Abstract
Polymeric expansile nanoparticles (eNPs) that respond to a mildly acidic environment by swelling with water and expanding 2–10 X in diameter represent a new responsive drug delivery system. Here, we use a variety of techniques to characterize the pH- and time-dependence of eNP swelling as this is a key property responsible for the observed in vitro and in vivo performance of eNPs. Results demonstrate a significant change in eNP volume (>350 X) at pH 5.0 as seen using: scanning electron microscopy (SEM), conventional transmission electron microscopy (TEM), freeze-fracture transmission electron microscopy (ff-TEM), fluorescence microscopy, and a new nanopore based characterization technology, the qNano, which measures both individual particle size as well as overall particle concentration in situ using tunable resistive pulse sensing. eNP swelling occurs in a continuous and yet heterogeneous manner over several days and is pH dependent.
Keywords: pH-responsive, nanoparticles, qNano, swelling, electron microscopy, paclitaxel, drug delivery, resistive pulse sensing, hydrogel
Introduction
Nanoparticles (NPs) are extensively investigated as a means to increase drug solubility, alter biodistribution, target specific sites within the body, and minimize drug side effects.1–14 Of the various particle formulations, polymer-based systems offer a number of advantages such as biocompatibility, biodegradability, ease of preparation and availability: PLGA nanoparticles are one the most widely used polymer particle systems because they are readily prepared, and this specific copolymer of lactic and glycolic acid is used in current medical products. However, recent studies have reported that the control of drug release from PLGA NPs is limited, as this system delivers a significant portion of its payload extracellularly, lacks a triggering or responsive property to concentrate its payload at a specific site, and, often requires incorporation of a ligand to achieve cellular targeting.15 Even with these limitations, PLGA NPs have demonstrated some success in a number of in vivo studies.16 Nonetheless, significant research efforts are focused on many areas in order to achieve better overall NP performance, including the development of NPs that respond to external (e.g., light) or internal biological (e.g. pH or oxidative stress) cues.17–19
Responsive nanoparticle systems can be synthesized from a variety of materials, each imparting a unique functionality to the system.17, 18 Fig. 1 outlines several broad categories of stimuli, such as pH, temperature, light, oxidation/reduction, or osmolality increases/decreases, that may be used to trigger various nanoparticle responses, such as degradation, swelling/shrinking, particle inversion/shape change, or hydrophobic/hydrophilic conformational changes. Recent examples of such responsive systems include pH-responsive polyacids, such as poly(acrylic acid) (PAAc), or polybases, such as poly(N,N’-dimethyl aminoethyl methacrylate) (PDMAEMA), that are protonated or de-protonated depending on local pH with the resulting ionic interactions frequently resulting in net swelling or collapse of the material.14, 20, 21 The pH- profile and hydrophobic/hydrophilic characteristics of these polymers can be tuned by selection of the appropriate monomer units.22 Numerous temperature responsive polymers stem from the poly(N-isopropylacrylamide) (PNIPAAm) family of materials having lower critical solution temperatures (LCSTs) in the physiological range (32–42 °C) at which the material experiences a net volume contraction or expansion.20, 21 Photo-responsive particles are obtained by introducing photo-chromic molecules, such as azobenzene, which can undergo cis-trans isomerization.20 Reactive oxygen species, which are generally up-regulated in pathologic tissues and are found in cell lysosomes, may also trigger conformational, hydrophobic/hydrophilic or size/shape changes.23–25 Enzymatic cleavage (e.g. lysosomal glutathione cleavage of disulfide bonds)23 also represents a commonly used trigger.21
Of these many triggers, pH-responsive materials are frequently selected for applications involving delivery to tumors or sites of inflammation due to the lower pH profile ubiquitous in these tissues.23, 26, 27 Several pH-responsive nanoparticle delivery systems have been developed. Among others, Murthy and collaborators have used pH-responsive polymeric particles to increase the uptake and endosomal release of oligonucleotides in hepatic cells.28 Jabr-Milane and coworkers used poly(β-amino esters) (PbAE), which are hydrophobic at physiologic pH but rapidly dissolve at pH < 6.5 (i.e., pH found in tumor microenvironments and endosomes), to achieve intracellular delivery of Pax.29 Frechet and coworkers synthesized pH-responsive nanoparticles from acetylated dextran polymers as delivery agents for vaccines.30–33 Almutairi et. al. have utilized dual pH-/oxidative-stress responsive particles to modulate intracellular burst release of drugs.25 These examples illustrate several applications of pH-responsive nanoparticles. For an in depth review, we refer the reader to reviews by Ganta23 (2008), Motornov21 (2010), and Colson19 (2012).
Our interest is in responsive polymeric nanoparticles that respond to a pH-trigger in order to deliver drug intracellularly. However, instead of relying on particle degradation or dissolution to achieve drug release, we utilize particle swelling. Due to their pH-induced expansion at mildly acidic conditions, such as those found within the cellular endosome, we refer to these particles as “expansile nanoparticles” or eNPs. The mechanism of action of these particles is outlined in Fig. 2. The motivation for the studies described herein stems from recently published results demonstrating that eNPs, when loaded with the chemotherapeutic agent paclitaxel (Pax), are superior to traditional methods of Pax delivery using Cremophor/ethanol (Pax-C/E) (or non-expansile particles) in several in vivo models. Specifically, paclitaxel loaded expansile nanoparticles (Pax-eNPs) are efficacious in murine models of Lewis Lung Carcinoma, breast carcinoma, and human malignant peritoneal mesothelioma.34–37 The ability of Pax-eNPs to prevent tumor establishment and/or delay tumor recurrence is hypothesized to be a consequence of: 1) local delivery to the site of the primary cancer and uptake by the tumor;34 2) lymphatic trafficking of the eNPs and delivery of Pax to lymph nodes;37, 38 and 3) the expansion of the eNP following cellular uptake and its resulting ability to act as an intracellular depot for Pax.39 Given the importance of eNP composition, reactivity, and resulting swelling for its in vivo performance, we now investigate these characteristics by studying the size, structure, and morphology of eNPs in the condensed and expanded state. We use a combination of traditional particle characterization modalities such as scanning electron microscopy (SEM), conventional transmission electron microscopy (TEM), freeze-fracture transmission electron microscopy (ff-TEM) and fluorescence microscopy, as well as a new nanopore-based characterization technology, the qNano, which measures both individual particle size as well as overall particle concentration in situ using tunable resistive pulse sensing.
Results and Discussion
Expansile Nanoparticle Synthesis and Control of Particle Size
Expansile nanoparticle monomer and crosslinker were prepared according to a previously reported method40 with slight modifications to improve yield and purity (see Figure 2, SI, Fig. S1). In brief, 2,4,6-trimethoxybenzaldehyde and 1,1,1-tris(hydroxymethyl)ethane were reacted in the presence of a catalytic amount of H2SO4 to produce an intermediate, (5-methyl-2-(2,4,6-trimethoxyphenyl)-1,3-dioxan-5-yl)methanol, which was further reacted with methacryloyl chloride in the presence of TEA to produce the monomer unit, (5-methyl-2-(2,4,6-trimethoxyphenyl)-1,3-dioxan-5-yl)methyl methacrylate. The crosslinker, 1,4-phenylene bis(2-methylacrylate), was synthesized by reacting hydroquinone with methacryloyl chloride in the presence of TEA.
Nanoparticles were prepared using a miniemulsion oil-in-water suspension created via sonication followed by polymerization of the acrylic monomer and crosslinker in the presence of a free radical initiator (Fig. 3). Three different polymerization reactions were investigated including: tetramethylethylenediamine (TEMED) with ammonium peroxydisulfate (APS), azobisisobutyronitrile (AIBN) in the presence of heat, and eosin Y with 1-vinyl-2-pyrrolidinone under exposure to unfiltered light from a xenon arc lamp. Due to ease of use and consistency of particle polymerization, the TEMED/APS method was used to prepare eNPs for all of the subsequent nanoparticle characterization studies.
Nanoparticle size is controlled by adjusting the amount of surfactant, in this case sodium dodecyl sulfate (SDS), in the aqueous phase as well as the amount of sonication energy used to form the miniemulsion (Fig. 3). Dynamic light scattering (DLS) results show that the average size of eNPs depends logarithmically (R2 = 0.99) on the ratio of surfactant to monomer with higher ratios of SDS:monomer leading to smaller particles (size range 20 nm – 300 nm) (see SI, Fig. S2 a). Similarly, eNP size also depends logarithmically (R = 0.95) on the sonication energy used to create the mini-emulsion with higher sonication energies leading to smaller particles (size range 150 nm – 900 nm) (see SI, Fig. S2 b).
In order to confirm the size measurements obtained via DLS, we performed scanning electron microscopy (SEM) on the nanoparticle suspensions. In all cases, SEM images revealed polydisperse size distributions with the largest population of particles (by number) between 20 nm – 50 nm (Fig. 4, left). By filtering the particles with a 0.22 µm filter, the smaller population of particles was isolated from the larger population (Fig. 4, bottom). The discrepancy in particle size measured using DLS and SEM can be explained by DLS’s bias toward disproportionately weighting the size of larger particles.41
Expansile Nanoparticle Swelling
Previously published characterizations of eNP swelling using DLS demonstrate a ~10X increase in eNP diameter after 1 day of exposure to pH 5.40 Particle swelling closely follows the hydrolysis of the pH-labile protecting group (Fig. 2), and release of Pax from the eNP is triggered by particle swelling at pH 5.40 To further characterize the differences in size and morphology of eNPs in the unswollen v. swollen state, we used a variety of techniques including scanning electron microscopy, conventional transmission electron microscopy, freeze-fracture transmission electron microscopy, fluorescence microscopy, and tunable resistive pulse sensing. Cumulatively, these characterization modalities allow interrogation of particle size and morphology both in aqueous suspension and in dried or lyophilized form.
Electron Microscopy
Expansile nanoparticles maintained at pH 5.0 or pH 7.4 for up to 3 days were examined using SEM (Fig. 5). Particles maintained at neutral pH ranged from 20 nm - 200 nm in diameter whereas those exposed to acidic pH 5.0 increase in diameter with sizes ranging from 200 nm to 2 µm, or more. In addition to SEM, freeze-fracture transmission electron microscopy (ff-TEM) was used to examine the nanoparticles. ff-TEM was chosen because it allows preservation and replication of the state of the particle in solution. Expansile nanoparticles maintained at pH 7.4 appeared as spherical 50 nm - 200 nm structures, while eNPs maintained at pH 5.0 appeared as larger irregular structures approximately 1 µm in diameter (Fig. 6). In the pH 5.0 samples, a distinct lack of smaller 50 nm - 200 nm structures was observed
Lastly, we employed conventional transmission electron microscopy (TEM) to visualize eNPs labeled with a covalently bound iodine co-monomer. The results revealed that, following 3 days at pH 5.0, eNPs swell into a heterogeneous population of particles spanning a wide range of swollen or semi-swollen states and, as before, controls maintained at pH 7.4 show no significant changes (Fig. 7). Of particular note are four broadly defined phases of swelling observed in the “swollen” population of eNPs outlined in Fig. 7, including, from least to most swollen: 1) particles showing little or no swelling; 2) particles with an unswollen core and an expanding corona that accounts for ≤50% of the particle diameter; 3) particles with a small unswollen core and large swollen corona ≥50% of the particle diameter; and, 4) particles that have swollen so far they lack a solid core and exhibit a diminishing overall diameter, which we hypothesize occurs as the ester crosslinks are cleaved and polymer chains diffuse away. It is expected that, eventually, all particles will swell to this fourth state. The morphology and ringed structure of the unswollen-core/swollen-shell (or corona) eNPs seen in Fig. 7 is reminiscent of core-shell nanoparticles imaged with TEM, such as gold-core silica-shell nanoparticles, platinum-maghemite core-shell nanoparticles, and nanocapsules synthesized by Chen, Teng, Wang, and Liz-Marzan, respectively.42–45 These results demonstrate that eNP swelling is a continuous, prolonged, process which, due to particle-to-particle heterogeneity, results in a broad distribution of particle features, morphologies and sizes at any single time point.
Fluorescence Microscopy
In order to optically observe and characterize individual eNPs, we prepared larger particles of ~450 nm in diameter doped with a fluorescent, covalently incorporated, rhodamine co-monomer. Particle diameters were measured after 20 hours using post-acquisition size calibration in Image J. Results showed eNPs held at neutral pH 7.4 have an average diameter of 479 ± 257 nm. In contrast, eNPs maintained at acidic pH 5.0 for 20 hours are significantly (p < 0.001) larger with an average diameter of 1556 ± 1274 nm and a distribution of diameters ranging from several hundred to several thousand nanometers (Fig. 8). Similarly to the TEM data above, these results once again reinforce the heterogeneous nature of eNP swelling, wherein particles swell to various degrees and at different absolute rates.
It is of note that the swollen structures observed in both ff-TEM (Fig. 6) and fluorescence microscopy studies (Fig. 8, insert) have a similar, characteristic irregularity to their surface features. This ruffled appearance was not observed using SEM; rather, the swollen particles appeared smooth and spherical. It is possible that the vacuum drying/sputter coating process required for SEM preparation smoothed over and homogenized the particle surface. These data illustrate the need for using multiple modalities when investigating nanoparticle features and morphology.
qNano: Two Point Swelling Measurement
Having studied the morphological changes in eNPs as they swell with time, we next quantified the overall population dynamics of eNP swelling, and, specifically, further probed the heterogeneity of swelling observed using TEM and fluorescence microscopy regarding absolute size and rate of swelling. For these studies, we used a qNano, which utilizes a tunable resistive pulse sensing technique to characterize particles. Specifically, this technology uses a single, tunable nanopore cut into an elastomeric membrane to size nano- and microparticles on a particle-by-particle basis.46–50 The lower limit of size detection on the qNano is ~70 nm while the upper limit is tens of microns. Relative size is determined in an aqueous environment by measuring the decrease in electrical current passing through the nanopore as particles obstruct the pore during translocation from one side to the other. Absolute size is determined by comparing the magnitude of the decrease in electrical current of an unknown particle and a known, standard calibration particle at the same running conditions (i.e., pore type, stretch, electrolyte, pressure, and applied voltage).
To determine concentration, an unknown sample is measured at several different externally applied pressures, each of which produces different rates of particle translocation through the pore—greater applied pressures result in nanoparticles passing through the pore faster and thus higher translocation rates. A standard particle is similarly measured at multiple applied pressures using the same running conditions. A linear plot of pressure vs. translocation rate is then constructed for each sample. Particle concentration is directly related to the slope of the pressure vs. translocation rate graph and, by taking the ratios of the slopes of the known and unknown samples, the concentration of the standard is used to determine the concentration of the unknown sample.47
Pilot studies to investigate eNP swelling by qNano demonstrated results consistent with electron and fluorescence microscopy experiments. Expansile nanoparticles showed a significant (p < 0.001) change in size between those exposed to pH 7.4 (98 ± 51 nm) and those exposed to pH 5.0 (673 ± 461 nm) for 3 days (Fig. 9).
qNano: Time Course Swelling Measurement
Each qNano nanopore has a dynamic range over which its performance is optimal. If it is stretched too little, the pore will not open to fill with electrolyte and allow translocation of particles. If stretched too far, the pore will tear and become unusable. Table 1 gives the available pores and their optimal dynamic ranges.51 In practice, the dynamic range for larger pores is greater than the values given in Table 1; i.e., an NP800 can be stretched wide enough to measure 2,000 nm particles without causing damage. However, due to the limits of the dynamic range on any given pore, it is impossible to measure the size of both small (100 nm - 200 nm) and large (500 nm - 2,000 nm) particles simultaneously. The qNano measurements of eNP swelling in Fig. 9 were performed using a single NP400 nanopore. It was, therefore, possible that unswollen particles smaller than 200 nm were present in the pH 5.0 population but were not being detected.
Table 1.
Nanopore | Dynamic Range (nm) |
---|---|
NP100 | 70–200 |
NP200 | 100–400 |
NP400 | 200–800 |
NP800 | 400–1,600 |
NP1000 | 500–2,000 |
To address this concern, as well as to investigate the time course evolution of eNP swelling with a focus on particle sub-populations, we performed a further investigation. Slightly larger particles (250 nm), which are more easily measured on the qNano, were exposed to acidic and neutral conditions as before and the diameter measured by qNano at time 0 and at subsequent time points after 3 days and 5 days of swelling. At each time point, particle size was measured on both a small (NP400) and large (NP800) nanopore (combined dynamic range 200 nm - 2,000 nm) to ensure neither large nor small particles were excluded from the measurement. Additionally, particle concentration was determined at each time point and on each nanopore using a previously published technique described in the previous section and supporting information.47
Expansile nanoparticles showed an overall increase in diameter upon exposure to pH 5.0 (Fig. 10) from an average diameter of 221 ± 36 nm to 418 ± 112 nm and 1,230 ± 469 nm on day 0, 3, and 5, respectively (Fig. 10, right). No change in size was observed at neutral pH 7.4 (data not shown). Furthermore, the initial eNP concentration was 8.5 × 1011 particles/mL and this decreased to 2.3 × 1011 and 3.9 × 1010 particles/mL on day 3 and day 5, respectively (Fig. 11, bottom). This decrease in overall particle concentration may be indicative of eNP degradation following swelling, as noted in previous sections. By measuring both small and large particles separately, on suitable pores, at each time point, it was confirmed that particle swelling occurs heterogeneously within the eNP population. The percent of particles measured on the small pore, as determined by particle concentration, decreased with time from > 99% to 72% to 10% at 0, 3, and 5 days, respectively, while the average diameter measured on this pore did not change significantly (215 ± 35 nm, 240 ± 55 nm, and 246 ± 40 nm respectively). Concurrently, the percent of particles measured on the large pore increased from < 1% to 28% to 90% at 0, 3, and 5 days, respectively, with the average diameter increasing significantly (p < 0.0001) from 876 ± 259 nm to 1339 ± 516 nm from day 3 to 5 (Fig. 11, top).
qNano: Deformation of Swollen eNPs
The data presented thus far demonstrate that eNP swelling is a continuous process wherein the particle transforms from a condensed, hydrophobic particle into a swollen, more hydrophilic, hydrogel-like structure. We therefore hypothesized that, in the swollen state, the eNP may become soft and deformable with the mass of polymer that is in a ~100 nm diameter structure now spread throughout a ~1000 nm diameter structure. To test this hypothesis, we used the dynamic tunability of the qNano pores.
Figures 12 and 13 illustrate the premise behind this study. Particles, either swollen or unswollen, will not translocate the pore if the pore is much smaller than the diameter of the particle; in these cases a flat current trace is observed. In contrast, when the pore is opened to a size larger than the eNP, either swollen or unswollen, the particle will translocate the pore resulting in a single, narrow downward spike in the basal current. For swollen eNPs, if the pore is decreased to approximately the same diameter as the particle, the eNP can be squeezed through the pore. Due to the interaction between the particle and pore, the translocation occurs more slowly, resulting in a broadened downward peak in the basal current. This intermediate behavior is not observable for unswollen eNPs thus confirming that the peak broadening is due to the softened, hydrogel-like state of the water swollen eNP. A representative signal trace from eNPs at pH 5.0 that are either “squeezed” through a small pore or allowed to pass cleanly through a large pore (broad v. narrow translocation peaks, respectively) is given in Fig. 13.
To quantify the difference between these translocation behaviors, we measured the full-width half-maximum (FWHM) translocation duration (TD) for unswollen-eNPs, swollen-eNPs, and swollen-eNPs squeezed through the nanopore (Fig. 14). The mean TD for particles at pH 7.4 (0.13 ± 0.17 ms) and un-squeezed eNPs at pH 5.0 (0.15 ± 0.13 ms) were not significantly different (p = 0.42). However, the TD of eNPs at pH 5.0 squeezed through the pore (26.4 ± 47.8 ms) was significantly (p < 0.0001) longer than for either of the previous cases.
It is known that particle TD increases with decreasing pore size due to the increased excluded particle volume as well as the decreased velocity of the fluid (and, hence, the particle) through the pore.52 However, this increase in TD is typically no greater than 50% of the duration for a 20% change in pore size,53 and, therefore, another mechanism must be responsible for the observed increase of over two orders of magnitude in TD.
Unusual signal traces from resistive pulse sensing measurements have been attributed to multiple phenomena, however the reported observations do not satisfactorily explain the current eNP swelling data. For example, Willmott et al. used magnetic fields to cause aggregation and increased translocation rates of super-paramagnetic beads with blockades characterized by multiple overlapped peaks and thus overall increased TD.54 However, in the current study, the overall particle concentration (and thus translocation rate) decreases with eNP swelling (Fig. 11) thus making the likelihood of multiple, nearly simultaneous, overlapping translocations negligible. Furthermore, the signal traces in Fig. 13 resemble a single translocation event rather than multiple superimposed translocations observed by Willmott et al. In a different study, Ang et. al. observed increased translocation durations due to increased particle aggregate size.55 However, the increase in duration (~4X) is significantly less than the increase observed in the current study (over two orders of magnitude different).
Another study by Platt et. al. suggests possible confirmation of the proposed mechanism of particle squeezing. The authors demonstrate that rod shaped nanoparticles have a dramatically increased TD.56 While swollen eNPs appear generally spherical, as seen via SEM, TEM, ff-TEM, and fluorescence microscopy, it is reasonable to suggest that squeezing a swollen eNP through a narrow pore elongates it therefore changing its aspect ratio to become more rod-like.
While it is possible that a combination of factors, such as those noted above and others (e.g. viscous boundary effects) contribute to the observed trend, it is clear that there is both an empirical and a quantitative difference in the way in which swollen eNPs translocate the pore.
Hydrophobicity of eNPs with Particle Swelling
Having characterized the change in eNP diameter and morphology as a function of both time and pH, we next investigated the relative hydrophobicity of the eNP interior as a function of these variables. Previous studies have shown that the hydrophobic drug paclitaxel (Pax) can be readily loaded into eNPs with an efficiency over 80%.40 Additionally, Pax has an affinity for the swollen eNP that causes it to partition into these particles in a 4:1 ratio over an aqueous environment.39 These results suggest that, even as the eNP becomes more hydrophilic during swelling in a mildly acidic aqueous environment, it maintains sufficient hydrophobic character to preferentially sequester hydrophobic drugs from aqueous solution.
To characterize the hydrophobic nature of the eNP interior, we encapsulated covalently-bound pyrene—a hydrophobic, solvochromatic fluorophore—in the eNPs. Four peaks characterize pyrene’s fluorescence emission spectrum with the ratio of the peak I area to peak III area serving as an indicator of the hydrophobicity of pyrene’s environment (SI, Fig. S4, a). For example, in an aqueous environment, this ratio is 2.34 ± 0.11 while in dimethylsulfoxide (DMSO), which is slightly more hydrophobic, it is 2.05 ± 0.01 and in n-hexane, which is significantly more hydrophobic, it is 1.29 ± 0.01 (SI, Fig. S4, b). Monitoring the hydrophobicity of pyrene-eNPs revealed a significant (p < 0.001) increase in hydrophilicity with swelling at pH 5.0 from a ratio of peak I area to peak III area of 1.73 ± 0.01 to 1.83 ± 0.02; no significant change was observed at pH 7.4 (Fig. 15). Using a standard curve of pyrene-emission v. polarity index for various solvents (SI, Fig. S4, b), these changes correlate to an increase in hydrophilicity from an environment with a polarity similar to dichloromethane (DCM) or toluene to an environment with polarity between that of ethyl acetate and acetone.
This quantification supports our previously proposed method of eNP swelling (Fig. 2). Specifically, prior to swelling, hydrophobic eNPs form a suspension in water and are “immiscible” with their environment, much the same way DCM and water are immiscible. As eNPs swell and achieve a more hydrophilic character similar to that of acetone, they also become “miscible” with their environment and water infiltrates the polymer network forming a hydrogel-like structure. However, even as eNP hydrophilicity increases, the final character of the particles is still more hydrophobic than the aqueous environment at large. This persistent hydrophobicity is likely the origin of the drug-depot effect previously noted and published.39 Specifically, eNPs can act as intracellular depots for separately administered hydrophobic drugs and, therein, provide improved and sustained release of these drugs over prolonged periods of time.
Conclusions
In summary, we have characterized polymeric nanoparticles that expand at mildly acidic conditions using a variety of techniques. We observed a significant change in eNP volume (>350 X) using SEM, conventional TEM, ff-TEM, fluorescence microscopy, and responsive pulse sensing. eNP swelling occurs in a continuous and yet heterogeneous manner over several days. Lastly, the hydrophobicity of eNPs changes with swelling in accordance with the proposed mechanism of action leading to the drug-depot effects previously observed. Continued research with eNPs will increase our understanding of how this nanomaterial functions in vitro and in vivo. It will also advance the development of new materials that significantly improve the local delivery of chemotherapy to the sites of tumor with the goals of preventing tumor growth and recurrence while minimizing exposure to non-tumor bearing soft tissues.
Supplementary Material
Acknowledgments
The authors would like to acknowledge A. Griset, K. Zubris, M. Ericsson, and S. Yu for their support as well as J. Sutin and A. Meller for the use of their microscope.
This work was supported in part by funding from: National Science Foundation (DMR-1006601), Center for Integration of Medicine and Innovative Technologies (CIMIT #07-004), Boston University’s Nanomedicine Program and Cross-Disciplinary Training in Nanotechnology for Cancer (NIH R25 CA153955), Brigham and Women’s Hospital, and Boston University.
Footnotes
The authors have no conflicts of interest to declare.
Supporting information includes: chemical synthesis schemes for eNP monomer and crosslinker; DLS characterization of particle size as a function of synthesis parameters; pyrene fluorescence spectra and solvent standard curve; and, qNano operating parameters. This material is available free of charge via the Internet at http://xxxxxxxx.
Contributor Information
Yolonda L. Colson, Email: Yolonda_Colson@dfci.harvard.edu.
Mark W. Grinstaff, Email: mgrin@bu.edu.
Notes and references
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