Real-time analysis of composite magnetic nanoparticle disassembly in vascular cells and biomimetic media

Jillian E. Tengood; Ivan S. Alferiev; Kehan Zhang; Ilia Fishbein; Robert J. Levy; Michael Chorny

doi:10.1073/pnas.1324104111

Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved February 11, 2014 (received for review December 27, 2013)

Significance

Disassembly rates and patterns of nanoparticles used as drug carriers or diagnostic tools are important determinants of their performance and biocompatibility. Thus, understanding them is essential for the design and optimization of nanoparticle formulations for clinical use. Currently available strategies are not applicable to real-time, quantitative nanoparticle disassembly analysis directly in environments of interest, such as the cell interior or biomimetic media. In the present study, a quantitative strategy using Förster resonance energy transfer for monitoring integrity status of nanoparticles in situ was developed and applied for characterizing disassembly of biodegradable polymer-based magnetic nanoparticles in vascular cells and different types of model milieu. This methodology and study results may be relevant for a broad range of biomedical and analytical applications.

Abstract

The fate of nanoparticle (NP) formulations in the multifaceted biological environment is a key determinant of their biocompatibility and therapeutic performance. An understanding of the degradation patterns of different types of clinically used and experimental NP formulations is currently incomplete, posing an unmet need for novel analytical tools providing unbiased quantitative measurements of NP disassembly directly in the medium of interest and in conditions relevant to specific therapeutic/diagnostic applications. In the present study, this challenge was addressed with an approach enabling real-time in situ monitoring of the integrity status of NPs in cells and biomimetic media using Förster resonance energy transfer (FRET). Disassembly of polylactide-based magnetic NPs (MNPs) was investigated in a range of model biomimetic media and in cultured vascular cells using an experimentally established quantitative correlation between particle integrity and FRET efficiency controlled through adjustments in the spectral overlap between two custom-synthesized polylactide–fluorophore (boron dipyrromethene) conjugates incorporated in MNPs. The results suggest particle disassembly governed by diffusion–reaction processes with kinetics strongly dependent on conditions promoting release of oligomeric fragments from the particle matrix. Thus, incubation in gels simulating the extracellular environment and in protein-rich serum resulted in notably lower and higher MNP decomposition rates, respectively, compared with nonviscous liquid buffers. The diffusion–reaction mechanism also is consistent with a significant cell growth-dependent acceleration of MNP processing in dividing vs. contact-inhibited vascular cells. The FRET-based analytical strategy and experimental results reported herein may facilitate the development and inform optimization of biodegradable nanocarriers for cell and drug delivery applications.

Nanoparticles (NPs) of different compositions and designs are emerging as versatile diagnostic tools and promising carriers for delivery of small molecule drugs and biotherapeutics (1, 2). Among the prerequisites for their safe and effective use, an understanding of the fate of NPs intended for diagnostic or therapeutic applications is an obvious requirement, as it ensures the absence of acute or chronic toxicity caused by foreign materials retained in the body (3). Biodegradation of NPs developed as drug delivery carriers also plays a key role in their therapeutic performance by contributing to the biodistribution and fate of the cargo; thus, it is of essential importance for optimizing the site specificity and duration of the pharmacological effect for a given application (4).

Despite the recognized need for definitive studies of NP degradation and factors governing its kinetics (5), investigative strategies providing reliable in situ measurements of NP disassembly are lacking. The few in vitro studies examining the stability and degradation of biodegradable polyester-based submicronial particles point to the complexity of the breakdown process (6 ⇓–8). Degradation of NPs presents itself as simultaneous changes in particle size, molecular weight of the matrix-forming polymer and acidity, generation of increasingly hydrophilic degradation products, etc. Notably, the evolutions of these parameters in the course of NP degradation exhibit highly dissimilar kinetics and distinct patterns of dependence on formulation properties, polymer chemistry, medium composition and pH, temperature, and other experimental variables, making any single parameter inapplicable as a sole metric of the multifactorial NP degradation process (9, 10). Reliable measurements of nanocarrier disassembly additionally are hampered by the time and sample manipulations required for analysis, adversely affecting the accuracy of available techniques (11). The development of novel approaches enabling continuous, noninvasive monitoring of NP disassembly directly in living cells, as well as in environments modeling exposure to various types of biological milieu, may inform the design and optimization of biodegradable NP formulations for a wide range of biomedical applications and facilitate their translation into the clinic (12).

In the present study, an approach using real-time measurements of Förster resonance energy transfer (FRET) was characterized and implemented for elucidating disassembly patterns of polylactide (PLA)-based magnetic NPs (MNPs) in cultured arterial cells chosen because of their relevance to injury-triggered arterial renarrowing (restenosis) therapy, as well as in a range of biomimetic media. Biodegradable composite MNPs applied in combination with a recently reported two-source magnetic guidance strategy have shown promise as carriers for targeting cells, genes, and small molecule therapeutics to sites of vascular injury (13 ⇓–15). Although the properties of MNPs optimized for site-specific delivery of these different classes of therapeutics vary significantly, the capacity for disintegration into bioeliminable products when the task of guiding the cargo to its site of action is completed is a common prerequisite in the design of these formulations. The objective of the present investigations was to evaluate a FRET-based approach enabling quantitative, longitudinal measurements of MNP degradation status directly in the studied medium, without distortions imposed by sample processing. FRET involves a radiationless, distance-dependent transfer of energy between spectrally complementary fluorophores that may provide the basis for a highly sensitive assay of particle disintegration with a nanometer-scale spatial resolution and subsecond time response (11). A progressive disruption of the colocalization of two properly chosen fluorophores covalently linked to the chains of the MNP matrix-forming polymer was hypothesized to result in readily quantifiable, real-time changes in the measured fluorescent spectra, thus enabling continuous monitoring of the disassembly process in situ, without separating the particles from their environment (Fig. 1). As a first step, PLA-based MNPs covalently labeled with spectrally overlapping boron dipyrromethene (BODIPY) probes were formulated and their FRET efficiency was established in model experiments as a function of labeling intensity and extent of fluorophore colocalization. The applicability of FRET analysis as an assay for particle integrity next was evaluated in forced degradation experiments using optimized MNP formulations exhibiting an extended dynamic range of response. The fluorimetric assay was used further for studying the disassembly of composite MNPs under conditions simulating exposure to the intracellular and extracellular milieu of the arterial tissue. The disintegration kinetics of MNPs was investigated in contact-inhibited and dividing endothelial cells and A10, a vascular cell line exhibiting the defining characteristics of neointimal smooth muscle cells (16), as well as in liquid and semisolid media mimicking the composition and viscoelasticity of the arterial wall.

Fig. 1.

Schematic showing the correlation between emission spectra and integrity status of MNPs colabeled with donor and acceptor PLA-Gly-BODIPY conjugates, illustrating the principle of the FRET-based disassembly assay.

Results

End-group modification of the particle-forming PLA with FRET donor (don) and acceptor (acc) fluorophores was carried out using a poly(dl-lactide) glycinate precursor (PLA-Gly) and N-hydroxysuccinimide esters of BODIPY_558/568 and BODIPY_630/650-X, providing the PLA-Gly-BODIPY conjugates with respective labeling densities of 5.7 and 4.6 µmol/g as determined by ¹H NMR analysis (Figs. S1 and S2). PLA-based MNPs colabeled (MNP[don/acc]) or individually labeled with the PLA-Gly-BODIPY derivatives (MNP[don] and MNP[acc]) were formed by using a modification of a formulation strategy providing uniformly sized, superparamagnetic particles with an average diameter of 270 ± 10 nm and a magnetite loading of 38 ± 2% wt/wt (17) (shown schematically in Fig. S3). N_FRET, a parameter developed for global FRET analysis (18) and sensitively reflecting changes in FRET efficiency while exhibiting stability within a wide range of donor/acceptor ratios (19), was chosen for quantitatively investigating disassembly of MNPs in vascular cells and biomimetic media in the present studies. In model experiments examining the expected dynamic range of N_FRET changes resulting from the dissociation of the FRET pair fluorophores during the MNP disassembly process, colabeled and individually labeled MNPs were mixed at ratios representing different extents of the particle-forming polymer degradation (modeled degradation statuses). The limited scale of FRET response observed with MNPs formed using 10% of labeled PLA (0 ≤ N_FRET ≤ 0.25 for all modeled conditions, Fig. S4A), was extended remarkably by increasing the labeled polymer fraction to 40% (Fig. 2A). The dependence of N_FRET on the simulated decomposition of the particle-forming polymer was similar for MNPs suspended in liquid media (PBS or FBS) or internalized by cultured rat aortic smooth cells stably expressing GFP (A10, Fig. 2A). Notably, endocytosed MNPs caused no changes in cell morphology or GFP fluorescence compared with untreated cells and exhibited the characteristic perinuclear localization in the cell interior (Fig. 2 B and C) (17). The evolution of N_FRET as a function of the modeled MNP degradation status was accompanied by changes in the donor and acceptor emission intensities shown in Fig. 2D. The increase in the labeled PLA fraction to 40% was found to cause a shift in the fluorescence pattern of MNP[don], revealing, in addition to the expected emission peak at 575 nm, a prominent new peak at 612 nm (Fig. S4B), possibly as a result of the formation of a dimeric form of the fluorophore exhibiting a red-shifted spectrum, as described previously for another BODIPY derivative (20, 21). This shift in the emission spectrum due to the denser packaging of the donor fluorophore in the MNP matrix increases the overlap with the BODIPY_630/650 acceptor excitation spectrum (Fig. 2E), which likely contributed to the substantial extension of the N_FRET scale. Thus, based on their superior FRET response (Fig. 2 A and D), MNPs with the high labeling ratio were used in all subsequent studies.

Fig. 2.

N_FRET and spectral properties of MNP formulations. The evolution of N_FRET and emission spectra as a function of the donor–acceptor colocalization progressively disrupted in the process of MNP disassembly was simulated by combining MNP[don/acc] with respective fractions of a 1:1 mixture of MNP[don] and MNP[acc] (A and D). Data in A are presented as mean ± SD. Some error bars are too small to be shown. The characteristic perinuclear localization pattern is observed following magnetically driven cell uptake of MNP[don/acc], (B) exhibiting no interference with GFP fluorescence of A10 cells stably expressing GFP (C). Fluorescent microscopic cell images were taken after a 24-h exposure to MNPs under the magnetic conditions specified in the text. Original magnification: ×200. The emission spectrum of MNP[don] formulated at a high labeling density (labeled PLA fraction, 40%) exhibits prominent peaks at 575 nm and 612 nm, the latter overlapping extensively with the excitation peak of MNP[acc] (E). The effect of labeling density on the emission pattern of MNP[don] and the N_FRET scale is shown in Fig. S4.

The correlation between the simulated stages of MNP degradation and N_FRET values observed in the model experiments first was confirmed qualitatively by showing the disappearance of FRET concomitant with donor fluorescence recovery after disrupting the fluorophore colocalization by MNP digestion with acetonitrile (Fig. S5A). Notably, in agreement with the proposed reversible, concentration-dependent association of BODIPY_558/568 into complexes exhibiting red-shifted emission, the spectrum of acetonitrile-digested MNPs lacked the peak at 612 nm, consistent with the dissociation of these complexes upon disassembly of the MNP polymeric matrix. In another experiment, the validity of the established N_FRET correlation with the extent of MNP disassembly was addressed quantitatively by subjecting MNPs to accelerated degradation with proteinase K, an enzyme previously shown to cleave PLA effectively (22, 23). The progressive fragmentation of the enzymatically degraded polymer monitored by measuring the dissociated fluorophore fraction was paralleled by changes in the emission spectra and N_FRET (Fig. S5 B and C), with patterns similar to those seen in the model experiments (compare with Fig. 2 A and D). The enzymatic breakdown of the MNP matrix led to a decrease in the chloroform-extractable polymer fraction by 70.6 ± 0.3% between nondegraded and extensively degraded MNP[don/acc]s (0% vs. 92% degradation) with respective N_FRET values of 1.23 and 0.08 (Fig. S5D). A similar decrease in polymer organophilicity (73.3 ± 1.6%) also was observed with enzymatically degraded MNP[don]s (Fig. S5D).

The confirmed correlation between N_FRET and MNP degradation status next was applied to study the disassembly of MNPs in model media. MNPs maintained under stirring in albumin-supplemented PBS and citrate buffers with a corresponding pH of 7.4 and 4.7 exhibited similar disassembly kinetics, reaching 13.2 ± 4.1% and 15.9 ± 5.3% degradation at 2 wk and 44.7 ± 0.9% and 49.3 ± 4.2% at 132 d, respectively (Fig. 3A), in agreement with the results of previous studies showing that acceleration of the acid-catalyzed PLA degradation is triggered at pH ≤3.1, the pK_a of oligomeric PLA (24). MNPs incubated in citrate buffer started to flocculate after 3 wk, likely because of the effect of acidic conditions weakening the electrosteric stabilization provided by MNP surface-associated albumin. In contrast, MNPs incubated at pH 7.4 degraded by 34.2 ± 2.2% over 80 d without significantly changing their size, consistent with the bulk hydrolysis degradation mechanism previously demonstrated for PLA-based nanospheres (7). In comparison with the decelerating degradation in PBS and citrate buffers, MNP disassembly in serum occurred at a steady rate (0.51–0.61% per day, P < 0.001; Fig. 3A).

Fig. 3.

MNP disassembly in model liquid and semisolid media. The FRET-based assay was applied to examine the disassembly kinetics of MNPs incubated at 37 °C with stirring in buffers (Dulbecco’s PBS, pH 7.4, or citrate buffer, pH 4.7, supplemented with 1% albumin) or in FBS (A). The effect of restricted mobility experienced by MNPs in the extracellular microenvironment on their disassembly rates was studied using Matrigel (B) or collagen I gels (C) prepared according to the respective manufacturer’s instructions with specified modifications. MNPs incubated in unstirred Dulbecco’s PBS, pH 7.4, were included in B as a control. Data are presented as mean ± SD.

The effect of restricted mobility experienced by MNPs in the viscous milieu of the arterial tissue was modeled by placing MNPs in Matrigel or collagen I-based gels. The disassembly rate of MNPs embedded in Matrigel, which has viscoelastic properties comparable with those of the arterial wall (25, 26), decreased notably compared with that of MNPs in liquid media (19.1 ± 2.2% vs. 31.2 ± 1.8% degradation over 45 d in albumin-supplemented PBS; Fig. 3 A and B). The magnitude of this effect remained unchanged when the Matrigel was diluted threefold vs. the standard 9-mg/mL concentration (Fig. 3B). Similar deceleration of MNP degradation was observed in gels formed with collagen I. Notably, in contrast to liquid buffered media, acidification notably increased the disassembly rate of MNPs immobilized in the semisolid medium (27.8 ± 1.7% vs. 19.7 ± 2.3% degradation at 70 d in collagen I gels with pH 5.5 and 7.0, respectively; Fig. 3C). MNP incubation in unstirred PBS, included as a nonviscous control in this series of experiments, resulted in the slowest disassembly at a near-constant rate of 0.25 ± 0.02% degradation per day (P < 0.001; Fig. 3B).

Intracellular disassembly of endocytosed MNPs was investigated in cultured rat aortic smooth muscle cells (A10) and bovine aortic endothelial cells (BAECs). In the two cell types cultured under serum conditions providing comparable rates of cell expansion (i.e., 10% and 2% FBS, resulting in an average of four population doublings per week for A10 and BAECs, respectively), MNP disassembly occurred at a similar rate in the first 7 d (1.0 ± 0.1% for both cell types; Fig. 4A). Although MNP degradation in A10 continued at a slower rate, reaching a total of 27.2 ± 8.6% at day 55, a more complex kinetic pattern was observed in BAECs, with a rapid phase (54.1 ± 7.1% degradation at day 30) followed by a period of slower MNP degradation (12.6% between days 30 and 61). In an additional series of experiments examining initial degradation rates of MNPs endocytosed by the two cell types, a strong and serum concentration-dependent increase in MNP degradation kinetics was observed in dividing vs. confluent (contact-inhibited) cells (Fig. 4B). Notably, the significant enhancement of MNP degradation in high-serum conditions evident in proliferating cells (P = 0.014) was absent in contact-inhibited cells (P = 0.45), suggesting an association of this effect with cell division. This association also was supported by a direct correlation between MNP degradation status and the expansion of the dividing cells after 3 d of incubation at different serum concentrations (Fig. 4C; P = 0.023 and 0.046 for BAECs and A10, respectively).

Fig. 4.

MNP disassembly in cultured vascular cells. Magnetically driven, quantitative internalization of MNPs was achieved over 24 h in confluent rat aortic smooth muscle cells (A10) or BAECs (see text for details). MNP disassembly was monitored continuously by global FRET analysis in cells maintained using culture conditions providing an average of four population doublings per week for each cell type (A). The effect of serum-stimulated cell proliferation on the initial rate of MNP disassembly measured over 3 d is shown in comparison with contact-inhibited cells of the respective types in B. The overall MNP degradation status of BAECs and A10 at day 3 is shown as a function of % of cell expansion in C. Data are presented as mean ± SD.

Discussion

Reliable quantitative analysis of degradation kinetics is a prerequisite for the rational design of NP formulations intended for both experimental and clinical use, and especially for optimizing their performance for specific therapeutic and diagnostic applications. Despite the recognized importance of elucidating the factors controlling the structural and chemical stability of nanocarriers and understanding their role in the fate of NP formulations in various environments and living systems, there still is a critical unmet need for effective approaches providing undistorted real-time measurements of NP degradation directly in the medium of interest. In the present studies, a FRET-based strategy is introduced for real-time in situ measurements of biodegradable NP disassembly, and is applied to examining the degradation patterns of composite PLA-based MNPs in liquid and semisolid biomimetic media and in cultured vascular cells. Because of their unique advantages, including nanometer-scale spatial resolution and subsecond time response (11), FRET-based techniques are particularly well-suited for studies of molecular disassembly processes, such as intracellular decondensation of gene vectors or drug release from colloidal systems (27 ⇓–29). However, the use of FRET for investigating disassembly of biodegradable polymer-based nanocarriers poses several significant challenges, including (i) identifying chemically and spectrally compatible donor and acceptor fluorophores providing an adequate range of FRET response and exhibiting strong, environment-insensitive fluorescence; (ii) developing chemical strategies for covalently attaching the fluorophores to a particle-forming polymer without adversely affecting the colloidal stability and other properties of the carrier; (iii) identifying conditions providing rapid, effective, and synchronized cell uptake in studies focusing on intracellular disassembly of NPs; and (iv) enabling continuous, quantitative measurements in cells or biomimetic media on a protracted time scale ranging from several days to several weeks.

In the present investigations, small molecule BODIPY fluorophores combining suitable chemical properties with robust, environment-insensitive fluorescence (30, 31) were used as the donor–acceptor pair, and a new synthetic strategy was used for their high-yield attachment to PLA via a poly(dl-lactide) glycinate precursor. Notably, this synthetic route does not require de novo polymer synthesis and may be applied directly to create fluorescent labeled derivatives of preformed aliphatic polyesters, thus making the labeled formulations an accurate model of the prototype unlabeled NPs. The relatively ineffective energy transfer due to a limited spectral overlap of the two probes having a small Stokes shift, a persistent and remarkable shortcoming of the BODIPY fluorophores often detrimental to their use as fluorescent probes (30), was overcome in this study by increasing the labeling density of MNPs above the threshold of BODIPY_558/568 complex formation. This complex exhibiting excitation and emission maxima at 565 nm and 612 nm, respectively, and presumably representing a dimer with red-shifted fluorescence (21) contributed to improving the sensitivity of the FRET-based degradation assay through participating in a chain of energy transfer events: BODIPY_558/568 → [BODIPY_558/568]₂ → BODIPY_630/650. A similar energy transfer between the monomeric and dimeric forms of the fluorophore was demonstrated previously for another BODIPY derivative (20). The implementation of the multistep FRET phenomenon enabling the effective use of the unique photophysical properties of BODIPY for developing highly sensitive measurement techniques may be of relevance to a broad range of quantitative analytic applications.

The results obtained using the FRET-based particle disassembly assay monitoring the progressive spatial dissociation of the donor–acceptor pair fluorophores may not reflect the very early stages of NP degradation, in which polymer fragments generated through random scission are still too large to leach out of the particle. The presence and expected duration of a “lag” (i.e., time to the commencement of the actual disassembly) will depend on multiple factors, related to the characteristics of the surrounding medium (32) as well as the properties of the particles (6 ⇓–8). Thus, although our experiments did not reveal a significant delay in the onset of MNP disintegration, a detectable lag may be anticipated for NPs formed from a polymer with a larger initial molecular weight or a higher degree of crystallinity (6, 8). Composite PLA-based magnetic particles were chosen as a model biodegradable NP formulation for evaluation in the present studies because of their rapid and quantitative, magnetically controlled cell uptake (17), facilitating and synchronizing measurements of the internalized MNP disassembly in cultured cells, and also because of their direct relevance to magnetically targeted cell and drug delivery to sites of arterial injury, which is the therapeutic focus of our recent research (14, 15, 17). Particle-forming polymer was monitored in our experiments as the primary component determining structural integrity of the particles. However, the applicability of this approach is not limited to magnetically responsive NPs, and with the appropriate modifications, it may be extended to biodegradable polymer-based NPs of different designs and compositions.

The degradation of PLA-based NP previously was shown to be dominated by bulk alkaline hydrolysis with random chain scission at neutral as well as moderately acidic conditions (6, 7). The kinetics of the hydrolytic NP degradation is governed by several concomitant processes and largely depends on a balance between the elimination of oligomeric breakdown products with a resulting increase in particle porosity, making its core more accessible to water, and the formation of new carboxylic groups that in their deprotonated form can create a negatively charged shield hindering the catalytic action of hydroxyl anions (7). The interplay between these two oppositely contributing effects may account for the different patterns of MNP degradation in liquid and semisolid media observed in our studies. MNP disassembly in FBS, which in contrast to that in liquid buffers advanced to near-completion over 130 d, likely reflects the large capacity of serum proteins for solubilizing oligomeric PLA fragments and promoting their diffusion from the particles, thus maintaining the steady rate of the polymeric matrix decomposition. In accordance with this hypothesis, the retardation of MNP disassembly observed in gels modeling the viscous environment of the arterial wall likely is associated with an increasingly bound state of water in these systems (33), both affecting the amount of “free” water available for hydrolysis and preventing its access to the particle core by immobilizing and preventing the release of the cleaved fragments. The acceleration and further deceleration of MNP disassembly observed in acidified collagen I gel and unstirred PBS, respectively, suggest the importance of the aforementioned shielding effect of the newly formed, negatively charged carboxylic groups. Compared with MNPs embedded in a gel with neutral pH, the number of deprotonated carboxylic groups in the MNP matrix may be decreased at pH 5.5 but likely will be higher in the nonviscous unstirred PBS, preventing the release of the relatively large polymer fragments because of the formation of a stagnant, saturated layer around the particles but not restricting the penetration of the small ions, hydro- and dihydrophosphate, transporting the negative charge into the particle interior.

The results of experiments in cultured vascular cells indicate important differences in MNP disassembly rates in endothelial and smooth muscle cells. Although MNP disassembly initially may be driven primarily by the relatively slow, “abiotic” hydrolytic processes in both cell types, the change in kinetic pattern resulting in a notably accelerated MNP degradation in BAECs may be associated with the contribution of the “biotic,” presumably enzyme-mediated mechanisms specific to this cell type (34). The strong dependence of MNP degradation rates on the proliferation observed in both cell types is noteworthy. Dividing cells were shown to process MNPs many times faster than contact-inhibited cells, possibly as a result of the rapid cumulative expansion of the cytosol compartment promoting solubilization of the released oligomeric fragments and facilitating their diffusion from the particle matrix. Thus, in the therapeutic context of magnetically targeted drug or cell delivery as a therapeutic strategy for preventing arterial restenosis (35), our experiments offer several important observations. The increased rates of MNP disassembly in an acidified semisolid environment and in dividing smooth muscle cells suggest that the degradation of the respective fractions of the arterial wall-localized MNPs, i.e., those immobilized in the extracellular matrix or taken up by neointimal cells, may occur substantially faster early in the healing phase, characterized by rapid cell proliferation and inflammation, which may cause acidification of the local microenvironment (36). At the same time, it may be predicted that local delivery of antirestenotic drugs inhibiting the proliferation of the neointimal cells can reverse the accelerated MNP processing and slow the degradation of the cell-associated MNP fraction. Similar predictions may be made regarding the fate of MNPs used to generate magnetically responsive endothelial cells for targeted delivery and enhanced reendothelialization of injured arteries (15, 17). MNPs loaded into magnetically guided cells may exhibit faster disassembly at earlier time points, but their processing likely will occur at a slower rate when the cells reach the contact-inhibited state. These observations may be of practical importance for the design of nanocarriers with controllable degradation kinetics intended for treating different types of proliferative conditions, and they suggest that therapeutic combinations of nanocarriers and antiproliferative drugs may extend the time scale of NP degradation significantly, potentially leading to their accumulation, which in turn may affect the toxicity and effectiveness of the NP-mediated therapy.

In conclusion, a unique approach for direct, real-time monitoring of NP disassembly in various types of milieu and in the cell interior was characterized and used to examine the disassembly kinetics of composite biodegradable MNPs in biomimetic media and cultured vascular cells. Because of the difficulty in establishing in vivo the bleed-through controls required to correct and normalize FRET measurements, this method may be reserved primarily for model in vitro studies of NP disassembly. Although these studies do not fully reproduce the complexity and interplay of multiple factors governing nanoparticle disassembly in in vivo settings, the ability to delineate the effects of individual experimental variables and to elucidate their interactions, facilitating comparison between different formulations, types of milieu, and exposure conditions, may be instrumental in the development and optimization of nanocarriers for a variety of applications. A new synthetic scheme applied herein for efficient covalent modification of the particle-forming polymer with a pair of BODIPY fluorophores capable of engaging in a multistep energy transfer process made it possible to extend the scale of FRET response remarkably, which in turn allowed highly sensitive, undistorted in situ measurements of MNP disassembly in different biomimetic environments. The results of these investigations suggest several important observations regarding the fate of NP formulations, particularly those designed to treat proliferative conditions, and point to an important role of cell proliferation status as a factor governing disassembly kinetics of endocytosed nanocarriers.

Materials and Methods

Synthesis of PLA Derivatives and Formulation of Covalently Labeled MNPs.

Poly(dl-lactide) with the carboxylic end terminated with dodecyl groups (HO-PLA-OC₁₂H₂₅) with number average molecular weight = 50 kDa (Lakeshore Biomaterials) was acylated with N-(tert-butoxycarbonyl)glycine (Boc-Gly-OH) in dichloromethane by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as an activator and N,N-dimethylaminopyridine tosylate (DPTS) as a catalyst. After removal of the Boc-protection, the resulting amino-functionalized polymer was reacted with BODIPY_558/568 or BODIPY_630/650-X N-succinimidyl esters. The labeled polymer conjugates were reprecipitated repeatedly from dichloromethane solutions with methanol to remove any PLA-unbound fluorophores and were analyzed by ¹H NMR (Fig. S2). The labeled polymers contained 5.7 and 4.6 µmol/g of the covalently bound BODIPY_558/568 (don) or BODIPY_630/650 (acc), respectively.

Uniformly sized PLA-based MNPs were formulated by using a modification of the emulsification–solvent evaporation method (17). In brief, an ethanolic solution of ferric chloride hexahydrate and ferrous chloride tetrahydrate (170 and 62.5 mg, respectively, in 2.5 mL) was added to an equivalent amount of sodium hydroxide dissolved in deionized water (5 mL). The precipitate was maturated for 1 min at 90 °C, cooled on ice, and separated on a magnet. The obtained magnetite was stirred with a solution of oleic acid in ethanol (200 mg in 2 mL) at 90 °C for 5 min. Deionized water (4 mL) was added to separate free oleic acid. The magnetite was washed with ethanol, dispersed in 8 mL of chloroform, and used to dissolve a total of 200 mg of PLA including indicated fractions of the covalently labeled PLA-Gly-BODIPY conjugates, admixed either separately or in combination at a 1:1 weight ratio to provide individually labeled (MNP[don] and MNP[acc]) or colabeled (MNP[don/acc]) formulations. The organic dispersion was emulsified by sonication on ice in an aqueous solution of BSA (2% wt/vol, 10 mL), and the solvent was evaporated under reduced pressure. The MNPs were washed twice by magnetic decantation, resuspended in 6 mL of aqueous trehalose (10% wt/vol), passed through a sterile 5.0-µm polyvinylidene difluoride membrane (EMD Millipore), and lyophilized. Lyophilized MNPs were kept at −80 °C and resuspended in deionized water before use at 24 mg/mL. Particle size measurements were performed by dynamic light scattering. Magnetite content was determined spectrophotometrically against a suitable calibration curve (λ = 335 nm) in MNP samples digested for 30 min with sodium hydroxide (1 N) at 37 °C to produce precipitate, which subsequently was dissolved in hydrochloric acid (1 N) by heating to 90 °C for 5 min.

Spectral Properties of MNP and N_FRET/MNP Disassembly Correlation.

Fluorescence intensity and spectra of individually labeled and colabeled MNPs were taken after dilution at the indicated ratios in the respective media. For MNP fluorescence measurements in the cell interior, quantitative magnetically driven internalization was achieved over 24 h, as described previously (17), by positioning plates with confluent cells on a high-gradient field magnetic separator (LifeSep 96F; Dexter Magnetic Technologies). Emission spectra were taken using λ_ex = 540 and 590 nm, corresponding to the optimal excitation wavelengths for the donor and acceptor fluorophores determined in preliminary experiments using MNP[don] and MNP[acc], respectively. Normalized FRET efficiency, N_FRET, was calculated as in ref. 18 from MNP[don/acc] emission intensities at λ_ex/λ_em = 540 nm/640 nm, λ_ex/λ_em = 540 nm/575 nm, and λ_ex/λ_em = 590 nm/640 nm (, , and , respectively), using identically treated MNP[don] and MNP[acc] to obtain the bleed-through coefficients for each individual N_FRET measurement (/ and / ratios measured for MNP[don] and MNP[acc], respectively, and shown as a and b in the equation below):

A correlation between N_FRET and modeled MNP disassembly was established in media and cells by combining MNP[don/acc] with increasing fractions of a 1:1 mixture of MNP[don] and MNP[acc], thus simulating the progressive dissociation of the FRET pair fluorophores. Identical scales of N_FRET and transmittance >60% were confirmed in all studied media for MNP concentrations within the range of 12–240 µg/mL and for respective MNP doses applied to cells using magnetic conditions as in ref. 17.

As part of the validation of the approach, emission spectra of MNP[don/acc] before and after digestion in an organic solvent (acetonitrile applied at a 100:1 volume ratio to MNPs in suspension) were taken using λ_ex = 540 nm to confirm the complete reversal of the fluorescence pattern upon dissociation of the FRET pair-forming MNP components. In an additional series of experiments, N_FRET was monitored in MNP suspensions exposed to accelerated degradation with proteinase K (Sigma–Aldrich). The enzyme was dissolved in water and added to MNPs to provide the respective final concentrations of 150 and 240 µg/mL. Samples were incubated at 37 °C, and their emission spectra were taken at predetermined time points. Part of each sample was passed through an aluminum oxide membrane with a 0.02-µm pore size (Anotop; Whatman Inc.) impermeable to intact MNPs, and aliquots taken before and after filtration were digested with acetonitrile and analyzed by fluorimetry (λ_ex/λ_em = 540 nm/575 nm). N_FRET measured at each time point was plotted as a function of the respective extent of the fluorophore dissociation from MNPs representing the enzymatically decomposed fraction of the particle-forming polymer. Additionally, the fluorescence of nondegraded and extensively degraded MNP[don/acc]s extracted in chloroform and reconstituted after solvent removal in acetonitrile (i.e., the chloroform extractable fraction) was correlated with respective values of N_FRET and MNP-unbound fluorescence measured as above.

MNP Disassembly in Liquid and Semisolid Media.

Colabeled or individually labeled (bleed-through control) MNPs were diluted in FBS or buffered media containing 1% BSA (Dulbecco’s PBS, pH 7.4, or citrate buffer, pH 4.7). As a model of exposure to viscous extracellular milieu, MNPs were incorporated in Matrigel (BD Biosciences) or collagen I gels (Advanced BioMatrix) prepared according to the respective manufacturer’s instructions with indicated modifications. Samples were incubated at 37 °C with or without stirring as indicated and analyzed for N_FRET at predetermined time points.

Disassembly Kinetics of MNPs in Cultured Vascular Cells.

Rat aortic smooth muscle cells (A10) stably expressing GFP and BAECs were seeded at confluence on 96-well plates. For magnetically enhanced internalization, MNPs were applied to cells at 12 µg per well and the plates were positioned for 24 h on 96-well magnetic separators with an average field gradient of 32.5 T/m (LifeSep 96F; Dexter Magnetic Technologies) to achieve quantitative uptake (17). Cell culture then was continued using serum conditions providing an average of four population doublings per week for each cell type [DMEM supplemented with 10% and 2% (vol/vol) FBS for A10 and BAECs, respectively], and the cells were split weekly at a 1:16 ratio. N_FRET was monitored by using respective cells treated with individually labeled MNPs as bleed-through controls.

Alternatively, to determine the comparative effect of the serum-stimulated cell growth vs. contact inhibition, A10 and BAECs treated with MNPs as above were either kept confluent or split 1:10 and maintained in DMEM supplemented with 2%, 10%, 15%, or 25% (vol/vol) FBS. Their N_FRET values were measured repeatedly over 3 d, and the extent of cell expansion at the end of the incubation period was determined by GFP fluorescence (A10) or by using the Alamar Blue assay (37). N_FRET data were used to calculate the respective degradation statuses (% MNP disassembly at a given time point) and initial degradation rates (expressed as % MNP disassembly per day).

Statistical Analysis.

Experimental data were expressed as means ± SD. MNP disassembly data were examined by regression analysis. The Student's t test and ANOVA were used to compare between curves and to analyze the significance of individual curve slopes, respectively. Differences were termed significant at P < 0.05.

Acknowledgments

The authors acknowledge the secretarial assistance of Susan Kerns. This research was supported in part by National Institutes of Health Grants T32-HL007915 (to J.E.T.) and R01-HL111118 (to M.C.), American Heart Association Scientist Development grants (to I.F. and M.C.), the Foerderer Fund (J.E.T.), and the William J. Rashkind Endowment of The Children’s Hospital of Philadelphia (R.J.L.).

Footnotes

↵¹To whom correspondence should be addressed. E-mail: chorny{at}e-mail.chop.edu.

Author contributions: J.E.T., R.J.L., and M.C. designed research; J.E.T., I.S.A., K.Z., I.F., and M.C. performed research; I.S.A., K.Z., and M.C. contributed new reagents/analytic tools; J.E.T., I.S.A., I.F., and M.C. analyzed data; and J.E.T., R.J.L., and M.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1324104111/-/DCSupplemental.

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1. Polyak B,
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1. Rao RS,
2. Miano JM,
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OpenUrl Abstract/FREE Full Text
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1. Chorny M,
2. et al.
(2012) Formulation and in vitro characterization of composite biodegradable magnetic nanoparticles for magnetically guided cell delivery. Pharm Res 29(5):1232–1241.
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↵
1. Xia Z,
2. Liu Y
(2001) Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys J 81(4):2395–2402.
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↵
1. Lantsman K,
2. Tombes RM
(2005) CaMK-II oligomerization potential determined using CFP/YFP FRET. Biochim Biophys Acta 1746(1):45–54.
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1. Bergström F,
2. et al.
(2002) Dimers of dipyrrometheneboron difluoride (BODIPY) with light spectroscopic applications in chemistry and biology. J Am Chem Soc 124(2):196–204.
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1. Mikhalyov I,
2. Gretskaya N,
3. Bergström F,
4. Johansson LB-Å
(2002) Electronic ground and excited state properties of dipyrrometheneboron difluoride (BODIPY): Dimers with application to biosciences. Phys Chem Chem Phys 4:5663–5670.
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1. Tsuji H,
2. Miyauchi S
(2001) Enzymatic hydrolysis of poly(lactide)s: Effects of molecular weight, L-lactide content, and enantiomeric and diastereoisomeric polymer blending. Biomacromolecules 2(2):597–604.
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1. Williams DF
(1981) Enzymic hydrolysis of polylactic acid. Eng Med 10(1):5–7.
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↵
1. Siparsky GL,
2. Voorhees KJ,
3. Miao FD
(1998) Hydrolysis of polylactic acid (PLA) and polycaprolactone (PCL) in aqueous acetonitrile solutions: Autocatalysis. J Environ Polym Degrad 6(1):31–41.
OpenUrl CrossRef
↵
1. Soofi SS,
2. Last JA,
3. Liliensiek SJ,
4. Nealey PF,
5. Murphy CJ
(2009) The elastic modulus of Matrigel as determined by atomic force microscopy. J Struct Biol 167(3):216–219.
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↵
1. Hasehawa H,
2. Kanai H
(2004) Measurement of elastic moduli of the arterial wall at multiple frequencies by remote actuation for assessment of viscoelasticity. Jpn J Appl Phys 43(5B):3197–3203.
OpenUrl CrossRef
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1. Chen H,
2. et al.
(2008) Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Forster resonance energy transfer imaging. Proc Natl Acad Sci USA 105(18):6596–6601.
OpenUrl Abstract/FREE Full Text
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1. Martin-Fernandez M,
2. et al.
(2004) Adenovirus type-5 entry and disassembly followed in living cells by FRET, fluorescence anisotropy, and FLIM. Biophys J 87(2):1316–1327.
OpenUrl CrossRef PubMed
↵
1. Thibault M,
2. Nimesh S,
3. Lavertu M,
4. Buschmann MD
(2010) Intracellular trafficking and decondensation kinetics of chitosan-pDNA polyplexes. Mol Ther 18(10):1787–1795.
OpenUrl CrossRef PubMed
↵
1. Chen Y,
2. Zhao J,
3. Guo H,
4. Xie L
(2012) Geometry relaxation-induced large Stokes shift in red-emitting borondipyrromethenes (BODIPY) and applications in fluorescent thiol probes. J Org Chem 77(5):2192–2206.
OpenUrl CrossRef PubMed
↵
1. Karolin J,
2. Johansson LB-A,
3. Strandberg L,
4. Ny T
(1994) Fluorescence and absorption spectroscopic properties of dipyrrometheneboron difluoride (BODIPY) derivatives in liquids, lipid membranes, and proteins. J Am Chem Soc 116(17):7801–7806.
OpenUrl CrossRef
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1. Vert M,
2. Mauduit J,
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(1994) Biodegradation of PLA/GA polymers: Increasing complexity. Biomaterials 15(15):1209–1213.
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1. Maquet J,
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↵
1. Pandey AK
(2014) Recent advancements of biodegradable polylactic acid/polylactide: A review on synthesis, characterization and applications. Adv Matter Lett, in press.
↵
1. Chorny M,
2. Fishbein I,
3. Forbes S,
4. Alferiev I
(2011) Magnetic nanoparticles for targeted vascular delivery. IUBMB Life 63(8):613–620.
OpenUrl CrossRef PubMed
↵
1. Rajamäki K,
2. et al.
(2013) Extracellular acidosis is a novel danger signal alerting innate immunity via the NLRP3 inflammasome. J Biol Chem 288(19):13410–13419.
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1. O’Brien J,
2. Wilson I,
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Proceedings of the National Academy of Sciences: 111 (11)

Table of Contents

Submit

[1] ↵
Moghimi SM,
Peer D,
Langer R
(2011) Reshaping the future of nanopharmaceuticals: Ad iudicium. ACS Nano 5(11):8454–8458.
OpenUrl CrossRef PubMed

[2] Moghimi SM,

[3] Peer D,

[4] Langer R

[5] ↵
McNeil SE
(2011) Unique benefits of nanotechnology to drug delivery and diagnostics. Methods Mol Biol 697:3–8.
OpenUrl CrossRef PubMed

[6] McNeil SE

[7] ↵
Yildirimer L,
Thanh NT,
Loizidou M,
Seifalian AM
(2011) Toxicological considerations of clinically applicable nanoparticles. Nano Today 6(6):585–607.
OpenUrl CrossRef PubMed

[8] Yildirimer L,

[9] Thanh NT,

[10] Loizidou M,

[11] Seifalian AM

[12] ↵
Kamaly N,
Xiao Z,
Valencia PM,
Radovic-Moreno AF,
Farokhzad OC
(2012) Targeted polymeric therapeutic nanoparticles: Design, development and clinical translation. Chem Soc Rev 41(7):2971–3010.
OpenUrl CrossRef PubMed

[13] Kamaly N,

[14] Xiao Z,

[15] Valencia PM,

[16] Radovic-Moreno AF,

[17] Farokhzad OC

[18] ↵
McNeil SE
(2009) Nanoparticle therapeutics: A personal perspective. Wiley Interdiscip Rev Nanomed Nanobiotechnol 1(3):264–271.
OpenUrl CrossRef PubMed

[19] McNeil SE

[20] ↵
Coffin MD,
McGinity JW
(1992) Biodegradable pseudolatexes: The chemical stability of poly(D,L-lactide) and poly(ε-caprolactone) nanoparticles in aqueous media. Pharm Res 9(2):200–205.
OpenUrl CrossRef PubMed

[21] Coffin MD,

[22] McGinity JW

[23] ↵
Belbella A,
et al.
(1996) In vitro degradation of nanospheres from poly(D,L-lactides) of different molecular weights and polydispersities. Int J Pharm 129(1-2):95–102.
OpenUrl CrossRef

[24] Belbella A,

[25] et al.

[26] ↵
Lemoine D,
et al.
(1996) Stability study of nanoparticles of poly(ε-caprolactone), poly(D,L-lactide) and poly(D,L-lactide-co-glycolide) Biomaterials 17(22):2191–2197.
OpenUrl CrossRef PubMed

[27] Lemoine D,

[28] et al.

[29] ↵
Wallis KH,
Müller RH
(1993) Comparative measurements of nanoparticle degradation velocity using an accelerated hydrolysis test. Pharm Ind 55(2):168–170.
OpenUrl

[30] Wallis KH,

[31] Müller RH

[32] ↵
Mohammad AK,
Reineke JJ
(2013) Quantitative detection of PLGA nanoparticle degradation in tissues following intravenous administration. Mol Pharm 10(6):2183–2189.
OpenUrl CrossRef

[33] Mohammad AK,

[34] Reineke JJ

[35] ↵
Cho EJ,
et al.
(2013) Nanoparticle characterization: State of the art, challenges, and emerging technologies. Mol Pharm 10(6):2093–2110.
OpenUrl CrossRef

[36] Cho EJ,

[37] et al.

[38] ↵
Crist RM,
et al.
(2013) Common pitfalls in nanotechnology: Lessons learned from NCI’s Nanotechnology Characterization Laboratory. Integr Biol (Camb) 5(1):66–73.
OpenUrl CrossRef PubMed

[39] Crist RM,

[40] et al.

[41] ↵
Chorny M,
et al.
(2013) Site-specific gene delivery to stented arteries using magnetically guided zinc oleate-based nanoparticles loaded with adenoviral vectors. FASEB J 27(6):2198–2206.
OpenUrl Abstract/FREE Full Text

[42] Chorny M,

[43] et al.

[44] ↵
Chorny M,
et al.
(2010) Targeting stents with local delivery of paclitaxel-loaded magnetic nanoparticles using uniform fields. Proc Natl Acad Sci USA 107(18):8346–8351.
OpenUrl Abstract/FREE Full Text

[45] Chorny M,

[46] et al.

[47] ↵
Polyak B,
et al.
(2008) High field gradient targeting of magnetic nanoparticle-loaded endothelial cells to the surfaces of steel stents. Proc Natl Acad Sci USA 105(2):698–703.
OpenUrl Abstract/FREE Full Text

[48] Polyak B,

[49] et al.

[50] ↵
Rao RS,
Miano JM,
Olson EN,
Seidel CL
(1997) The A10 cell line: A model for neonatal, neointimal, or differentiated vascular smooth muscle cells? Cardiovasc Res 36(1):118–126.
OpenUrl Abstract/FREE Full Text

[51] Rao RS,

[52] Miano JM,

[53] Olson EN,

[54] Seidel CL

[55] ↵
Chorny M,
et al.
(2012) Formulation and in vitro characterization of composite biodegradable magnetic nanoparticles for magnetically guided cell delivery. Pharm Res 29(5):1232–1241.
OpenUrl CrossRef PubMed

[56] Chorny M,

[57] et al.

[58] ↵
Xia Z,
Liu Y
(2001) Reliable and global measurement of fluorescence resonance energy transfer using fluorescence microscopes. Biophys J 81(4):2395–2402.
OpenUrl CrossRef PubMed

[59] Xia Z,

[60] Liu Y

[61] ↵
Lantsman K,
Tombes RM
(2005) CaMK-II oligomerization potential determined using CFP/YFP FRET. Biochim Biophys Acta 1746(1):45–54.
OpenUrl PubMed

[62] Lantsman K,

[63] Tombes RM

[64] ↵
Bergström F,
et al.
(2002) Dimers of dipyrrometheneboron difluoride (BODIPY) with light spectroscopic applications in chemistry and biology. J Am Chem Soc 124(2):196–204.
OpenUrl CrossRef PubMed

[65] Bergström F,

[66] et al.

[67] ↵
Mikhalyov I,
Gretskaya N,
Bergström F,
Johansson LB-Å
(2002) Electronic ground and excited state properties of dipyrrometheneboron difluoride (BODIPY): Dimers with application to biosciences. Phys Chem Chem Phys 4:5663–5670.
OpenUrl CrossRef

[68] Mikhalyov I,

[69] Gretskaya N,

[70] Bergström F,

[71] Johansson LB-Å

[72] ↵
Tsuji H,
Miyauchi S
(2001) Enzymatic hydrolysis of poly(lactide)s: Effects of molecular weight, L-lactide content, and enantiomeric and diastereoisomeric polymer blending. Biomacromolecules 2(2):597–604.
OpenUrl CrossRef PubMed

[73] Tsuji H,

[74] Miyauchi S

[75] ↵
Williams DF
(1981) Enzymic hydrolysis of polylactic acid. Eng Med 10(1):5–7.
OpenUrl CrossRef

[76] Williams DF

[77] ↵
Siparsky GL,
Voorhees KJ,
Miao FD
(1998) Hydrolysis of polylactic acid (PLA) and polycaprolactone (PCL) in aqueous acetonitrile solutions: Autocatalysis. J Environ Polym Degrad 6(1):31–41.
OpenUrl CrossRef

[78] Siparsky GL,

[79] Voorhees KJ,

[80] Miao FD

[81] ↵
Soofi SS,
Last JA,
Liliensiek SJ,
Nealey PF,
Murphy CJ
(2009) The elastic modulus of Matrigel as determined by atomic force microscopy. J Struct Biol 167(3):216–219.
OpenUrl CrossRef PubMed

[82] Soofi SS,

[83] Last JA,

[84] Liliensiek SJ,

[85] Nealey PF,

[86] Murphy CJ

[87] ↵
Hasehawa H,
Kanai H
(2004) Measurement of elastic moduli of the arterial wall at multiple frequencies by remote actuation for assessment of viscoelasticity. Jpn J Appl Phys 43(5B):3197–3203.
OpenUrl CrossRef

[88] Hasehawa H,

[89] Kanai H

[90] ↵
Chen H,
et al.
(2008) Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Forster resonance energy transfer imaging. Proc Natl Acad Sci USA 105(18):6596–6601.
OpenUrl Abstract/FREE Full Text

[91] Chen H,

[92] et al.

[93] ↵
Martin-Fernandez M,
et al.
(2004) Adenovirus type-5 entry and disassembly followed in living cells by FRET, fluorescence anisotropy, and FLIM. Biophys J 87(2):1316–1327.
OpenUrl CrossRef PubMed

[94] Martin-Fernandez M,

[95] et al.

[96] ↵
Thibault M,
Nimesh S,
Lavertu M,
Buschmann MD
(2010) Intracellular trafficking and decondensation kinetics of chitosan-pDNA polyplexes. Mol Ther 18(10):1787–1795.
OpenUrl CrossRef PubMed

[97] Thibault M,

[98] Nimesh S,

[99] Lavertu M,

[100] Buschmann MD

[101] ↵
Chen Y,
Zhao J,
Guo H,
Xie L
(2012) Geometry relaxation-induced large Stokes shift in red-emitting borondipyrromethenes (BODIPY) and applications in fluorescent thiol probes. J Org Chem 77(5):2192–2206.
OpenUrl CrossRef PubMed

[102] Chen Y,

[103] Zhao J,

[104] Guo H,

[105] Xie L

[106] ↵
Karolin J,
Johansson LB-A,
Strandberg L,
Ny T
(1994) Fluorescence and absorption spectroscopic properties of dipyrrometheneboron difluoride (BODIPY) derivatives in liquids, lipid membranes, and proteins. J Am Chem Soc 116(17):7801–7806.
OpenUrl CrossRef

[107] Karolin J,

[108] Johansson LB-A,

[109] Strandberg L,

[110] Ny T

[111] ↵
Vert M,
Mauduit J,
Li S
(1994) Biodegradation of PLA/GA polymers: Increasing complexity. Biomaterials 15(15):1209–1213.
OpenUrl CrossRef PubMed

[112] Vert M,

[113] Mauduit J,

[114] Li S

[115] ↵
Maquet J,
et al.
(1986) State of water in gelatin solutions and gels: An 1H n.m.r. investigation. Polymer (Guildf) 27(7):1103–1110.
OpenUrl CrossRef

[116] Maquet J,

[117] et al.

[118] ↵
Pandey AK
(2014) Recent advancements of biodegradable polylactic acid/polylactide: A review on synthesis, characterization and applications. Adv Matter Lett, in press.

[119] Pandey AK

[120] ↵
Chorny M,
Fishbein I,
Forbes S,
Alferiev I
(2011) Magnetic nanoparticles for targeted vascular delivery. IUBMB Life 63(8):613–620.
OpenUrl CrossRef PubMed

[121] Chorny M,

[122] Fishbein I,

[123] Forbes S,

[124] Alferiev I

[125] ↵
Rajamäki K,
et al.
(2013) Extracellular acidosis is a novel danger signal alerting innate immunity via the NLRP3 inflammasome. J Biol Chem 288(19):13410–13419.
OpenUrl Abstract/FREE Full Text

[126] Rajamäki K,

[127] et al.

[128] ↵
O’Brien J,
Wilson I,
Orton T,
Pognan F
(2000) Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 267(17):5421–5426.
OpenUrl CrossRef PubMed

[129] O’Brien J,

[130] Wilson I,

[131] Orton T,

[132] Pognan F

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Real-time analysis of composite magnetic nanoparticle disassembly in vascular cells and biomimetic media

Significance

Abstract

Results

Discussion

Materials and Methods

Synthesis of PLA Derivatives and Formulation of Covalently Labeled MNPs.

Spectral Properties of MNP and N_FRET/MNP Disassembly Correlation.

MNP Disassembly in Liquid and Semisolid Media.

Disassembly Kinetics of MNPs in Cultured Vascular Cells.

Statistical Analysis.

Acknowledgments

Footnotes

References

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Significance

Abstract

Results

Discussion

Materials and Methods

Synthesis of PLA Derivatives and Formulation of Covalently Labeled MNPs.

Spectral Properties of MNP and NFRET/MNP Disassembly Correlation.

MNP Disassembly in Liquid and Semisolid Media.

Disassembly Kinetics of MNPs in Cultured Vascular Cells.

Statistical Analysis.

Acknowledgments

Footnotes

References

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Spectral Properties of MNP and N_FRET/MNP Disassembly Correlation.