Soft Discoidal Polymeric Nanoconstructs Resist Macrophage Uptake

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Soft Discoidal Polymeric Nanoconstructs Resist Macrophage Uptake and Enhance Vascular Targeting in Tumors Jaehong Key, Anna Lisa Palange, Francesco Gentile, Santosh Ayral, Cinzia Stigliano, Daniele Di Mascolo, Enrica De Rosa, Minjung Cho, Yeonju Lee, Jaykrishna Singh, and Paolo Decuzzi ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b04866 • Publication Date (Web): 21 Oct 2015 Downloaded from http://pubs.acs.org on October 29, 2015

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Soft Discoidal Polymeric Nanoconstructs Resist Macrophage Uptake and Enhance Vascular Targeting in Tumors

Jaehong Key1,2,*, Anna Lisa Palange1,4,*, Francesco Gentile4, Santosh Ayral1, Cinzia Stigliano1, Daniele Di Mascolo4, Enrica De Rosa1, Minjung Cho1, Yeonju Lee1, Jaykrishna Singh1, Paolo Decuzzi1,3,4♣

1

Department of Translational Imaging and Department of Nanomedicine, Houston Methodist Research

Institute, Houston, TX 77030, USA 2

Department of Biomedical Engineering, Yonsei University, 1 Yonseidae-gil, Wonju, Gangwon-do, 220-

710, South Korea 3

Department of Experimental and Clinical Medicine, University of Magna Graecia, Catanzaro, Italy.

4

Fondazione Istituto Italiano di Tecnologia (IIT), Laboratory of Nanotechnology for Precision Medicine,

via Morego 30, I16163 Genova, IT

♣

*

Corresponding author: E-mail address: [email protected] J.K. and A.P. contributed equally to this work.

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ABSTRACT Most nanoparticles for biomedical applications originate from the self-assembling of individual constituents through molecular interactions and possess limited geometry control and stability. Here, 1,000 x 400 nm discoidal polymeric nanoconstructs (DPNs) are demonstrated by mixing hydrophobic and hydrophilic polymers with lipid chains and curing the resulting paste directly within silicon templates. By changing the paste composition, soft- and rigid-DPNs (s- and rDPNs) are synthesized exhibiting the same geometry, a moderately negative surface electrostatic charge (−14 mV), and different mechanical stiffness (~ 1.3 and 15 kPa, respectively). Upon injection in mice bearing non-orthotopic brain or skin cancers, s-DPNs exhibit ~24h circulation half-life and accumulate up to ~20% of the injected dose per gram tumor, detecting malignant masses as small as ~ 0.1% the animal weight via PET imaging. This unprecedented behavior is ascribed to the unique combination of geometry, surface properties, and mechanical stiffness which minimizes s-DPN sequestration by the mononuclear phagocyte system. Our results could boost the interest in using less conventional delivery systems for cancer theranosis.

KEY WORDS: nanoparticle shape, nanoparticle stiffness, tumor imaging, macrophage uptake, iron oxide nanocubes.


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The encapsulation of therapeutic and contrast agents into nanoparticles has already had substantial impact in clinical medicine.1-5 Nanoparticles have been shown to modulate the biodistribution, organ specific accumulation, and circulation half-life of the original encapsulated agents providing enhanced therapeutic and imaging performance while limiting off-target effects.6-9 Clinical examples are given by PEGylated liposomal doxorubicin10, liposomal daunorubicin,11 and albumin nano-assemblies transiently associated with paclitaxel12 and gadolinium chelates13. Moreover, nanoparticles can carry and deliver multiple agents, attaining spatio-temporal control on their release, enhancing the efficacy of combinatorial drug therapies, multimodal imaging, and enabling the integration of diagnosis and therapy – theranosis.14-17 Building on these successes and on the enormous potential of nanotechnology, over the last decade, a myriad of nanoparticles have been developed exhibiting different geometrical features – size and shape; surface properties – electrostatic charge, type and density of ligand moieties; and composition – lipid, polymer, metal, and hybrid.9, 18-20 Most nanoparticles are synthesized following a bottom-up approach where the basic constituents are mixed together and self-assemble to form larger structures relying on weak, non-covalent molecular interactions. This strategy has been successfully used for the synthesis of micelles21, liposomes22, and polymeric nanoparticles23 typically with a spherical shape and a size ranging from tens to a few hundreds of nanometers. The simplicity of the bottom-up approach, together with the evidence that the tumor vasculature is fenestrated with endothelial openings of a few hundreds of nanometers, has dictated its success.24, 25 However, by relying only on self-assembly and the fine balance between surface energy and chemical potentials, the bottom-up methodologies cannot provide precise and independent control on the nanoparticle size and shape, as well as its stability over time.


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Within the last few years, technologies have been developed for synthesizing nano- and microparticles using top-down approaches.26-31 This was mostly fostered by the evidence that nanoparticle shape, together with size and surface properties, plays a pivotal role in controlling their behavior over multiple biological scales. At the vascular level, the authors and others have shown that non-spherical particles would more efficiently navigate the diseased microcirculation, recognize endothelial alterations, and preferentially lodge in vascular districts with low blood flow.8, 26, 32-34 At the cellular level, several authors have demonstrated that particle shape can modulate the rate of uptake in both professional phagocytic and non-phagocytic cells.35-37 Finally, at the sub-cellular level, more recent works have elaborated on the role of particle geometry in regulating intracellular trafficking and distribution, and exocytosis.38-40 Despite all this, only a handful of research laboratories are synthesizing non-spherical particles for biomedical applications. Indeed, a limiting factor is the increased complexity of the top-down fabrication methodologies which often require specialized equipment and facilities. In this work, a novel top-down approach is presented for the facile synthesis of particles with a precise control on size, shape, surface properties, and mechanical stiffness. The approach is demonstrated for the production of discoidal polymeric nanoconstructs (DPNs) presenting a diameter of ~ 1,000 nm; a height of ~ 400 nm; a surface electrostatic charge of – 14 mV; and a Young’s modulus (mechanical stiffness) of ~ 1.3 kPa. These nanoconstructs are formed by mixing two well-known polymers – poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) diacrylate – with lipid chains and other agents. In particular, the resulting polymer matrix incorporates three different agents, namely iron oxide nanocubes (NCs) with an edge size of ~ 20 nm; Cu64(DOTA) radioactive molecules, and red fluorescent rhodamine B dyes directly conjugated to the lipid chains. The step-by-step fabrication process, the physico-chemical


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properties, in vitro behavior, and the in vivo biodistribution and imaging performance of DPNs are described in the sequel.

RESULTS Discoidal polymeric nanoconstructs (DPN) were synthesized following four main steps, as schematically depicted in Figure.1. Step 1 deals with the definition of a pattern of wells in a silicon template. These wells represent the geometry of the desired nanoconstructs which in this case were circular discs with a ∼ 1,000 nm diameter and a ∼ 400 nm height (Figure.1A and Supporting Figure.1). The silicon template was obtained using a standard electron beam lithographic approach, as described in the Methods. In Step 2, the paste constituting the DPN polymer matrix was directly transferred over the template to carefully fill in the wells (red spots in Figure.1B and Supporting Figure.1). In the current configuration, the polymer paste comprises two conventional biodegradable and biocompatible polymers – poly(D,L-lactide-coglycolide) (PLGA, MW 38,000-54,000) and poly(ethylene glycol) diacrylate (PEG diacrylate); a photo-initiator (2-Hydroxy-40-(2-hydroxyethoxy)-2-methylpropiophenone) for curing the paste; and multiple agents providing different functionalities, namely 20 nm super-paramagnetic iron oxide nanocubes (NCs), for separation and purification purposes; lipid chains coupled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic

acid

(Lipid-DOTA)

for

the

stable

encapsulation of Cu2+-ions for positron emission tomography (PET) imaging; and lipid chains conjugated to Rhodamine B dyes (Lipid-RhB) for optical imaging. After carefully removing the excess of paste, the loaded template was exposed to UV-light for polymerization and then dried at room temperature for 10 min. In Step 3, a hydrophilic poly(vinyl alcohol) (PVA) solution was deposited over the template and dried in oven to form a film (Figure.1C). Weak, non-covalent


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bonds were originated at the interface between the polymerized DPNs and the PVA film, which was then peeled away extracting the DPNs from the wells of the template. In Step 4, the PVA film was immerged in an aqueous solution and dissolved after gentle stirring for 30 min (Figure.1D). The released DPNs were eventually rapidly collected via centrifugation, magnetic dragging, and membrane filtration to remove residual PVA fragments. Note that the proposed approach allows us to obtain different DPN configurations by simply using silicon templates with different well geometries and surface patterns, as shown in the Supporting Figure.2, in the case of rectangular cylinders, or rods.

Next, the DPNs were characterized for their physico-chemical properties using multiple techniques to confirm their geometrical and mechanical features. Scanning electron microscopy (SEM) images are presented in Figure.2A for DPNs on the PVA film and released (top-right inset). DPNs appear as orderly arranged in arrays with a pitch of ~ 3,000 nm and present a diameter slightly larger than 1,000 nm. These geometrical features are also confirmed by the atomic force microscopy (AFM) image of Figure.2B showing two DPNs, released from the PVA film, under hydrated conditions. The presence of Lipid-RhB chains is appreciated in Figure.2C where images of DPNs in aqueous solution were taken via a fluorescent microscope. As illustrated in the inset, the DPNs facing the objective presented a circular base with ∼ 1,000 nm diameter, whereas inclined DPNs showed a lateral thickness of ∼ 400 nm, precisely matching the geometry imparted in the silicon template. Given their discoidal shape, the DPN size distribution appeared as a single peak around 800 nm, in a multisizer instrument (Figure.2D). The surface electrostatic charge of DPNs was slightly negative with a zeta-potential of –14.0 mV. The transmission electron microscopy (TEM) image of Figure.2E reveals additional


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information on the inner properties of DPNs and shows the presence of iron oxide nanocubes (NCs) with an edge length of 20 nm (Figure.2F and Supporting Figure.3). In addition to controlling geometry, this fabrication process allows us to modulate the mechanical stiffness of DPNs. By changing the composition of the polymeric paste, as described in Methods, two types of DPNs have been synthesized exhibiting the same geometry and surface properties but different mechanical stiffness: soft DPNs (s-DPNs) and rigid DPNs (r-DPNs). The mechanical properties of DPNs were tested using an atomic force microscope and the Young’s modulus was calculated by analyzing the resulting force-displacement curves (Figure.2G). The average elastic moduli were of 1.28 ± 0.14 and 14.62 ± 9.09 kPa for the s-DPNs and r-DPNs, respectively (Figure.2H).

In order to perform quantitative biodistribution studies, lipid-DOTA chains, intimately mixed within the s-DPN polymer matrix, were reacted with copper chloride salts. The Cu2+-ions were transferred in the DOTA cage, under mild acidic conditions resulting in radiolabeled DPNs for positron emission tomography (PET) imaging. Only a negligibly small fraction of Cu2+-ions was estimated to leak out of the lipid-DOTA chains, demonstrating the stability of these polymeric nanoconstructs under physiological conditions (Supporting Figures.4, 5). Two different tumor models were considered, namely human primary glioblastoma cells (U87-MG) injected in the flank of the animal to develop a brain tumor model, and melanoma cells (B16-F10) injected subcutaneously to induce an orthotopic model for skin cancer. Radioactive s-DPNs were injected systemically in both experimental groups and mice were monitored longitudinally acquiring images at 1, 6, 24, and 48h post injection (p.i.). Representative PET/CT images for the transverse (top) and coronal (bottom) sections are presented in Figure.3A, and in Supporting Figure.6, 7.


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The PET images were converted to percentage injected dose per gram (%ID/g) and decaycorrected to the time of scanning. Within the first hour post injection, a moderate activity is detected in the lungs. This reduces over time, as the deformable DPNs are washed away from the pulmonary microcirculation, and already at 6h p.i., no more significant activity is observed in the lungs (Figure.3A). Higher is the signal detected from the liver and the abdominal cavity that continuously reduces over time as documented by the four insets of Figure.3A. Differently, the tumor uptake of DPNs grows steadily over time (red arrow in Figure.3A), as quantified in Figure.3B for both animal models. At 6h p.i., the dose of DPNs accumulating in the tumor is ∼ 8% ID/g. This becomes ∼12% ID/g at 24h p.i. and passes 15% ID/g at 48h p.i.. Notably, no significant difference in accumulation over time is observed for the two tumor models, although their biological properties and overall mass are different. The tumor size varies significantly for the presented experiments, ranging from 0.03 to 0.9 g, as deduced by direct weighting and CT imaging (Supporting Figure.6C). Consequently, the radiolabeled DPNs were capable of detecting tumor masses down to 0.1% of the total animal weight (0.03 g of tumor vs 25 g of mouse). The long circulation half-life of DPNs is confirmed by the data presented in Figure.3C where the DPN concentration in blood is measured over time using a scintillation counter. From this data, a circulation half-life of ∼24h is estimated for DPNs, which is also consistent with the observed progressive accumulation in the tumor tissue and residual activity in the liver. Then, the activity ratios between the tumor and the abdominal cavity (Figure.3D) and the tumor and the lungs (Figure.3E) were quantified using three regions of interest depicted in Figure.3F. The data show a progressive and continuous increase in the tumor-to-abdominal cavity ratio starting from zero, at the time of DPN injection, till about 0.65 at 48h p.i.. Differently, the tumor-to-lung ratio grows more rapidly and tends to plateau at about 24h p.i. around a value of 1.5. Given the


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blood longevity of s-DPNs and the large volume of blood processed in the abdominal cavity and lungs, it is perhaps not surprising to observe a residual activity at these sites even at 48h p.i.. Given the unprecedented longevity in blood and significant amount of iron loaded into s-DPNs, tumor accumulation was also quantified after exposing the malignant tissue to an external static magnetic field for about 1h p.i.. Tumor accumulation data are presented in Supporting Figure.7 demonstrating that s-DPNs can be efficiently guided towards the malignant mass and, in the presence of magnets, can reach already more than 15% ID/g at only 3h p.i.. Although the absolute increase in tumor accumulation at 24h is of only 30%, the rate of accumulation was drastically improved within the first few hours post injection.

In order to elucidate the biophysical mechanisms regulating the observed in vivo behavior, intravital microscopy was used to monitor the dynamics of soft and rigid DPNs in the liver and tumor. DPNs were systemically injected in Tie-2 mice bearing a skin cancer model. These mice are genetically engineered to express green fluorescent proteins in the endothelial cells, so that the vasculature lights up in green under the microscope. Representative images taken from full length movies are presented in Figure.4A-E for the liver and Figure.5A-D for the tumor. The original movies are available as Supporting Movies 1 – 3. In the same animals, DiD-labeled RBCs were injected prior imaging for quantifying blood flow and identifying phagocytic cells (Kupffer cells and tumor macrophages) which would turn blue upon the internalization of RBCs, following protocols previously reported by the authors.8 In the liver tissue (Figure.4), the upper row is related to soft-DPNs (Figure.4A,B), the central row is related to rigid-DPNs (Figure.4C,D), and the bar chart in the lower row (Figure.4E) reports on the number of DPNs accumulating in the liver per unit area. Two different


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accumulation compartments have been considered: “Kupffer cells” and “vascular”. In the first case, DPNs are co-localized with the Kupffer cells whereas in the second case DPNs are adhering to the vessel walls in the liver, away from Kupffer cells. Within 120 minutes post injection, multiple trains of DPNs (red spots) were observed to zig-zag within the complex vasculature (green spots) (Figure.4, Supporting Figure.8 and Supporting Movie.1), while only a few DPNs were not moving (dashed white ovals). The data of Figure.4E show a continuous deposition of DPNs over a 2h observation period via intravital microscopy. Specifically, r-DPNs deposit in the liver about 4 times more than s-DPNs (24,000 vs 6,000 #/mm2). This difference can be immediately observed in Supporting Figure.8c, where three different regions of interested are presented side by side for s-DPNs and r-DPNs. Interestingly, an almost equal number of r-DPNs (~12,000 #/mm2) are either uptaken by Kupffer cells or adhere nonspecifically to the vascular walls, away from Kupffer cells. Differently, a very minor portion of s-DPNs adheres non-specifically to the vascular walls in the liver. If the sole portion of DPNs uptaken by the Kupffer cells is considered, the ratio between internalized r-DPNs and s-DPNs is larger than 2. In the Supporting Information (Supporting Figure.9 – 11 and Supporting Movie.2), a direct comparison with 750 nm spherical, green fluorescent polystyrene particles (PSs) is also presented showing a much larger and rapid accumulation of PS in the liver tissue as compared with s-DPNs. As per the tumor tissue, trains of DPNs were also observed to move rapidly and eventually accumulate within the vasculature (Figure.5, Supporting Figure.12 and Supporting Movie.3). The intravital microscopy data document s-DPNs depositing at a higher level and rate as compared to r-DPNs. Within a 2h observation window post injection, s-DPNs accumulate continuously reaching at 120 min a surface density of ~ 1500 DPNs/mm2. Differently, r-DPNs


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tend to reach a maximum around 60 min with a surface density of ~ 250 DPNs/mm2 (Figure.5C). This leads to a tumor accumulation ratio s-DPN/r-DPN of about 6. The significantly higher tumor accumulation of s-DPNs over r-DPNs should, most likely, be associated with the longer circulation half-life of the former and increased likelihood of passing through tortuous vessels with lower blood flow and favorable vascular adhesion conditions, as shown by confocal intravital microscopy imaging (Figure.5D).

The unprecedented longevity in blood and tumor accumulation of s-DPNs should be associated with the minimal sequestration by phagocytic cells, particularly Kupffer cells and splenic macrophages, as documented by the nuclear imaging and intravital microscopy analysis (Figure.3 and 4). To further characterize such resistance to cell uptake, DPNs were incubated with macrophages and endothelial cells (HUVECs), which are the first cells encountered by any systemically injected nanoparticles. The interaction of DPNs with cells was analyzed via fluorescent confocal microscopy and flow cytometry (Figure.6, Supporting Figures.13– 16 and Supporting Movies.4 – 6). A side by side comparison between s-DPNs, r-DPNs and PSs, coincubated with bone marrow derived monocytes (BMDM) is provided in Figure.6A-D and Supporting Figure.14. The number of nanoconstructs internalized per cell was estimated and plotted at two different time points, namely 4 and 24h. The bar chart of Figure.6D quantifies the number of internalized nanoconstructs showing clearly that, at 24h, a significant difference in internalization propensity exists between PS, r-DPNs and s-DPNs. Specifically, s-DPNs are 2 to 3 times less internalized as compared to r-DPNs and about 5 times less than PS. At 4h, s-DPNs are two times less internalized as comparted to r-DPNs and no statistically significant difference exists between r-DPNs and PS. Notably, s-DPNs are observed to move around macrophages


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without activating any significant internalization process (Supporting Figures.14 - 16). The live-cell confocal microscopy results are in line with the FACS analysis performed on J774.A1 (Figure.6E). In this case, the internalization propensity of s-DPNs and r-DPNs was tested via flow cytometry upon incubation with J774.A1 macrophages (Figure.6E). This data continues to demonstrate that r-DPNs are uptaken by phagocytic cells in larger amounts and at higher rates as compared with s-DPNs. Particularly, at both 4 and 24h p.i., the number of r-DPNs associated with J774.A1 cells was ∼ 3 times larger than for s-DPNs, in agreement with Figure.6D. Furthermore, the ability of s-DPNs to transiently deform and squeeze through small orifices, just like circulating blood cells, was verified in vitro by using syringe filters with pores of 1,000, 800 and 400 nm (Supporting Figure.17, 18). Under these experimental conditions, s-DPNs were observed to cross the porous membrane in the syringe filters and fully recover their original shape even for the smallest 400 nm pores. Interestingly, the resistance to cell internalization demonstrated by s-DPNs is in agreement with experiments performed by Beningo and Wang, using polyacrylamide spherical beads of 1 – 6 µm in diameter and showing a diminished cell uptake with microparticle deformability.41 A similar cell internalization analysis was performed for s-DPNs interacting with HUVECs. No significant cell uptake was observed, even in the presence of an external magnetic field pulling sDPNs towards the cell membrane (Supporting Figure.19). All this would suggest that the deformability of these polymeric nanoconstructs could be responsible for their low macrophage uptake and blood longevity.

Finally, a preliminary analysis of the DPN toxicity profile was performed using standard in vitro MTT assay and quantifying in vivo the blood level of inflammatory related cytokines (IL-6, IL-


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10, and TNF-α) and concentration of serum enzymes (AST, ALT, and creatinine). This preliminary data presented in Supporting Figure.20 would suggest a favorable toxicity profile for DPNs in mice. It is also important to note that DPNs would not be directly filtered through the glomeruli of the kidneys, which have a characteristic size of a few tens of nanometers.42 Therefore their geometry and deformability would favor their long blood circulation followed by a progressive accumulation within the tumor vascular bed. Importantly, the three main components of DPNs (i.e. polymeric chains of PLGA; polymeric chains of PEGDA, and iron oxide nanocubes (NCs) would biodegrade over time and their products could be readily excreted through the kidneys or metabolized. 43, 44 44

DISCUSSIONS In the previous sections, the nuclear imaging and intravital microscopy data together with the cell-uptake analysis shed light on two unique features of DPNs: i) the in vivo longevity in the blood stream with a circulation half-life of ∼24h; ii) the capability of lodging within the tumor vasculature with accumulation doses as high as 20% ID/g. Such accumulation doses were documented in two different tumor models with malignant masses ranging from 0.03 to 0.9 g. Although, most nanomedicines perform significantly better than the corresponding free agents, the values of circulation half-life and tumor accumulation reported for s-DPNs are uncommon even among the most successful intravascular nanodelivery systems. It is here important to recall that other authors have also recently shown that deformable particles tend to circulate longer than rigid particles, possibly mimicking the vascular behavior of blood cells.45,

46

However, no

information was provided in these studies on tumor accumulation. Furthermore, the authors have extensively studied in previous works the in vitro and in vivo properties of rigid particles,


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similar in geometry to DPNs, documenting tumor accumulation levels of ~ 5% ID/g and circulation half-lives of a few hours.8 20 Recently, in the attempt to modulate uptake by immune cells, these same rigid particles have been coated with a leukocyte membrane showing tumor-toliver ratios much smaller than for DPNs (1:1,000), at 90 min p.i..47

The s-DPN longevity in blood should certainly be related to the minimal uptake by phagocytic cells, as documented in vitro (BMDM and J774.A1 cells, Figure.6) and in vivo (Kupffer cells in the liver, Figure.3 and 4). This results from DPN specific architecture and physico-chemical properties. The DPN matrix is obtained by direct entanglement of hydrophobic and hydrophilic polymer chains leading to spongy and deformable nanoconstructs with a mechanical stiffness of ∼ 1.3 kPa (Figure.2G) and a moderately negative surface electrostatic charge (- 14 mV). Interestingly, the low mechanical stiffness and surface charge are similar to those of most cells.48 Note that more rigid particles, such as PSs and r-DPNs, have shown over 3 – fold higher cell internalization, as compared to soft DPNs (Figure.6). Moreover, the authors have previously shown that rigid mesoporous silicon particles, with a geometry similar to DPNs, were rapidly internalized by different cell types, including macrophages and endothelial cells, even in the case of PEGylated or negatively charged surfaces.49 Therefore, the low mechanical stiffness and moderately negative surface charge of DPNs would contribute to their stealthiness with respect to the immune cells. In terms of tumor accumulation, it is important to recall that the authors have extensively shown in previous works that sub-micron discoidal nanoconstructs lodge more efficiently that conventional spherical nanoparticles within the diseased tumor vasculature. Sub-micron nanoconstructs are forced to navigate in proximity of the vessel walls by the fast moving red


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blood cells50, 51 and tend to preferentially adhere within the tumor capillaries, characterized by higher tortuosity and lower blood flow as compared to the healthy vasculature.8, 20, 32, 52 Under these biophysical conditions, the fine balance between hydrodynamic dislodging forces and nonspecific vascular adhesive interactions have shown to favor the firm deposition of sub-micron discoidal nanoconstructs, such as the DPNs (Figure.3 and 5). Note that smaller DPNs would behave differently from the current DPNs in that would accumulate in tumors mostly via the EPR effect, similarly to 100 – 200 nm spheres. Interestingly, the rate of tumor deposition of the long circulating

DPNs can be significantly enhanced using external static magnetic fields, applied to the malignant mass (Supporting Figure.7).

DPNs are synthesized via a top-down fabrication process that allows for the facile preparation of polymeric nanoconstructs with a precise control on size, shape, surface properties, and mechanical stiffness – the 4S parameters. Differently from other methodologies relying on sacrificial polymer templates and imprinting techniques26, 29, 30, here a rigid silicon template is directly filled with the polymer paste of interest. This reduces the fabrication time and, most importantly, prevents distortions of the nanoconstructs that may occur during the filling, cleaning, and polymerization in non-rigid templates, thus endowing for a precise particle geometry control. Although two different geometrical configurations were only presented in this work – ∼ 1,000×400 nm circular discs and ∼ 1,000×500×500 nm rectangular discs or short rods – discoidal nanoconstructs with any arbitrary contour can be prepared. Furthermore, the DPN matrix is here composed of PLGA, PEG diacrylate, and lipid chains intimately entangled one with the other, which is different from more conventional nanoparticle architectures where a uniform polymer core is stabilized by a superficial layer of lipid and


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polymer chains. Consequently, the DPNs released from the silicon template do not require any surface modification, differently from most nanomedicines. The intimate entanglement of hydrophobic and hydrophilic polymer chains facilitates the incorporation of molecules and nanoparticles with different properties and functionalities. This is the case of that iron oxide nanocubes, which could be also used for Magnetic Resonance Imaging and localized hyperthermia, as shown by the authors in previous works with different nanotechnological platforms.37-39 Similarly, the lipid-DOTA chains, which are here reacted with Cu salts to provide radioactive Cu64(DOTA) molecules, could be also used as chelators of gadolinium, in T1-weighted MR imaging; and other radioisotopes, such as Ga68, Y90, Lu177, for nuclear imaging and radiotherapy.

CONCLUSIONS In conclusion, a novel, flexible top-down approach was presented for the synthesis of polymeric nanoconstructs with precise size, shape, surface properties, and mechanical stiffness – the 4S design parameters. It has been shown that, once in the blood stream, soft DPNs would circulate with minimal sequestration by the mononuclear phagocyte system and, as they repeatedly cross the tumor vasculature, would eventually find proper hemodynamic conditions for stable deposition. The unprecedented tumor accumulation and longevity in the blood circulation of soft DPNs derives from the proper combination of the 4S parameters, particularly the discoidal shape and the low mechanical stiffness. It is the authors’ hope that the facile preparation together with the demonstrated multi-functionality and unprecedented in vivo performance of DPNs could boost the interest of several other laboratories in using non-spherical, sub-micron sized nanoconstructs for the imaging and therapy of tumors.


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METHODS AND EXPERIMENTAL Fabrication process for the Discoidal Polymeric Nanoconstructs (DPNs). The silicon master template was fabricated using Electron Beam Lithography (EBL) techniques. The master template is a silicon substrate with textures comprising an array of cylindrical holes with a fixed diameter (1.2 μm) and height (400 nm). (100) silicon wafers (from Jocam, Milan, Italy) were cleaned with acetone and isopropanol to remove possible contaminant and then etched with a 4% wet HF solution. The wafers were then rinsed with DI water and dried with N2. A high resolution positive electronic resist (PMMA-A2) was spin–coated for 60 s at 4000 rpm to obtain a 70 nm thick layer of resist on the substrate. The sample was therefore pre-baked at 170 °C for 2 min to remove any residual solvent from the resist. Patterns of ~ 105 discs with a diameter of 1.2 µm, packed in 1200×1200 µm fields, were written on the sample using an Electron Beam Lithography EBL system (CRESTEC), operating at 50 keV. For the exposure, a current of 10 nA, a dose of 220 µC/cm2, and a resolution of 20 nm/pixels were adjusted. After e-beam lithography, the silicon wafer was developed in 3:1 isopropanol:methyl isobutyl ketone (MIBK) solution for 60 s, and in 1:1 isopropanol: MIBK solution for 10 s, to remove exposed regions of the photoresist. The pattern was transferred to the underlying silicon substrate by deep reactive ion etching with SF6/O2 plasma. For soft DPN (s-DPN) synthesis, 30 mg of poly(lactic-co-glycolic acid) (PLGA) were dissolved in solvent in either dichloromethane (DCM) or chloroform (CHCl3) and the solution was mixed with 6 mg of polyethylene glycol (PEG) diacrylate and 10 µg of 2-Hydroxy-40-(2hydroxyethoxy)-2-methylpropiophenone (Photo-initiator). For rigid DPN (r-DPN) synthesis, 90 mg of PLGA was dissolved in the co-solvent describe above without PEG and photo-initiator.


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For imaging purposes, 30 µg of Rhodamine-lipid, 30 µg of iron oxide nanocube, or 3 mg of lipid-DOTA were mixed together. The final polymeric mixture was loaded into the wells engraved on the Si master templates. After loading, the surface of the templates was quickly cleaned by DCM solution and then exposed under the UV-light for further polymerization. The polymer loaded templates were completely dried at 60 °C. 6 % w/v of Poly(vinyl alcohol) (PVA) solution was prepared in deionized water. The PVA solution was cooled down and stored at room temperature. The PVA solution was loaded on the surface of the polymer loaded Si master templates and the solution was dried at 60 °C. The PVA solution became a thin film and the film was easily peeled off from the Si master template. The film also holds numerous identical discoidal polymeric nanoconstructs (DPNs) formed from the loaded polymer in the Si master template. Under magnetic stirring, the PVA templates were swollen and also dissolved in DI water at room temperature releasing a lot of DPNs. The released DPNs were separated from the PVA solution by centrifugation at 3,700 rpm for 20 minutes. The separation processes were repeated until pure pellet is collected. Finally, using 2 µm filter membranes, DPNs were collected. Physico-chemical characterization of the Discoidal Polymeric Nanoconstructs (DPNs). Scanning electron microscope (SEM) (Figure.2A) The size and shape characterization of DPNs was done by using FEI Nova_ Nano-SEM 230 (Hillsboro, OR, USA). Ultra-high resolution SEM images were acquired at high vacuum conditions after 5~7 nm platinum coating using a Cressington 208HR sputter-coater (Ted Pella Inc.). 5 ~ 15 keV of beam energies and corresponding electron currents of 0.98 pA ~ 0.14 nA were used. In some cases, the mode 2 configuration was used whereby SEM images were magnified over 2500 k.


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Size and zeta-potential measurement (Figure.2C, D) Particle size and distribution were obtained using a Multisizer 4 Coulter Particle Counter (Beckman Coulter, CA). DPNs were suspended in isotone solution for the size analysis after shortly sonicating for 30 s before the measurement. The DPN zeta potential was analyzed in phosphate buffer (pH 7.0) using Zetasizer Nano (Malvern, UK). Transmission electron microscope (TEM) (Figure.2E, F) The morphology and inner components of DPNs were observed by a JEOL 2100 field emission TEM (FE-TEM), operating at 200 kV with a single tilt holder. TEM sample was prepared by evaporating a drop of DPNs solution on 300 mesh copper grid (Ted Pella Inc.). Atomic Force Microscopy (AFM) imaging (Figure.2B, G) A petri dish (diameter: 6 cm) was coated with poly-l-lysine (Sigma Aldrich) for 20 min at room temperature and washed twice with purified water. A drop of 10 µL DPNs was spotted onto the poly-l-lysine coated petri dish and then incubated for 30 min to allow the DPNs to absorb onto the substrate. Then, the substrate was washed twice by purified water. Morphological shape of DPNs was acquired in liquid condition with the contact mode on a Bioscope Catalyst AFM using MLCT-C cantilever (k=0.01 N/m, Bruker Corporation, MA). Measurement of stiffness using AFM (Figure.2H) The stiffness of DPNs was measured by the force-curve technique on a Bioscope Catalyst AFM. 10 µL DPN colloids were spotted onto polyl-lysine coated petri dish at room temperature, and after completely drying, 10 µL DPN colloids were again spotted onto substrate. This procedure was repeated 3-4 times. The point and shoot mode of the AFM was utilized to measure the mechanical properties of DPNs. The force spectroscopy was precisely measured using the points on the topography image obtained right before this stiffness measurement. The silicon nitride cantilevers modified from silica


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microparticle of diameter: 5 µm (Novascan Technologies Inc., IA) were used for this measurement. The spring constant value (0.06 N/m) was measured by the thermal tuning method and applied for this experiment. Force curves were acquired at a sampling rate of 1 Hz. The Young’s modulus, E, was calculated from the force curve on the Hertz model (Eq. 1) using Nanoscope analysis program of Bruker corporation. =

4 √

3 (1 − )

(Eq. 1)

where F=force, E=Young’s modulus, ν=Poisson’s ratio (ν=0.5, in this study), R=radius of the indenter (R=2500 nm, in this study), and δ=indentation depth.

PET/CT imaging. PET/CT scanning was performed with a Dedicated PET (dPET, resolution: ≤ 1.4 mm) and Multi-Modality System (MM), together function of PET and CT with integration capabilities for in-line fusion imaging (Siemens, Inveon). Athymic nude female mice (8 weeks old) from Charles River Laboratories, (Wilmington, MA) were used. All tumors were generated by a one-time subcutaneous injection of 106 tumor cells typically on the left flank, following protocols reviewed and approved by the IACUC at the Houston Methodist Hospital. U87-MG and B16-F10 were inoculated to generate, respectively, a glioblastoma multiforme and a melanoma model (ATCC, Manassas, VA). When tumors reached 2–6 mm in diameter were prepared for imaging procedures. Before imaging, mice were anesthetized in a plastic chamber using isoflurane gas in 100% oxygen. For the imaging, the mice were positioned on the imaging bed equipped with a heating pad and the imaging system was connected with the isoflurane gas system. Following systemic administration of 10 µCi of Cu64(DOTA)-DPNs, mice images were acquired for 4 min by using high resolution CT imaging and CT image reconstruction was achieved using the scanner’s default common cone-bean reconstruction (COBRA) algorithm


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(Siemens). Subsequently, PET acquisition completed for 10 min at 1, 6 h post-injection and 20 min at 24, 48 h post-injection. PET images were reconstructed by 2D ordered subset expectation maximization (OSEM2D) algorithm (Siemens). PET and CT images were coregistered and viewed using the Inveon Research Workplace software (Siemens). For the circulation half-life of Cu64(DOTA)-DPNs, the Cu64 radioactivity in the blood was measured by using an automatic scintillation counter (PerkinElmer, 2470 Automatic Gamma counter, Wizard 2™). The measured radioactivity (counts per minutes, cpm) was converted by a known aliquot of the injected dose (µCi). 100 µCi of Cu64(DOTA)-DPNs were injected in nu/nu mice (n = 4) via tail vein using a catheter. At each time point (0, 2, 7, 24, and 48 h), 25 µL of blood were drawn via tail vein bleed. For the biodistribution study, 10–15 days after subcutaneous injection of the tumor cells, mice had tumor sizes of about 1.0 cm in diameter. The mice were injected intravenously with DPNs (10 µCi). PET/CT scans were performed as described above at 1, 6, 24, and 48 h post injection of DPNs. The tumor accumulation of DPNs was estimated by using the Inveon Research Workplace software (Siemens) where PET images were corrected based on the initial injection dose and the decay rate of Cu64. CT images co-localized with PET images were utilized to delineate the area of tumors in each slide. The accumulation values were presented as the mean and standard deviation from four animals (n = 4) of U87 tumor models and five animals (n = 5) for B16 tumor models. The unit of the accumulation was expressed as the percentage injected dose per gram tissue (% ID/g). All animal procedures were approved by the Institutional Animal Care and User Committee (IACUC) at Houston Methodist Research Institute. Pharmacokinetic experiments involving radioactive materials (64Cu) were approved by the Radiation Safety Committee at Houston


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Methodist Research Institute. n = 4 mice were used for the U87-MG cells, n = 5 mice were used for the B16-F10 cells, and n = 4 mice were used for the circulation half-life.

Intravital image acquisition. Transgenic Tie2-GFP mice (6 – 8 weeks old) from Jackson Laboratory (Bar Harbor, ME) were used for intra vital microscopy imaging. 106 malignant cells (B16 melanoma) suspended in 200 µl PBS were injected subcutaneously into the flank of the Tie2-GFP mice. The tumor bearing mice were ready to image in 2-3 weeks. Animals were housed and treated in accordance with the policies and procedures of the Animal Welfare Committee with all testing performed using IACUC approved protocols at the Houston Methodist Research Institute. The liver was exposed via a 0.5 cm midline incision in the abdomen. Mice received a one-time injection of fluorescently labeled autologous red blood cells 2 days prior to imaging to allow visualization of macrophages. Briefly, blood collected retroorbitally was stained with lipophilic carbocyanine DiD (Invitrogen) at 37 °C using the manufacturer's recommended protocol and immediately re-injected retro-orbitally. Live animals were imaged on an upright Nikon A1R laser scanning confocal microscope platform equipped with a resonance scanner, isoflurane anesthesia system, heated stage, and custom coverslip mount. Two-channel images were acquired per animal using 4X or 20X high-NA, LWD objectives. Anesthetized mice were injected intravenously (either via tail vain or retro-orbitally) with 109 particles in 150 µl PBS and monitored for up to 2 hours. Using this delivery route, all particles were able to circulate within the mouse and were observed to reach the liver in approximately 10 seconds. n = 3 transgenic Tie2-GFP mice were used and 4 to 5 different regions of interests were considered for statistical analysis.


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Cell culture. Human umbilical vein endothelial cells (HUVECs) were obtained from PromoCell, and grown until the 6th passages in endothelial growth medium at 37°C in a humidified 5% CO2 atmosphere. J774.A1 macrophage and B16-F10 mouse melanoma cell lines were obtained from ATCC and cultured in DMEM medium supplemented with 10% FBS and antibiotics (100 U/ml penicillin G, 100 mg/ml streptomycin), and grown at 37°C in a humidified 5% CO2 atmosphere. U87-MG human glioblastoma cells were obtained from ATCC and cultured in EMEM medium supplemented with 10% FBS and antibiotics (100 U/ml penicillin G, 100 mg/ml streptomycin), and grown at 37°C in a humidified 5% CO2 atmosphere. Bone marrow derived monocytes (BMDM) were obtained by flushing out bone marrow from mice femurs. Bone marrow was then filtered with 70 µm filters and plated in 10 cm dish. Cells were grown in basal media (HG-DMEM medium with 15% FBS, 2 mM L-glutamine, 1% penicillin (100 UI/ml)-streptomycin (100 mg/ml), and 0.25 mg/ml amphotericin B) supplemented with 10ng/ml M-CSF to allow monocytes activation. On day 3, media was replaced with fresh media containing 10ng/ml M-CSF and on day 7 cells were ready for internalization studies.

Live cell microscopy and Confocal Microscopy. Live cell imaging with DPNs was acquired by IX81 Inverted Microscope (Olympus, Japan). 5x104 J774.A1 macrophages were seeded into glass bottom dishes and incubated for 2 hours with lipid-rhodamine-DPNs at a concentration of 5 DPNs per cell. Cells were imaged at Live Cell Confocal Image System using 40X objective for all the incubation period to follow DPNs dynamics and interaction with macrophages. Pictures were taken every 5 minutes till the end of the experiment. Cell nuclei were stained with Hoechst 33342 dye.


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7 x 104 BMDM were seeded onto 8 well cover slides and incubated for 4 and 24 h with sDPN, rDPNs and fluorescent Polystirene Beads (PS) at concentration of 5 particles per cell. Cell were then washed and fixed using 4% paraformaldehyde and DPNs uptake by BMDMs was studied through Z-stacks analysis performed on Nikon confocal microscopy. For these studies, rhodamine lipids were added in the polymeric mixture. Flow cytometry. In vitro DPNs and 750 nm Fluorescent Polystyrene Beads internalization into macrophage was analyzed with flow cytometer. J774.A1 cells were plated in 6-well dishes at a density of 4x105 and incubated with rhodamine-Lipid-DPNs and Fluorescent Polystyrene Beads at a concentration of 5 particles per cell for 3 hours. After that, cells were detached from the dishes using PBS-EDTA 0.5 mM and processed following standard procedures. Briefly, cells were washed twice in PBS buffer to remove free particles, suspended in a final volume of 300 µl of PBS + 3 % of FBS and then analyzed on LSRII FACS from BD-Biosciences.

Statistical Analysis. All data were processed using Excel 2010 software (Microsoft). Results are expressed as mean ± standard deviation. Linear regression analysis was performed to obtain the correlation coefficient. Comparisons of successive scans within the same animal and with control animals were performed using a paired and unpaired 2-sided Student t test, respectively. The Dunnett’s test has been performed for the cell viability results where 5 groups (different cell:DPN ratios) are compared to a single control.

ACKNOWLEDGMENTS This work is partially supported by the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant agreement n° 616695” and the


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Houston Methodist Research Institute. The authors acknowledge the help of Matt Landry for the graphical work; the Advanced Cellular & Tissue Imaging Core and the Preclinical Imaging Core at HMRI (TX – USA); and the Microfabrication Facilities at the Istituto Italiano di Tecnologia (GE – ITA).

AUTHOR CONTRIBUTIONS J.K. synthesized DPNs and participated in all experiments; A.L.P. helped with the synthesis of DPNs, cell uptake and intravital microscopy experiments; F.G. fabricated the silicon templates for the precise synthesis of DPNs; S.A. synthesed the lipid-DOTA chains, helped with the PET/CT and half-life circulation time experiments; C.S. performed the nanotoxicological experiments, D.D. helped with the flow cytometry analysis; E.D.R. performed the intravital microscopy experiments; M.C. synthesized the iron oxide nanocubes; Y.L. performed the atomic force microscopy experiments; J.V.S. performed the mathematical analysis; P.D. designed and coordinated the research, analyzed the data, and wrote the manuscript. All authors analyzed and discussed the results.

SUPPORTING INFORMATION Supporting information is available free of charge at http://pubs.acs.org. The Supporting Information includes additional Methods and Experimental on the synthesis and characterization of DPNs in vitro and in vivo. Correspondence and requests for materials should be addressed to P.D.

COMPETING FINANCIAL INTERESTS


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The authors declare no competing financial interests.


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Figure 1. Preparation steps and constituents of Discoidal Polymeric Nanoconstructs (DPNs). The facile preparation of DPNs follows four sequential steps: A. Filling the cylindrical wells (1000×400 nm) in a silicon template with a polymer paste, obtained by intimately mixing together poly(lactic-co-glycolic acid) (PLGA); polyethylene glycol-diacrylate (PEG-Diacrylate); Lipid-DOTA; Lipid-Rhodamine B; and 20 nm iron oxide nanocubes (NCs). B. Cleaning the excess polymer paste (scum layer) deposited over the template and curing the polymer paste under UV light. C. Forming a hydrophilic, thin PVA film on top of the silicon template and extracting DPNs from the silicon template via peeling off the PVA film; D. Dissolving the PVA film in water and efficiently collecting DPNs by centrifugation, magnetic dragging, and filtration. For PET/CT imaging, the purified DPNs are reacted with Cu salts to form Cu64(DOTA) molecules stably entangled with the polymer matrix.


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Figure 2. Physico-Chemical properties of Discoidal Polymeric Nanoconstructs (DPNs). A. SEM image of DPNs on a thin PVA film, before separation, showing a characteristic diameter of ∼ 1,000 nm. A single DPN is shown in the top-right inset. B. Atomic Force Microscopy (AFM) image of DPNs confirming their discoidal shape and size. C. Fluorescent microscopy image of an aqueous solution of DPNs. The magnified image in the top-right inset shows a DPN with a diameter of ∼ 1,000 nm (front view) and a height of ∼ 400 nm (later view). D. Distribution of the DPN hydrodynamic diameter as measured by a multi-sizer instrument presenting a peak around 800 nm. E. TEM image showing 20 nm iron oxide nanocubes loaded in the DPN polymer matrix. F. Magnified view of the DPN inner core clearly documenting the size and shape of the iron oxide nanocubes. G. Atomic force microscopy image of a DPN showing the indentation points (crosses= for the mechanical stiffness measurements, and H. Young’s modulus analysis for soft and rigid DPNs.


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Figure 3. PET/CT imaging and circulation half-life of Discoidal Polymeric Nanoconstructs (DPNs). A. Transversal (top) and coronal (bottom) PET/CT images of a U87-MG tumor bearing mouse acquired at 1, 6, 24, and 48h p.i. of radiolabeled DPNs. The PET images are converted to percentage injected dose per gram (%ID/g) and decay-corrected to the time of scanning. As the deformable DPNs squeeze out of the pulmonary microcirculation, the initial activity in the lungs and abdomen reduces while the tumor activity grows steadily over time. B. Quantification of DPN accumulation in U87-MG and B16-10 tumor bearing mice, expressed in terms of percentage injected dose per gram tissue (%ID/g). C. DPN concentration in blood measured via scintillation counter, at different time points (0, 2, 7, 24, 48h p.i.). D. Tumor to abdominal cavity activity ratio over time. E. Tumor to lungs activity ratio over time. F. Regions of interest for the estimation of the activity ratios.


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Figure 4. Intravital Microscopy analysis of liver accumulation of Discoidal Polymeric Nanoconstructs (DPNs). The DPN sequestration in the liver was investigated via intravital microscopy (IVM) in Tie-2 GFP+ mice. In the representative IVM images and Supporting Movies, the endothelial cells lining the blood vessel walls appear as green spots; DPNs loaded with lipid-rhodamine B chains appear as red spots; red blood cells (RBCs) labeled with DiD appear as “moving” blue spots; Kuppfer cells in the liver that have internalized RBCs appear as “firm” blues spots. A.-D. Representative image showing s-DPNs and r-DPNs moving within the complex liver microvasculature and not moving DPNs (dashed white ovals around red spots). E. Bar chart summarizing the level of DPN accumulation with the liver, both as sequestered by the Kupffer cells and adhering to the vasculature. (n = 3, 4 to 5 regions of interest per mice). (** p < 0.001).


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Figure 5. Intravital Microscopy analysis of tumor deposition of Discoidal Polymeric Nanoconstructs (DPNs). The DPN deposition in the tumor vascular bed was investigated via intravital microscopy (IVM) in Tie-2 GFP+ mice. In the representative IVM images and Supporting Movies, the endothelial cells lining the blood vessel walls appear as green spots; DPNs loaded with lipid-rhodamine B chains appear as red spots; red blood cells (RBCs) labeled with DiD appear as “moving” blue spots; macrophages that have internalized RBCs appear as “firm” blues spots. A.-B. Representative image showing s-DPNs and r-DPNs within the tortuous and abnormal tumor microvasculature. C. Bar chart summarizing the level of DPN accumulation with the tumor microvasculature. (n = 3, 4 to 5 regions of interest per mice). D. Representative confocal IVM images acquired over different z-planes demonstrating that DPNs are homed within the blood vessel walls.


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Figure 6. Analysis of macrophages interaction with Discoidal Polymeric Nanoconstructs (DPNs). DPNs were incubated with Bone Marrow Derived Monocytes (BMDM) and J-774.A1 murine macrophages. A.-C. Representative fluorescent confocal microscopy images of BMDM co-incubated with 750 nm spherical polystyrene beads (PS; green spots); r-DPNs (red spots) and s-DPNs (red spots). D. Bar chart quantifying the number of DPNs and PS uptaken per unit area as compared to the initial number of incubated particles. E. Flow cytometry analysis for the cellular uptake of DPNs by J-774.A1 macrophages as a function of time (4 and 24h incubation) and mechanical stiffness (s-DPNs vs r-DPNs).


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References:

1.

Hubbell, J. A.; Chilkoti, A. Chemistry. Nanomaterials for Drug Delivery. Science 2012,

337, 303-305. 2.

Petros, R. A.; DeSimone, J. M. Strategies in the Design of Nanoparticles for Therapeutic

Applications. Nat Rev Drug Discovery 2010, 9, 615-627. 3.

Ferrari, M. Cancer Nanotechnology: Opportunities and Challenges. Nat Rev Cancer

2005, 5, 161-171. 4.

Davis, M. E.; Chen, Z. G.; Shin, D. M. Nanoparticle Therapeutics: an Emerging

Treatment Modality for Cancer. Nat Rev Drug Discovery 2008, 7, 771-782. 5.

Peer, D.; Karp, J. M.; Hong, S.; FaroKHzad, O. C.; Margalit, R.; Langer, R. Nanocarriers

as an Emerging Platform for Cancer Therapy. Nat Nanotechnol 2007, 2, 751-760. 6.

Mitragotri, S.; Lahann, J. Physical Approaches to Biomaterial Design. Nat Mater 2009, 8,

15-23. 7.

Chauhan, V. P.; Jain, R. K. Strategies for Advancing Cancer Nanomedicine. Nat Mater

2013, 12, 958-962. 8.

van de Ven, A. L.; Kim, P.; Haley, O.; Fakhoury, J. R.; Adriani, G.; Schmulen, J.;

Moloney, P.; Hussain, F.; Ferrari, M.; Liu, X.; Yun, S. H.; Decuzzi, P. Rapid Tumoritropic Accumulation of Systemically Injected Plateloid Particles and their Biodistribution. J Controlled Release 2012, 158, 148-155. 9.

Wang, J.; Byrne, J. D.; Napier, M. E.; DeSimone, J. M. More Effective Nanomedicines

through Particle Design. Small 2011, 7, 1919-1931.


Page 39 of 44

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10.

Northfelt, D. W.; Dezube, B. J.; Thommes, J. A.; Miller, B. J.; Fischl, M. A.; Friedman-

Kien, A.; Kaplan, L. D.; Du Mond, C.; Mamelok, R. D.; Henry, D. H. Pegylated-Liposomal Doxorubicin versus Doxorubicin, Bleomycin, and Vincristine in the Treatment of AIDS-Related Kaposi's Sarcoma: Results of a Randomized Phase III Clinical Trial. J Clin Oncol 1998, 16, 2445-2451. 11.

Gill, P. S.; Wernz, J.; Scadden, D. T.; Cohen, P.; Mukwaya, G. M.; vonRoenn, J. H.;

Jacobs, M.; Kempin, S.; Silverberg, I.; Gonzales, G.; Rarick, M. U.; Myers, A. M.; Shepherd, F.; Sawka, C.; Pike, M. C.; Ross, M. E. Randomized Phase III Trial of Liposomal Daunorubicin versus Doxorubicin, Bleomycin, and Vincristine in AIDS-Related Kaposi's Sarcoma. J Clin Oncol 1996, 14, 2353-2364. 12.

Gradishar, W. J.; Tjulandin, S.; Davidson, N.; Shaw, H.; Desai, N.; Bhar, P.; Hawkins,

M.; O'Shaughnessy, J. Phase III Trial of Nanoparticle Albumin-Bound Paclitaxel Compared with Polyethylated Castor Oil-Based Paclitaxel in Women with Breast Cancer. J Clin Oncol 2005, 23, 7794-7803. 13.

Goyen, M.; Edelman, M.; Perreault, P.; O'Riordan, E.; Bertoni, H.; Taylor, J.; Siragusa,

D.; Sharafuddin, M.; Mohler, E. R., 3rd; Breger, R.; Yucel, E. K.; Shamsi, K.; Weisskoff, R. M. MR Angiography of Aortoiliac Occlusive Disease: a Phase III Study of the Safety and Effectiveness of the Blood-Pool Contrast Agent MS-325. Radiology 2005, 236, 825-833. 14.

Torchilin, V. P. Multifunctional Nanocarriers. Adv Drug Delivery Rev 2006, 58, 1532-

1555. 15.

McCarthy, J. R.; Weissleder, R. Multifunctional Magnetic Nanoparticles for Targeted

Imaging and Therapy. Adv Drug Delivery Rev 2008, 60, 1241-1251.


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16.

Page 40 of 44

Khemtong, C.; Kessinger, C. W.; Gao, J. Polymeric Nanomedicine for Cancer MR

Imaging and Drug Delivery. Chem Commun (Cambridge, U. K.) 2009, 3497-3510. 17.

Massoud, T. F.; Gambhir, S. S. Molecular Imaging in Living Subjects: Seeing

Fundamental Biological Processes in a New Light. Genes Dev 2003, 17, 545-580. 18.

Mitragotri, S.; Lahann, J. Physical Approaches to Biomaterial Design. Nat Mater 2009, 8,

15-23. 19.

Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers

as an Emerging Platform for Cancer Therapy. Nat Nanotechnol 2007, 2, 751-760. 20.

Decuzzi, P.; Godin, B.; Tanaka, T.; Lee, S. Y.; Chiappini, C.; Liu, X.; Ferrari, M. Size

and Shape Effects in the Biodistribution of Intravascularly Injected Particles. J Controlled Release 2010, 141, 320-327. 21.

Cabral, H.; Matsumoto, Y.; Mizuno, K.; Chen, Q.; Murakami, M.; Kimura, M.; Terada,

Y.; Kano, M. R.; Miyazono, K.; Uesaka, M.; Nishiyama, N.; Kataoka, K. Accumulation of sub100 nm Polymeric Micelles in Poorly Permeable Tumours Depends on Size. Nat Nanotechnol 2011, 6, 815-823. 22.

Moghimi, S. M.; Szebeni, J. Stealth Liposomes and Long Circulating Nanoparticles:

Critical Issues in Pharmacokinetics, Opsonization and Protein-Binding Properties. Prog Lipid Res 2003, 42, 463-478. 23.

Zhang, L.; Chan, J. M.; Gu, F. X.; Rhee, J. W.; Wang, A. Z.; Radovic-Moreno, A. F.;

Alexis, F.; Langer, R.; Farokhzad, O. C. Self-Assembled Lipid-Polymer Hybrid Nanoparticles: a Robust Drug Delivery Platform. ACS Nano 2008, 2, 1696-1702. 24.

Jain, R. K.; Stylianopoulos, T. Delivering Nanomedicine to Solid Tumors. Nat Rev Clin

Oncol 2010, 7, 653-664.


Page 41 of 44

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

ACS Nano

25.

Albanese, A.; Tang, P. S.; Chan, W. C. The Effect of Nanoparticle Size, Shape, and

Surface Chemistry on Biological Systems. Annu Rev Biomed Eng 2012, 14, 1-16. 26.

Gratton, S. E.; Pohlhaus, P. D.; Lee, J.; Guo, J.; Cho, M. J.; Desimone, J. M.

Nanofabricated Particles for Engineered Drug Therapies: a Preliminary Biodistribution Study of PRINT Nanoparticles. J Controlled Release 2007, 121, 10-18. 27.

Tasciotti, E.; Liu, X.; Bhavane, R.; Plant, K.; Leonard, A. D.; Price, B. K.; Cheng, M. M.;

Decuzzi, P.; Tour, J. M.; Robertson, F.; Ferrari, M. Mesoporous Silicon Particles as a Multistage Delivery System for Imaging and Therapeutic Applications. Nature Nanotechnol 2008, 3, 151157. 28.

Champion, J. A.; Katare, Y. K.; Mitragotri, S. Particle Shape: a New Design Parameter

for Micro- and Nanoscale Drug Delivery Carriers. J Controlled Release 2007, 121, 3-9. 29.

Glangchai, L. C.; Caldorera-Moore, M.; Shi, L.; Roy, K. Nanoimprint Lithography Based

Fabrication of Shape-Specific, Enzymatically-Triggered Smart Nanoparticles. J Controlled Release 2008, 125, 263-272. 30.

Acharya, G.; Shin, C. S.; McDermott, M.; Mishra, H.; Park, H.; Kwon, I. C.; Park, K.

The Hydrogel Template Method for Fabrication of Homogeneous Nano/Microparticles. J Controlled Release 2010, 141, 314-319. 31.

Tao, L.; Zhao, X. M.; Gao, J. M.; Hu, W. Lithographically Defined Uniform Worm-

Shaped Polymeric Nanoparticles. Nanotechnology 2010, 21, 095301. 32.

Decuzzi, P.; Pasqualini, R.; Arap, W.; Ferrari, M. Intravascular Delivery of Particulate

Systems: Does Geometry Really Matter? Pharm Res 2009, 26, 235-243. 33.

Muro, S.; Garnacho, C.; Champion, J. A.; Leferovich, J.; Gajewski, C.; Schuchman, E.

H.; Mitragotri, S.; Muzykantov, V. R. Control of Endothelial Targeting and Intracellular


ACS Nano

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

Page 42 of 44

Delivery of Therapeutic Enzymes by Modulating the Size and Shape of ICAM-1-Targeted Carriers. Mol Ther 2008, 16, 1450-1458. 34.

Huang, X.; Li, L.; Liu, T.; Hao, N.; Liu, H.; Chen, D.; Tang, F. The Shape Effect of

Mesoporous Silica Nanoparticles on Biodistribution, Clearance, and Biocompatibility in vivo. ACS Nano 2011, 5, 5390-5399. 35.

Gratton, S. E.; Ropp, P. A.; Pohlhaus, P. D.; Luft, J. C.; Madden, V. J.; Napier, M. E.;

DeSimone, J. M. The Effect of Particle Design on Cellular Internalization Pathways. Proc Natl Acad Sci USA 2008, 105, 11613-11618. 36.

Sharma, G.; Valenta, D. T.; Altman, Y.; Harvey, S.; Xie, H.; Mitragotri, S.; Smith, J. W.

Polymer Particle Shape Independently Influences Binding and Internalization by Macrophages. J Controlled Release 2010, 147, 408-412. 37.

Agarwal, R.; Singh, V.; Jurney, P.; Shi, L.; Sreenivasan, S. V.; Roy, K. Mammalian Cells

Preferentially Internalize Hydrogel Nanodiscs over Nanorods and Use Shape-Specific Uptake Mechanisms. Proc Natl Acad Sci USA 2013, 110, 17247-17252. 38.

Zhao, F.; Zhao, Y.; Liu, Y.; Chang, X.; Chen, C. Cellular Uptake, Intracellular

Trafficking, and Cytotoxicity of Nanomaterials. Small 2011, 7, 1322-1337. 39.

Ferrati, S.; Mack, A.; Chiappini, C.; Liu, X.; Bean, A. J.; Ferrari, M.; Serda, R. E.

Intracellular Trafficking of Silicon Particles and Logic-Embedded Vectors. Nanoscale 2010, 2, 1512-1520. 40.

Chithrani, B. D.; Chan, W. C. Elucidating the Mechanism of Cellular Uptake and

Removal of Protein-Coated Gold Nanoparticles of Different Sizes and Shapes. Nano Lett 2007, 7, 1542-1550.


Page 43 of 44

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

ACS Nano

41.

Beningo, K. A.; Wang, Y. L. Fc-Receptor-Mediated Phagocytosis is Regulated by

Mechanical Properties of the Target. J Cell Sci 2002, 115, 849-856. 42.

Choi, H. S.; Liu, W.; Misra, P.; Tanaka, E.; Zimmer, J. P.; Itty Ipe, B.; Bawendi, M. G.;

Frangioni, J. V. Renal Clearance of Quantum Dots. Nat Biotechnol 2007, 25, 1165-1170. 43.

Browning, M. B.; Cereceres, S. N.; Luong, P. T.; Cosgriff-Hernandez, E. M.

Determination of the in vivo Degradation Mechanism of PEGDA Hydrogels. J Biomed Mater Res Part A 2014, 102, 4244-4251. 44.

Lartigue, L.; Alloyeau, D.; Kolosnjaj-Tabi, J.; Javed, Y.; Guardia, P.; Riedinger, A.;

Pechoux, C.; Pellegrino, T.; Wilhelm, C.; Gazeau, F. Biodegradation of Iron Oxide Nanocubes: High-Resolution in situ Monitoring. ACS Nano 2013, 7, 3939-3952. 45.

Merkel, T. J.; Jones, S. W.; Herlihy, K. P.; Kersey, F. R.; Shields, A. R.; Napier, M.;

Luft, J. C.; Wu, H.; Zamboni, W. C.; Wang, A. Z.; Bear, J. E.; DeSimone, J. M. Using Mechanobiological Mimicry of Red Blood Cells to Extend Circulation Times of Hydrogel Microparticles. Proc Natl Acad Sci USA 2011, 108, 586-591. 46.

Anselmo, A. C.; Zhang, M.; Kumar, S.; Vogus, D. R.; Menegatti, S.; Helgeson, M. E.;

Mitragotri, S. Elasticity of Nanoparticles Influences their Blood Circulation, Phagocytosis, Endocytosis, and Targeting. ACS Nano 2015, 9, 3169-3177. 47.

Parodi, A.; Quattrocchi, N.; van de Ven, A. L.; Chiappini, C.; Evangelopoulos, M.;

Martinez, J. O.; Brown, B. S.; Khaled, S. Z.; Yazdi, I. K.; Enzo, M. V.; Isenhart, L.; Ferrari, M.; Tasciotti, E. Synthetic Nanoparticles Functionalized with Biomimetic Leukocyte Membranes Possess Cell-Like Functions. Nature Nanotechnol 2013, 8, 61-68.


ACS Nano

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

48.

Sokolov, I.; Dokukin, M. E.; Guz, N. V. Method for Quantitative Measurements of the

Elastic Modulus of Biological Cells in AFM Indentation Experiments. Methods 2013, 60, 202213. 49.

Serda, R. E.; Gu, J.; Bhavane, R. C.; Liu, X.; Chiappini, C.; Decuzzi, P.; Ferrari, M. The

Association of Silicon Microparticles with Endothelial Cells in Drug Delivery to the Vasculature. Biomaterials 2009, 30, 2440-2448. 50.

Lee, T. R.; Greene, M. S.; Jiang, Z.; Kopacz, A. M.; Decuzzi, P.; Chen, W.; Liu, W. K.

Quantifying Uncertainties in the Microvascular Transport of Nanoparticles. Biomech Model Mechanobiol 2014, 13, 515-526. 51.

Lee, T. R.; Choi, M.; Kopacz, A. M.; Yun, S. H.; Liu, W. K.; Decuzzi, P. On the Near-

Wall Accumulation of Injectable Particles in the Microcirculation: Smaller is not Better. Sci Rep 2013, 3, 2079. 52.

Godin, B.; Chiappini, C.; Srinivasan, S.; Alexander, J. F.; Yokoi, K.; Ferrari, M.;

Decuzzi, P.; Liu, X. Discoidal Porous Silicon Particles: Fabrication and Biodistribution in Breast Cancer Bearing Mice. Advanced functional materials 2012, 22, 4225-4235.


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