Coupling of HPLC with Electrospray Ionization Mass Spectrometry for

Oct 25, 2013 - Sub-5 nm multimodal nanoparticles have great potential for theranostic applications due to their easy renal elimination combined with ...
0 downloads 0 Views 2MB Size
Download PDF
Article pubs.acs.org/ac

Coupling of HPLC with Electrospray Ionization Mass Spectrometry for Studying the Aging of Ultrasmall Multifunctional GadoliniumBased Silica Nanoparticles Charles Truillet, François Lux, Olivier Tillement, Philippe Dugourd, and Rodolphe Antoine* Institut Lumière Matière, UMR5306 Université Lyon 1-CNRS, Université de Lyon 69622 Villeurbanne cedex, France S Supporting Information *

ABSTRACT: Sub-5 nm multimodal nanoparticles have great potential for theranostic applications due to their easy renal elimination combined with complementary imaging properties and therapeutic facilities. Their potential clinical use requires the full characterization of not only the nanoparticle but also all its possible degradation products. We have recently proposed new ultrasmall gadolinium-based nanoparticles for multimodal imaging and radiosensitization. The aim of this article is to describe an analytical tool to characterize degradation products in a highly diluted medium. We demonstrate that HPLC coupled to electrospray ionization mass spectrometry (ESI-MS) can be used in order to determine precisely the composition of nanoparticles and their degradation fragments during aging.

D

superior to 5 nm avoiding complete renal elimination. To reach such small sizes while conserving multimodality, our group has proposed an original top-down method consisting in the fragmentation of sub-10 nm structures made of core/shell nanoparticles with a gadolinium oxide core and polysiloxane shell. The grafting of chelating species at the surface leads to the formation in water of multimodal small rigid platforms (SRP).16,17 These SRPs are likely to take advantage of abnormalities of tumor vasculature, e.g., porous vessels,18 to reach tumor tissue via the enhanced permeability and retention (EPR) effect.19 Active targeting can be performed by covalent conjugation of an effective targeting peptide at the surface of the SRP. Thus, SRPs have been functionalized by different molecule vectors such as the ανβ3 integrin vector.20 Although such SRPs are small and allow rapid renal excretion and limit toxic effects, their kinetic degradation depends on their surface modification and must be characterized. Accurate characterization tools for determining the exact composition of the fragments resulting from degradation are required to monitor nanoparticle aging and biocompatibility as a function of time. For instance, the self-destruction of optically active nanomaterials can be easily traced by luminescence observation.13,21 For SRPs, this means that it would be necessary to graft fluorophores, which would change the surface and the size of the nanoparticles. On the other hand, there has been substantial growth in the application of mass spectrometry (MS) methods for the analysis of nanoclusters.22−30 Insights

uring the past decade, nanoparticles have fully invested the life sciences and biotechnology for the diagnosis and treatment of diseases.1−3 The clinical use of the nanoparticles or their fragments requires their harmless elimination from the human body within controlled periods of time. Although carbon nanotubes and quantum dots have demonstrated their potential for biological applications,4−6 their adoption for clinical use is difficult due to their intrinsic toxicity or that of their degradation products.7 Significant residual heavy metals or other toxic constituents can be released in the organism, provoking damaging effects during clearance. Moreover, nanoparticle size is a major parameter for toxicity concerns. Particle sizes must be below 100 nm to avoid undesirable macrophage uptake8,9 which is a key parameter regarding elimination from the body. Renal clearance seems to be the most reproducible and most efficient route of excretion. However, to be rapid and free of toxicity, the size of the particles should be below 5.5 nm.10,11 Ensuring the multimodality of such small nanoparticles is a considerable challenge. One of the most promising strategies relies on the formulation of silica- or polymeric-based structures that incorporate different functional entities, such as dyes for fluorescence imaging,12,13 magnetic complexes for MRI, radioactive elements for scintigraphy14 or curie-therapy, heavy elements for interaction with X- or γ-rays, neutron absorbers for neutron therapy, and sensitizers for photodynamic therapy. Silica nanoparticles are one of the most common probes described in the literature.15 The inconvenience of the classical method of silica nanoparticle synthesis is to form a core potentially small enough for renal elimination but the addition of each imaging or therapeutic functionality increases drastically the size of the nanoparticle leading to particles with hydrodynamic diameter © 2013 American Chemical Society

Received: August 2, 2013 Accepted: October 7, 2013 Published: October 25, 2013 10440

dx.doi.org/10.1021/ac402429p | Anal. Chem. 2013, 85, 10440−10447

Analytical Chemistry

Article

± 0.1 nm and an average molecular mass of 8.8 ± 1.0 kDa (see Figure S1 in the Supporting Information). The composition of the nanoparticles is given in Table 1.

into composition in terms of molecular weight can be addressed by time-of-flight mass spectrometry (MALDI TOF MS) and electrospray ionization-mass spectrometry (ESI-MS) after high-pressure liquid chromatography (HPLC)-separated fractions are collected.31−34 Studies relating to degradation or modification of the surface functionalization of nanoparticles have been reported, using HPLC and HPLC-MS.35,36 However, no attempt to analysis by these techniques the nanoparticles in combination with their degradation products has been done yet. In this article, we propose a new method to study nanoparticle aging continuously through time. We present the coupling between mass spectrometry (ESI-MS) and HPLC adapted to nanoparticles. This method allows determining the degradation kinetics, the nanoparticle structure, and the constitution of the fragments. The study was carried out on native SRP and derivatized SRP, such as SRP functionalized by ανβ3 integrin vector, i.e., cRGDfK molecules. To the best of our knowledge, these results represent the first online coupling of HPLC-MS for studying nanoparticle aging, by simultaneously characterizing the nanoparticles and their degradation products providing crucial information on the destruction of nanoparticles in vitro.

Table 1. Chemical Composition of the Nanoparticles Deduced from Elemental Analysis by ICP-MS with a Standard Error of 0.55% and the Chemical Composition of the Nanoparticles Deduced by Calculation elements

Gd

Si

N

C

mass ratio (%) ICP-MS mass ratio (%) calculated

11.1 11.2

8.4 8.5

8.6 8.5

31.1 30.7

The APTES/TEOS-ratio (0.6/0.4) remains constant during the further steps of functionalization and purification. SRP contains around 1.5 DOTAGA ligands per gadolinium atom. Thus, some ligands do not chelate gadolinium ions. The average chemical formula of a single construct is deduced from the global formula given by Table 1 and the mass of the structure (8.8 kDa). We can propose the following average composition for one individual SRP: Gd7APTES18TEOS12DOTAGA10.5, in agreement with the composition obtained previously.17 The mass ratio calculated is in a good agreement with the mass ratio deduced by ICP-MS with a deviation inferior to the standard error. These SRPs exhibit several carboxylic acid functions provided by free DOTAGA ligands available for grafting peptides such as cRGDfK. Only DOTAGA ligands that do not chelate gadolinium ions can be used to perform grafting on the carboxylic function. The particles, i.e., SRPs and SRP−cRGDfK, were purified by visvapins tangential membrane ultrapurification (MWCO = 5 kDa) in order to obtain only pure nanoparticles without precursors and fragmentations resulting from degradation. The particles were then freeze-dried for stabilization over time. HPLC-UV. Gradient HPLC analysis was done by using the Shimadzu Prominence series UFLC system with a CBM-20A controller bus module, an LC-20AD liquid chromatograph, a CTO-20A column oven, and an SPD-20A UV−vis detector. Gradient LC elution was carried out with two mobile phases: (A) Milli-Q water/TFA 99.9:0.1 v/v and (B) acetonitrile (CH3CN)/Milli-Q water/TFA 90:9.9:0.1 v/v/v. An amount of 20 μL of sample was loaded thanks to an injection valve into the solvent injection ratio: 95% solvent A−5% solvent B (A = Milli-Q water/TFA 99.9:0.1 v/v; B = CH3CN/Milli-Q water/ TFA 90:9.9:0.1 v/v/v) into a Jupiter C4 column (150 mm × 4.60 mm, 5 μm, 300 Å, Phenomenex) at a flow rate of 1 mL/ min over 5 min. The concentration of the injected nanoparticles was 0.3 mM per gadolinium atoms at pH 7.4. Then the elution was programmed as follows: samples were eluted by a gradient increased from 5% to 90% of solvent B in solvent A over 30 min. The concentration of solvent B was maintained over 10 min. Then, the concentration of solvent B was decreased to 5% over a period of 10 min to re-equilibrate the system, followed by additional 10 min at this final concentration. Before the measurement of each sample, a baseline was obtained under the same conditions by loading Milli-Q water into the injection loop. The column temperature was maintained at 25 °C. The UV absorption spectrum of the synthesized nanoparticles (NPs) was acquired using a Cary 50 spectrometer from Agilent Technologies. Quartz cells (Hellma Analytics) with an optical length of 10 mm were used for all optical

■

EXPERIMENTAL SECTION Chemicals. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, >98.0%), N-hydroxysuccinimide (NHS, >97.0%), gadolinium chloride hexahydrate ([GdCl3· 6H2O], 99%), sodium hydroxide (NaOH, 99.99%), hydrochloric acid (HCl, 36.5−38%), sodium chloride (NaCl, >99.5%), dimethyl sulfoxide (DMSO, >99.5%), acetonitrile (ACN, >99.9%), trifluoroacetic acid (TFA, >99%), tetraethoxysilane (Si(OC2H5)4, TEOS, 98%), and aminopropyl triethoxysilane (H2N(CH2)3−Si(OC2H5)3, APTES, 99%) were purchased from Aldrich Chemical (France) and used without further purification. The cyclic RGDfK peptides were purchased from GeneCust (Luxembourg). SRP synthesis and SRPs functionalized with cRGDfK have been described already elsewhere.17,20 The core/shell nanoparticles (core, gadolinium oxide; shell, polysiloxane) and the SRPs were purchased from Nano-H SAS (Saint-Quentin Fallavier, France). DOTAGA (1,4,7,10-tetraazacyclododecane-1-glutaric anhydride-4,7,10-triacetic acid) chelate was purchased from ChemaTech (Dijon, France). For the preparation of an aqueous solution of nanoparticles, only Milli-Q water (ρ > 18 MΩ·cm) was used. Each synthesis step was performed at room temperature. SRP concentrations are stated in mol/L of gadolinium. Small Rigid Platform Synthesis. The synthesis of the SRPs is a four-step synthesis. The first step is the synthesis of a gadolinium oxide core by addition of soda on gadolinium trichloride in diethylene glycol. The growth of a polysiloxane shell around the oxide cores is then ensured by the addition of silane precursors: APTES and TEOS in convenient ratio (APTES/TEOS ratio = 0.6/0.4) and catalyzed by triethylamine. The third step is the covalent grafting of DOTAGA anhydride on the free amino functions issued from APTES. The nanoparticles are precipitated and then dispersed in water before purification. During the purification process in water a top down process is observed by dissolution of the gadolinium oxide core and chelation of gadolinium ions by DOTAGA. Then a collapse of the hollow polysiloxane shell occurs leading to the SRPs. Classically, this pathway leads to the synthesis of SRPs exhibiting an average hydrodynamic diameter (HD) of 4 10441

dx.doi.org/10.1021/ac402429p | Anal. Chem. 2013, 85, 10440−10447

Analytical Chemistry

Article

v) into a Jupiter C4 column (150 mm × 4.60 mm, 5 μm, 300 Å, Phenomenex) at a flow rate of 0.4 mL/min over 5 min. The concentration of the nanoparticles injected was determined by the preparation of the nanoparticles for aging, i.e., 0.3 mM per gadolinium atoms at pH 7.4. Then the elution was programmed as follows: samples were eluted by a gradient increased from 5% to 90% of solvent B in solvent A over 30 min. The concentration of solvent B was maintained over 10 min. Then, the concentration of solvent B was decreased to 5% over a period of 10 min to re-equilibrate the system, followed by additional 10 min at this final concentration for the next injection. A baseline was obtained under the same conditions by loading Milli-Q water into the injection loop. The LC was coupled to a mass spectrometer interfaced by an electrospray source. Mass experiments were performed using a linear quadrupole ion trap mass spectrometer (LTQ, Thermo Fisher Scientific, San Jose, CA) with enlargement for the high 2000−4000 Th range. Two different settings were used: one for high-mass observation (setting 1 between 200 and 4000 m/z) and the other for low mass (setting 2 between 150 and 2000 m/z). The setting parameters are given in the Supporting Information. The low-mass setting allows studying fragments. Isotopic distributions of fragment ions were recorded using the zoom scan mode of the LTQ ion trap mass spectrometer. The highmass setting was optimized for the NPs. SRP NPs contain multiple groups capable of being ionized (e.g., DOTAGA(Gd)

measurements. The absorbance chromatogram was monitored by the SPD-20A UV−vis detector at 295 nm which corresponds to the maximum absorption of the nanoparticles (see Figure S2 in the Supporting Information). Coupling of HPLC with ESI-MS. The experimental setup developed in this work is composed of two devices: an HPLC instrument with an external column for NP analysis and a mass spectrometer interfaced by an electrospray source (see Figure 1).

Figure 1. General online HPLC/ESI-MS setup.

Chromatographic separation was conducted on an Agilent 1260 Infinity HPLC with a vacuum degasser, pump, and an autosampler. The sample to analyze was inserted into vials into the autosampler. Gradient LC elution was carried out with two mobile phases: (A) Milli-Q water/TFA 99.9:0.1 v/v and (B) acetonitrile (CH3CN)/Milli-Q water/TFA 90:9.9:0.1 v/v/v. An amount of 40 μL of sample was loaded in the solvent injection ratio: 95% solvent A−5% solvent B (A = Milli-Q water/TFA 99.9:0.1 v/v; B = CH3CN/Milli-Q water/TFA 90:9.9:0.1 v/v/

Figure 2. HPLC spectra of the nanoparticles degraded at different times and detected due to their absorption properties for ultraviolet light at 295 nm: the chromatogram for the particles at 0, 2, and 6 h after synthesis, then after 20 h of aging. The concentration of the nanoparticles was 0.3 mM per gadolinium atom at pH 7.4. The intensity of the peak at 13 min, corresponding to the entire SRPs, decreased with aging time. It was also slightly shifted to the left, as a few molecules seemed to detach from the particles due to the hydrolysis of Si−O−Si bonds, and the average mass of the particles seemed to decrease slightly. On the contrary, the peak at 3 min, corresponding to small particle fragments, increased strongly with aging. The inset figure is the kinetics curve of the mean peak at 13 min. Each point represents the area of the peak. 10442

dx.doi.org/10.1021/ac402429p | Anal. Chem. 2013, 85, 10440−10447

Analytical Chemistry

Article

Figure 3. (a) Relative abundance of the total ion signal as a function of retention time. (1−5) MS spectra recorded at different elution times defined in red in panel a. Insets in spectra 3−5 correspond to deconvoluted ESI mass spectra. The particles were aged for 3 h at a concentration of 0.3 mM per gadolinium atom at pH 7.4.

where f(m) is the intensity calculated in the spectrum for mass m, g(m/z) is the intensity in the mass-to charge spectrum, and grms is the root-mean-square (rms) value of the signal measured. Nanoparticle Preparation for Aging. The lyophilized SRPs were first dispersed in aqueous solutions at physiological pH at very high concentration (200 mM in Gd, pH = 7.4, room temperature). And then SRPs were diluted in aqueous solutions at physiological pH (0.3 mM in Gd, pH = 7.4, 37 °C) for the aging test. At this pH, ζ-potential measurements showed that the SRPs had a negative potential, i.e., −5.5 mV. Samples of aged nanoparticle solutions were extracted and analyzed at different times from 1 h to 1 month. Those samples were directly injected into the HPLC-UV analyzer or HPLC-MS analyzer. In order to homogenize the fresh solutions, the nanoparticles were dispersed by a vortex facility. Then before each injection of the nanoparticles into the analysis system, the particles from aged solutions were also dispersed by a vortex facility in order precisely to avoid adhesion to the vials side walls of the nanoparticles.

complexes), and the ESI source can produce highly charged ions. For those molecules that can sustain multiple charges, a distribution of charge states is often observed in the mass-tocharge spectrum. This multiplicity of states gives rise to an “envelope” of peaks in the spectrum. The distribution of charges was observed in the m/z range of 1000−4000 after electrospraying in positive mode water solution containing the nanoparticles. Although the position of the peaks in the mass spectra was stable, the relative abundance of the different peaks was found to be slightly dependent on the ESI-MS conditions and the synthetic procedure. A multiplicative correlation algorithm (MCA)37 was used to estimate the mass of nanoparticles from the mass-to-charge spectra produced by electrospray ionization mass spectrometry. The multiplicative correlation was designed to enhance the deconvoluted signal when the parent molecule was distributed into several charge states in the spectrum measured. This approach is summarized by the following formula:

■

RESULTS AND DISCUSSION Aging of SRPs by HPLC-UV. The SRP aging was studied using HPLC coupled with a UV detector. The flow rate was

z1

f (m ) =

∏ g(m/z)/grms z=z0

10443

dx.doi.org/10.1021/ac402429p | Anal. Chem. 2013, 85, 10440−10447

Analytical Chemistry

Article

Figure 4. Low m/z part of the ESI mass spectrum of the nanoparticles after 1 month of aging recorded for an elution time of 6 min (±1.25). The assignment of the principal peaks is given, and the corresponding structures are given in Figure S5 in the Supporting Information.

maintained at 1 mL/min−1 to obtain a retention factor equal to k = 0.25. k is the ratio of the adjusted retention volume and the hold-up volume.38 Figure 2 presents the chromatogram for the particles immediately after synthesis, then after 2, 6, and 20 h of aging. The chromatograms show a first peak centered at around 3 nm and a broad peak centered at 12 min (±1). The intensity of the peak at 12 min (±1) corresponding to the pure SRPs decreases with aging time. During the aging process of the nanoparticles, the peak is also slightly shifted to the left: at the initial time, the nanoparticles were retained at 12 min (±1), and 1 month later, the elution peak corresponding to the NP was located at 11.3 min (±1.5). This slight shift is due to the fact that a few molecules seem to detach from the particles due to the hydrolysis of Si−O−Si bonds, leading to a slight decrease in the average mass of the particles. The first elution peak at 3 min (±0.2) in all the chromatograms had similar retention times in each spectrum regardless of aging time. This peak corresponds to nonretained species under reversible phase HPLC (RP-HPLC) conditions. It is the time spent by the degradation products to travel the injection loop of the HPLC system from the column to the UV detector. It was speculated that these degradation fragments were composed of Gd chelates, which was confirmed by mass spectrometry. The evolution of the nanoparticle peak (12 min) with time allowed determining a kinetic curve (see the inset in Figure 2). This curve was fitted by a first-order exponential decrease. Lifetime was defined as the time required for the quantity of pure nanoparticles to fall to half its value measured at the beginning of the time period. The lifetime determined by the fitting was 3 h. In order to address the influence of nanoparticles functionalization, the same aging conditions were imposed as for the SRP−cRGDfK (Figure S3 in the Supporting Information). A kinetic curve was drawn, and aging lifetime was 5 h. Thus, the particle was stabilized by extra grafting on the SRP surface. In highly diluted medium, the polysiloxane matrix was less affected by water hydrolysis due to the distance

of the water from the matrix ensured by the peptide grafted on the surface of the nanoparticles. HPLC-MS. The particles can be purified by HPLC-UV in order to obtain pure nanoparticles. However, degradation fragments can be hidden during HPLC-UV due to different absorption properties between the nanoparticles and the different fragments. At 295 nm, when maximum nanoparticle absorption is reached, the fragments may not be detectable by UV−vis methods. Moreover, any structural information on possible degradation products is lost after nanoparticle purification. Additional information could be obtained by MS analysis. For HPLC-MS the gradient elution program was slightly changed regarding the elution program for HPLC-UV. Instead of 1 mL/min−1, the flow rate was reduced to 0.4 mL· min−1 allowing good dispersion of the liquid by electrospray into a fine aerosol for ionization and a slight increase of the retention factor (k = 0.33). The increase of the retention factor led to better resolution of the peaks. MS Spectra at Different Elution Times. Figure 3a presents the relative intensity of the total ion signal recorded on the mass spectrometer ion detector as a function of retention time. The MS setting was Setting 1 (the one optimized for nanoparticles at high mass). The SRPs were observed after 3 h of aging. The chromatogram is quite similar to the one obtained with HPLC-UV with longer elution time due to the longer elution protocol. The two peaks at 5.5 and 6.7 min correspond to the degradation fragments (detected at 3 min in the HPLC-UV protocol), and the peak at 18 min corresponds to the nanoparticles (detected at 12 min in HPLC-UV protocol). The first peak at 5.5 min corresponds to the time for the fragments (partially retained by the column) to travel the LC system. The corresponding mass spectra are shown in Figure 3 (spectra 1 and 2). The mass spectra (in particular spectrum 2) show the presence of several peaks in the low m/z range. They correspond to singly charged polysiloxane− DOTAGA species, some bearing a Gd3+ ion and others not. These ions are assigned in Figure 4. The global composition of the fragments can be written as (DOTAGA)x(DOTAGA10444

dx.doi.org/10.1021/ac402429p | Anal. Chem. 2013, 85, 10440−10447

Analytical Chemistry

Article

Figure 5. (a) Chromatograms (HPLC-MS) recorded at different aging times of the solution. (b) m/z spectra of the second elution time (between 17 and 19 min) at different degradation times. (c) Deconvoluted ESI mass spectra from m/z spectra in panel b. The SRPs were at a concentration of 0.3 mM per gadolinium atom at pH 7.4.

(Gd))y(APTES)z(TEOS)w. The DOTAGA chelate was always grafted on the silicon matrix composed of APTES with additional TEOS in some cases. No free Gd3+ ions were observed in the mass-to-charge spectrum due to the high stability constant of the DOTAGA with a gadolinium atom (complexation constant of the DOTAGA on the nanoparticles, log β110 = 24.78).17 In the mass spectrum recorded with a fresh solution without aging, the amount of ions detected at a retention time of 6 min (±1.25) was very low (Supporting Information Figure S4), in agreement with the assignment of this elution peak to degradation products. The second elution peak at 18 min corresponds to the retention time of the nanoparticles. The peak is broad, and mass spectra were recorded at different elution times within the elution peak. As the SRPs contain multiple ionized groups (e.g., [DOTAGA(Gd) complexes), a distribution of charge states was observed in the mass-to-charge spectrum, and the MCA was used to estimate the mass of the SRPs from the mass-to-charge spectra produced by the ESI-MS. The insets in Figure 3 (spectra 3 and 4) show the deconvoluted ESI mass spectra. The distribution is centered at 8680 Da at a retention time of 15 min (±1). For a longer retention time, the mass slightly

increased to 8810 Da (retention time 17.5 min (±1) and 8930 Da (retention time 21.3 (±1). Since SRP retention by the nonpolar C4 column was expected to increase with nanoparticle size, the results were consistent. The average mass from these MS spectra was in agreement with the HD results. Aging Characterization. The aim of this analytical development was to probe the evolution of the SRP as a function of time to assess toxicology in a highly diluted medium17 and the stability of surface functionalization. An additional study was first carried on the bare SRP nanoparticles and then on SRP− cRGDfK, SRP nanoparticles functionalized by a peptide. The HPLC-MS chromatograms recorded for solutions at different aging times are shown in Figure 5a. As already observed for HPLC-UV, the retention peak of the pure nanoparticles seems to vanish as degradation time increases. On the other hand, the first elution peak corresponding to fragments resulting from the nanoparticles increases. The m/z spectra shown in Figure 5b were obtained by data integration between 17 and 19 min retention time. They represent the m/z spectrum of the nanoparticles. During aging time, the intensity of the mass-to-charge spectrum decreases, corresponding to the degradation of the nanoparticles. 10445

dx.doi.org/10.1021/ac402429p | Anal. Chem. 2013, 85, 10440−10447

Analytical Chemistry

Article

Moreover the global mass is reduces slowly from ∼9 to 8.7 kDa, as evidenced by deconvoluted ESI spectra (see Figure 5c). This analysis and the analysis of the different fragment components made it possible to monitor the exact aging mechanism of the nanoparticles. Hydrolysis of the Si−O−Si bonds of the polysiloxane matrix reduced the global structure of the nanoparticles. After 3 h of aging, mass distribution was more dispersed. There was still considerable distribution centered at 8.7 kDa, but some particles also appeared with a mass between 5 and 7.5 kDa. This shows that homogeneous degradation occurred for these particles with, in addition, hydrolysis of Si−O−Si fission of the nanoparticles in several heavy parts of the DOTAGA(Gd) complexes. Each secondary distribution (at 5900, 7700, and 6900 Da, for instance, in Figure 3, spectrum 5) corresponded to nanoparticles with more or less DOTAGA(Gd) with part of the polysiloxane matrix around the nanoparticles. Nanoparticle lifetime was deduced from HPLC-MS following the same method as that used with HPLC-UV. Lifetime was 3 h, in agreement with the UV analysis results. In order to determine the influence of surface modification on nanoparticles, HPLC-MS was carried out for functionalized SRP (SRP−cRGDfK). The lifetime determined was 4.7 h versus 5 h by HPLC-UV (see the Supporting Information). The small difference between lifetime deduced by MS and lifetime deduced by UV can be explained by the sensitivity of the MS measurements. Since MS measurements are more sensitive, the degradation kinetics could also be measured by the kinetics of appearance of the first elution peak (around 6 min), Figure 6a. Following this peak, the lifetime obtained for the nanoparticles without postfunctionalization was 3.4 h, while the lifetime for the postfunctionalized nanoparticles (SRP−cRGD) was 6.6 h. The two peaks (at around 6 and 18 min) follow opposing kinetics, which was expected as the first peak resulted from the degradation of the second one. Figure 6 gives a clear overview of the degradation. The functionalization of the nanoparticles by cRGD considerably reduces degradation kinetics, as already observed for the UV analyses. This study should be linked to the observation of SRP evolution by MS of three main fragments at 1567, 1033, and 795, resulting from the first elution peak at 6 min. This evolution is presented in Figure 6b. It shows that the proportion of the fragment composed of polysiloxane matrix with one DOTAGA(Gd) and one free DOTAGA (m/z 1567) increases in the first 6 h and then decreases. The smallest fragments at 795 always increase and seem to stabilize after a few days. This phenomenon can be explained by the fact that the polysiloxane matrix of the largest fragment with several DOTAGA(Gd) or free DOTAGA continues to be hydrolyzed and forms several smaller fragments, such as the fragment at m/ z 795. The fragment at m/z 1033 increased and stabilized after 6 h. This fragment is composed of one DOTAGA(Gd) and the APTES/TEOS polysiloxane matrix. The augmentation of this fragment’s proportion in the first hours is due to the degradation of the pure nanoparticles. The nanoparticles in a very diluted environment corresponding to physiological phenomena appear to evolve in stable complexes of DOTAGA(Gd) containing only a small quantity of polysiloxane matrix.

Figure 6. (a) Evolution of the bare SRP and SRP functionalized by cRGDfK during aging, according to the first elution peak (at 6 min) corresponding to the fragments resulting from the nanoparticles and according to the second elution (at 18 min) peak corresponding to the pure nanoparticles (HPLC-MS data). (b) Evolution of kinetics of three main fragments at 1567, 1033, and 795 Da resulting from the first elution peak at 6 min. The nanoparticles were at a concentration of 0.3 mM per gadolinium atom at pH 7.4.

and functionalized multimodal SRP nanoparticles. Although HPLC-UV can be used to determine the degradation kinetics for such particles, this technique fails to provide molecular details on the aging of SRP NPs. Moreover, UV absorption of the degradation fragments can differ from that of pure nanoparticles and is nonexistent for the case of silicon or free chelate. In the present article, we proposed original coupling between mass spectrometry (ESI-MS) and HPLC, using C4 columns, in order to determine precisely the composition of nanoparticles and of their degradation fragments during aging. Although initially designed to analyze and purify intact proteins (molecular weight >10 kDa), such columns were found to be very suitable for our SRP nanoparticles. Degradation products were determined, and we obtained insights into the mass distribution of entire SRP NPs and its evolution with aging. Kinetics studies were conducted on both the SRP particles and their low-mass fragments. It is quite possible that such coupling between HPLC and ESI-MS could be applied with other small

■

CONCLUSION In brief, in this work, we aimed at developing analytical methods able to provide detailed analysis of the aging of bare 10446

dx.doi.org/10.1021/ac402429p | Anal. Chem. 2013, 85, 10440−10447

Analytical Chemistry

Article

Duc, G.; Roux, S.; Tillement, O.; Perriat, P. Chem.Eur. J. 2013, 19, 6122−6136. (18) Narang, A. S.; Varia, S. Adv. Drug Delivery Rev. 2011, 63, 640− 658. (19) Choi, H. S.; Frangioni, J. V. Mol. Imaging 2010, 9, 291−310. (20) Morlieras, J.; Dufort, S.; Sancey, L.; Truillet, C.; Mignot, A.; Dentamaro, M.; Laurent, S.; Elst, L. V.; Muller, R. N.; Antoine, R.; Dugourd, P.; Roux, S.; Perriat, P.; Lux, F.; Coll, J.-L.; Tillement, O. Bioconjugate Chem. 2013, 24, 1584−1597. (21) Gallach, D.; Sanchez, G. R.; Noval, A. M.; Silvan, M. M.; Ceccone, G.; Palma, R. J. M.; Costa, V. T.; Duart, J. M. M. Mater. Sci. Eng., B 2010, 169, 123−127. (22) Angel, L. A.; Majors, L. T.; Dharmaratne, A. C.; Dass, A. ACS Nano 2010, 4, 4691−4700. (23) Bonacic-Koutecky, V.; Kulesza, A.; Gell, L.; Mitric, R.; Antoine, R.; Bertorelle, F.; Hamouda, R.; Rayane, D.; Broyer, M.; Tabarin, T.; Dugourd, P. Phys. Chem. Chem. Phys. 2012, 14, 9282−9290. (24) Gaumet, J. J.; Strouse, G. F. J. Am. Soc. Mass. Spectrom. 2000, 11, 338−344. (25) Gies, A. P.; Hercules, D. M.; Gerdon, A. E.; Cliffel, D. E. J. Am. Chem. Soc. 2007, 129, 1095−1104. (26) Guan, B.; Lu, W. G.; Fang, J. Y.; Cole, R. B. J. Am. Soc. Mass. Spectrom. 2007, 18, 517−524. (27) Hamouda, R.; Bellina, B.; Bertorelle, F.; Compagnon, I.; Antoine, R.; Broyer, M.; Rayane, D.; Dugourd, P. J. Phys. Chem. Lett. 2010, 1, 3189−3194. (28) Hamouda, R.; Bertorelle, F.; Rayane, D.; Antoine, R.; Broyer, M.; Dugourd, P. Int. J. Mass Spectrom. 2013, 335, 1−6. (29) Lo, C. K.; Paau, M. C.; Xiao, D.; Choi, M. M. F. Anal. Chem. 2008, 80, 2439−2446. (30) Qian, H. F.; Jin, R. C. Chem. Commun. 2011, 47, 11462−11464. (31) Khitrov, G. A.; Strouse, G. F. J. Am. Chem. Soc. 2003, 125, 10465−10469. (32) Wilcoxon, J. P.; Martin, J. E.; Provencio, P. Langmuir 2000, 16, 9912−9920. (33) Xie, S. P.; Paau, M. C.; Zhang, Y.; Shuang, S. M.; Chan, W.; Choi, M. M. F. Nanoscale 2012, 4, 5325−5332. (34) Zhang, Y.; Shuang, S. M.; Dong, C.; Lo, C. K.; Paau, M. C.; Choi, M. M. F. Anal. Chem. 2009, 81, 1676−1685. (35) Phogat, A.; Kumar, M. S.; Mahadevan, N. Int. J. Recent Adv. Pharm. Res. 2011, 3, 31−36. (36) Chen, T.; Zheng, Y.; Lin, J.-M.; Chen, G. J. Am. Soc. Mass. Spectrom. 2008, 19, 997−1003. (37) Hagen, J. J.; Monig, C. A. Anal. Chem. 1994, 66, 1877. (38) Ettre, L. S. Pure Appl. Chem. 1993, 65, 819.

rigid platforms or liganded nanoclusters with molecular weights ranging between 10 and 50 kDa.

■

ASSOCIATED CONTENT

S Supporting Information *

Glossary of acronyms, setting parameters for ESI-MS experiments, hydrodynamic size distribution of the SRPs, UV−vis absorption spectrum of the SRP NPs, HPLC spectra of the SRP−cRGD nanoparticles degraded at different times, mass spectrum of the first elution peak of the nanoparticles recorded for a fresh solution without aging, and chemical structures for different low-mass fragments of SRP NPs. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Gao, X. H.; Cui, Y. Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. M. Nat. Biotechnol. 2004, 22, 969−976. (2) Lee, J. H.; Huh, Y. M.; Jun, Y.; Seo, J.; Jang, J.; Song, H. T.; Kim, S.; Cho, E. J.; Yoon, H. G.; Suh, J. S.; Cheon, J. Nat. Med. 2007, 13, 95−99. (3) Huang, W. Y.; Davis, J. J. Dalton Trans. 2011, 40, 6087−6103. (4) Liu, Z.; Davis, C.; Cai, W. B.; He, L.; Chen, X. Y.; Dai, H. J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 1410−1415. (5) Ballou, B.; Lagerholm, B. C.; Ernst, L. A.; Bruchez, M. P.; Waggoner, A. S. Bioconjugate Chem. 2004, 15, 79−86. (6) Chang, E.; Thekkek, N.; Yu, W. W.; Colvin, V. L.; Drezek, R. Small 2006, 2, 1412−1417. (7) Derfus, A. M.; Chan, W. C. W.; Bhatia, S. N. Nano Lett. 2004, 4, 11−18. (8) Geiser, M.; Casaulta, M.; Kupferschmid, B.; Schulz, H.; SemmlerBehnke, M.; Kreyling, W. Am. J. Respir. Cell Mol. Biol. 2008, 38, 371− 376. (9) Wei, A.; Mehtala, J. G.; Patri, A. K. J. Controlled Release 2012, 164, 236−246. (10) Choi, H. S.; Liu, W.; Misra, P.; Tanaka, E.; Zimmer, J. P.; Ipe, B. I.; Bawendi, M. G.; Frangioni, J. V. Nat. Biotechnol. 2007, 25, 1165− 1170. (11) Choi, H. S.; Liu, W. H.; Liu, F. B.; Nasr, K.; Misra, P.; Bawendi, M. G.; Frangioni, J. V. Nat. Nanotechnol. 2010, 5, 42−47. (12) Burns, A. A.; Vider, J.; Ow, H.; Herz, E.; Penate-Medina, O.; Baumgart, M.; Larson, S. M.; Wiesner, U.; Bradbury, M. Nano Lett. 2009, 9, 442−448. (13) Park, J. H.; Gu, L.; von Maltzahn, G.; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J. Nat. Mater. 2009, 8, 331−336. (14) Benezra, M.; Penate-Medina, O.; Zanzonico, P. B.; Schaer, D.; Ow, H.; Burns, A.; DeStanchina, E.; Longo, V.; Herz, E.; Iyer, S.; Wolchok, J.; Larson, S. M.; Wiesner, U.; Bradbury, M. S. J. Clin. Invest. 2011, 121, 2768−2780. (15) Wang, L.; Zhao, W.; Tan, W. Nano Res. 2008, 1, 99. (16) Lux, F.; Mignot, A.; Mowat, P.; Louis, C.; Dufort, S.; Bernhard, C.; Denat, F.; Boschetti, F.; Brunet, C.; Antoine, R.; Dugourd, P.; Laurent, S.; Elst, L. V.; Muller, R.; Sancey, L.; Josserand, V.; Coll, J.-L.; Stupar, V.; Barbier, E.; Rémy, C.; Broisat, A.; Ghezzi, C.; Le Duc, G.; Roux, S.; Perriat, P.; Tillement, O. Angew. Chem., Int. Ed. 2011, 50, 12299−12303. (17) Mignot, A.; Truillet, C.; Lux, F.; Sancey, L.; Louis, C.; Denat, F.; Boschetti, F.; Bocher, L.; Gloter, A.; Stéphan, O.; Antoine, R.; Dugourd, P.; Luneau, D.; Novitchi, G.; Figueiredo, L. C.; de Morais, P. C.; Bonneviot, L.; Albela, B.; Ribot, F.; Van Lokeren, L.; DéchampsOlivier, I.; Chuburu, F.; Lemercier, G.; Villiers, C.; Marche, P. N.; Le 10447

dx.doi.org/10.1021/ac402429p | Anal. Chem. 2013, 85, 10440−10447