) Ferrari, R.; Yu, Y. C.; Morbidelli, M.; Hutchinson, R. A.; Moscatelli, D. epsilon-Caprolactone-Based Macromonomers Suitable for Biodegradable Nanoparticles Synthesis through Free Radical Polymerization. Macromolecules 2011, 44 (23), 9205−9212.

Further parameters of efficacy, such as the ability to load, transport, and release putative drugs in cells and mice should be determined. However, this promising distribution profile shown by PCL3 NPs paves the way for a deep exploitation of this material. Finally, this approach can be easily applied to a wide range of polymeric NPs. The creation of such multimodal platform is still an unmet need in nanopharmacology and might guide scientist choice in selecting the most promising nanocarriers to be developed for clinical uses.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b01422. Additional data for NP characterization (1H NMR spectra, GPC characterization, fluorescence characterization of HEMA-RhB macromonomer, DLS distribution), in vitro quantitative fluorescence measurements, immunohistochemistry staining, and ex vivo free RhB biodistribution (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +39-02-39014221. Fax: +39-0239014744. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research has been totally financed by AIRC Special Program Molecular Clinical Oncology “5 per mille”. Authors are also indebted to Prof. Alberto Diaspro (IIT Genova), Dr. Lauretta Galeno, Dr Cristiano Rumio, and Nikon Italia for the nanosopic analysis (N-SIM Super-Resolution Microscope System) and the section acquisition through Virtual slide scanner NanoZoomer.

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REFERENCES

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DOI: 10.1021/acs.biomac.5b01422 Biomacromolecules XXXX, XXX, XXX−XXX