Article pubs.acs.org/Langmuir

Carbodiimide versus Click Chemistry for Nanoparticle Surface Functionalization: A Comparative Study for the Elaboration of Multimodal Superparamagnetic Nanoparticles Targeting αvβ3 Integrins Julie Bolley,† Erwann Guenin,† Nicole Lievre,‡ Marc Lecouvey,† Michael Soussan,§ Yoann Lalatonne,†,§ and Laurence Motte*,† †

Laboratoire CSPBAT, CNRS (UMR 7244), Université Paris 13, Sorbonne Paris Cité, 74 avenue M. Cachin, 93017 Bobigny, France UPRES 3410 Biothérapies Bénéfices et Risques, Université Paris 13, Sorbonne Paris Cité, 74 avenue M. Cachin, 93017 Bobigny, France § Department of Nuclear Medicine, APHP, Hospital Avicenne, 125 Route de stalingrad, F-93009 Bobigny, France ‡

S Supporting Information *

ABSTRACT: Superparamagnetic fluorescent nanoparticles targeting αvβ3 integrins were elaborated using two methodologies: carbodiimide coupling and click chemistries (CuACC and thiol− yne). The nanoparticles are first functionalized with hydroxymethylenebisphonates (HMBP) bearing carboxylic acid or alkyne functions. Then, a large number of these reactives functions were used for the covalent coupling of dyes, poly(ethylene glycol) (PEG), and cyclic RGD. Several methods were used to characterize the nanoparticle surface functionalization, and the magnetic properties of these contrast agents were studied using a 1.5 T clinical MRI. The affinity toward integrins was evidenced by solidphase receptor-binding assay. In addition to their chemoselective natures, click reactions were shown to be far more efficient than the carbodiimide coupling. The grafting increase was shown to enhance targeting affinity to integrin without imparing MRI and fluorescent properties.

■

of conditions with stereospecificity and high efficiency.13,14 The most widely adopted reaction of this type is the Cu(I)-catalyzed terminal alkyne−azide cycloaddition (CuAAC).15 But a variety of other click reactions have been reported molecules grafting onto nanoparticles: other cycloadditions, Michael additions,16 thiol−ene, and thiol−yne reaction.17,18 Thiol−yne reaction is a water-compatible photochemical reaction under UV irradiation, and in the presence of a radical initiator, two thiol functions are added successively on one alkyne function. We recently shown that the thiol−yne reaction was of promising interest for NPs functionalization.19 While several examples of the use of CuAAC on iron oxide nanoparticle could be found in the literature,10−12 relatively few studies20−23 gave precise quantifications of the grafting, and only one proposed a comparison with classical carbodiimide reaction.24 So ongoing with our research on the development of multimodal nanoplatform,25 we evaluated the efficiency of conjugation using carbodiimide coupling and two click

INTRODUCTION Superparamagnetic iron oxide nanoparticles (SPIO) NPs are promising nanomaterials in biomedicine due to their ultrasmall size, biocompatibility, and superparamagnetic property. NPs are currently being developed as T2(*)-weighted contrast for magnetic resonance, immunoassays, hyperthermal therapies, drug delivery, tissue repair, and cell manipulation, among other applications.1−3 For this wide range of applications, SPIO NPs surface must be tailored to insert molecules on the NP surface with control of their architecture and surface density to improve affinity and targeting efficiency or to impart additional properties. The design of multifunctional nanoparticles is an exciting future challenge in the field.4−8 Several chemical methodologies are described for the NP surface functionalization.9 One of the most popular strategies involves amide bond formation through carbodiimide coupling with the 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) cocktail. But recently, the emergence and adoption of click chemistry have had a large impact on nanomaterials surface functionalization.10−12 Click chemistry refers to modular chemical conjugations with an emphasis on simple reactions that can take place under a range © 2013 American Chemical Society

Received: August 26, 2013 Revised: October 30, 2013 Published: October 30, 2013 14639

dx.doi.org/10.1021/la403245h | Langmuir 2013, 29, 14639−14647

Langmuir

Article

Scheme 1. Carbodiimide (A) vs Click Chemistry (B); Elaboration of Multimodal Imaging Contrast Agents (C)

particles’ synthesis and analyses, all other commercially available reagents were purchased from Sigma-Aldrich. The FTIR spectra were recorded as thin films on KBr pellets on a Thermo Scientific Nicolet 380 FTIR and are reported in frequency of absorption (cm−1). UV−vis spectra were recorded on a Varian Cary 50 Scan UV−vis spectrophotometer. The size and the zeta potential of the nano complex were determined by dynamic laser light scattering (DLS) on a Nano-ZS (Red Badge) ZEN 3600 device (Malvern Instruments, Malvern, UK). TEM images were obtained using a FEI CM10 microscope (Philips), and samples were prepared by depositing a drop of nanoparticles suspension on carbon-coated copper grids placed on a filter paper. The median diameter is deduced from TEM data measurements, simulating the diameter distribution with a log-normal function. The magnetic behavior at room temperature of the as-synthesized nanoparticles is characterized using MIAplex reader (Magnisense SA). Miaplex reader is a miniaturized chip detector system, measuring a signal corresponding to the second derivative of the magnetization around zero field.26 With this sensor, measurements are performed on microliter sample volumes, in a handled portable device, without liquid helium cooling to operate. Moreover, MIAplex signal measurement (∼30 s) is about 102 times shorter than the VSM (∼1 h), and the sample quantity is 103 times lower (1 μg). The transverse nuclear relaxation times, T2, were measured from axial T2-weighted SE images obtained with a time repetition (TR) of 2000 ms and increasing time echos (TEs) of 20, 40, 60, and 80 ms with a 1.5 T MRI scanner (Philips intera 1.5T/Philips Healthcare) at room temperature for various iron concentrations.

reactions (CuAAC and thiol−yne) (Scheme 1). This evaluation is done on the same nanoparticle batch (composition, size), with the complexing agent only differing by the functionality at the outer surface. We for the first time quantitatively compared grafting with both methodologies with three different types of molecules (fluorophores, PEG chain, and a targeting peptide). We moreover evaluated the impact of grafting efficiency on fluorescence, MRI, and targeting properties. Thus, choosing cycloRGD peptides, we elaborate magnetofluorescent nanoparticles targeting αvβ3 integrins. Integrins αvβ3 are cell adhesion molecules known to be involved in multiple steps of angiogenesis and metastasis. The high expression of integrin αvβ3 during tumor growth, invasion, and metastasis makes it interesting molecular target for the development of αvβ3 targeted therapeutic drugs and molecular imaging probes.

■

EXPERIMENTAL SECTION

Chemical and Apparatus. The dyes Rhodamine123 (Rh123) and Rhodamine B and the HS-(PEG)4-COOH were purchased from Acros, Sigma-Aldrich, and Irish Biotech, respectively. The fluoraldehyde reagent solution (OPA) and carboxyl(tetraethylene glycol)ethylamine (H2N-(PEG)4-COOH) were purchased from ThermoScientific. The cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGDfK) and cyclo(Arg-Gly-Asp-D-Phe-Lys(PEG-PEG)) and its analogue with an azide terminal function cyclo(Arg-Gly-Asp-D-Phe-Lys(PEG−PEG)-Azide) were purchased from Peptides International. The cyclo(Arg-Gly-AspD-Phe-Lys) with an N-terminal azide function was purchased from Caslo. The integrin αvβ3 was purchased from Merck. Regarding 14640

dx.doi.org/10.1021/la403245h | Langmuir 2013, 29, 14639−14647

Langmuir

Article

°C, t = 5 min for cRGD derivatives coupling). The procedure was realized with various molar ratios R = nN3/nCCH. The modified particles were isolated with a magnet at pH 2 and washed several times with deionized water. The particles are redispersed in water at physiological pH for various physicochemical characterizations. The coupling of HS-(PEG)4-COOH onto the HMBP-CCH functionalized maghemite was performed in a water/DMF (50/50) mixture in a one-step procedure under UV irradiation (360 nm). Briefly, the γFe2O3@HMBP-CCH in deoxygenized water at pH 7 and DMF was mixed with 1-hydroxycyclohexylphenyl ketone (10%) and the thiol reactant. The procedure is realized for different time under UV irradiation and various molar ratios R = nSH/nCCH. The modified particles were isolated with a magnet at pH 2 and washed several times with deionized water. The particles were redispersed in water at physiological pH for various physicochemical characterizations. Coupling Characterizations. The efficiency of the dyes (Rh123, Rh-N3) conjugation was investigated qualitatively using various measurement methods: infrared spectroscopy, dynamic light scattering, and quantitatively using fluorescence spectroscopy. The number of dyes per nanoparticle was determined after molecule desorption using alkaline medium (pH 12, 24 h) using a previously described procedure.29 The magnetic particles were then separated from the supernatant by magnetic decantation. The pH of the supernatant was adjusted to physiological pH, and the concentration of the dye was deduced from fluorescence measurements. The quenching phenomenon was then evaluated using the ratio between the fluorescence measurement obtained from a solution of nanoparticles dispersed in water at pH 7.4, over the fluorescence measurement on supernatant after molecule desorption. The average number of molecules of H2N-(PEG)4-COOH and cRGD derivatives per nanocrystal was measured by OPA method and deduced from a calibration curve. 50 μL of the sample was diluted in 50 μL of NaOH (2 N) and stirred all night at 60 °C. 1 mL of OPA reagent was added to the mixture, and fluorescence measurement was recorded. The average number of HS-(PEG)4-COOH was deduced by EDX, measuring the ratio of iron to sulfur signals. Affinity Measurement by Solid-Phase Binding Assay. The receptor binding assay was performed as described previously.30,31 αvβ3 was diluted at 500 ng/mL in coating buffer (20 mM tris, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2), and an aliquot of 100 μL/well was added to a 96-well microtiter plate (Microlite 2+, Thermo Scientific) and incubated overnight at 4 °C. The plate was washed once with blocking/binding buffer (20 mM tris, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 1% bovine serum albumin) and incubated an additional 2 h at room temperature. The plate was rinsed twice with the same buffer and incubated with the radiolabeled ligand at the indicated concentrations for 3 h at room temperature. For coincubations, unlabeled competitor was included at the concentrations described. After an additional three washes with blocking/binding buffer, counts were solubilized with boiling 2 N NaOH and subjected to γ-counting. Nonspecific binding of ligand to the receptor was determined with molar excess (200-fold) of the unlabeled ligand. Each data point is a result of the average of triplicate wells. MRI. The 1H NMR relaxometric characterization was performed by measuring the transverse nuclear relaxation times T2 on a 1.5 T MRI scanner. The measurements were performed at room temperature for various iron concentrations between 0.061 and 0.25 mM. The efficiency of MRI contrast agent was determined by measuring the relaxivities r2 defined as r2 = (1/T2)meas/C, where (1/T2)meas is the value measured with the sample at concentration C of iron. Biological Stability. The biological stability of γFe2O3@HMBPCOOH-Rh123 and γFe2O3@HMBP-CCH-RhN3 was evaluated by fluorescence spectroscopy. Briefly, the particles were dispersed in 10% or 50% of FBS (fetal bovine serum) at an iron concentration of 0.1 mM. The fluorescence intensity was recorded as a function of time.

The thermogravimetric analysis (TGA) curves were recorded using a LabsSys evo TG-DTA-DSC 16000 device manufactured by Setaram Instrumentation. Energy-dispersive X-ray (EDX) microanalysis were performed using a TM 3000 tabletop microscope equipped with a Swift EDX-ray 3000 microanalysis system (Oxford Instruments). Samples were deposited as powder on a copper surface, and data were collected using a 15 kV accelerating voltage. Quantification of coating and grafting per particle was evaluated by studying ratio of iron vs phosphorus or sulfur and knowing the average number of iron atoms/ particles. The different couplings under microwave energy were performed on a Monowave 300 from Anton Paar. The fluorescence measurements were performed with a Spex FluoroMax spectrofluorometer (HORIBA Jobin-Yvon, France) equipped with a Hamamatsu 928 photomultiplier. The 125I echistatin radioactivity was counted in a gamma counter Wizard 1470 (PerkinElmer). Chemical Synthesis of Bisphosphonates and Rh-N3. The chemical syntheses of the bisphosphonates HMBP-COOH and HMBP-CCH and azido-modified rhodamine B (Rh-N3) were prepared according to previously reported procedures.27,28 Synthesis of γFe2O3 Nanocrystals. The maghemite NPs and γFe2O3 nanocrystals were synthesized according to a procedure already described.25,27 Briefly, bare γFe2O3 particles were synthesized by the reaction of ferrous dodecyl sulfate with dimethylamine. After 2 h at 28 °C, the solution was rinsed under acidic conditions. The magnetic particles were then separated from the supernatant via a permanent magnet. After 10 washings, the particles were dispersed in water at appropriate pH and concentration conditions. Synthesis of γFe2O3@HMBP Nanocrystals. 10 mL of an aqueous HMBP solution (15 mg mL−1) at pH 2 was directly mixed with 10 mL of bare γFe2O3 nanoparticle suspension (6 mg mL−1) at pH 2. After 2 h at room temperature, the HMBP in excess was removed from the coated particles using a magnetic field at pH 2. The magnetic γFe2O3@HMBPyne particles were dispersed in distilled water and adjusted to pH 7. The average number of molecules of HMBP per nanocrystal was measured by TGA or EDX. Carbodiimide Coupling onto γFe2O3@HMBP-COOH Nanocrystals. In the following procedures, the coupling of the molecule of interest (Rh123 dye, H2N-(PEG)4-COOH, or cRGD derivatives with a terminal amine function) onto the HMBP-COOH functionalized maghemite was performed in water in a two-step procedure (activation and conjugation) at room temperature (2 h for the activation step and one night for the conjugation step) or assisted by microwaves (Tmax = 150 °C, t = 3 min for dye coupling, and 3 cycles of Tmax = 65 °C, t = 5 min for cRGD derivatives coupling). First, the carboxylic acid functions at the outer surface of the nanocrystals were activated using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC, nEDC = 5nCOOH) and N-hydroxysuccinimide (NHS, nNHS= 5nCOOH) at pH 4. The second step was the reaction of the amine function of the interest molecule with the activated carboxylic acid functions on the nanocrystals. Prior to addition, the pH of the γFe2O3@HMBPCOOH ferrofluid was adjusted to pH = 9 with N,N-diisopropylethylamine. The molecule of interest was dissolved in water at pH 9 and then added to the ferrofluid. The procedure was realized with various molar ratios R = nNH2/ nCOOH. The modified particles were isolated with a magnet at pH 2 and washed several times with deionized water. The particles are redispersed in water at physiological pH for various physicochemical characterizations. Click Coupling onto γFe2O3@HMBP-CCH Nanocrystals. In the following procedures, the coupling of the molecule of interest (Rh−N3 dye or cRGD derivatives with a terminal azide function) onto the HMBP-CCH functionalized maghemite was performed in water in a one-step procedure at room temperature or assisted by microwaves. Briefly, the azido reactant, copper sulfate hexahydrate (5%), and sodium ascorbate (20%) were added to the γFe2O3@ HMBP-CCH solution (pH 7). The resulting solution was either stirred one night at room temperature or submitted to microwaves (Tmax = 150 °C, t = 3 min for dye coupling, and 3 cycles of Tmax = 65 14641

dx.doi.org/10.1021/la403245h | Langmuir 2013, 29, 14639−14647

Langmuir

Article

Figure 1. γFe2O3@HMBP nanoplatform characterizations. Hydrodynamic diameter (blue) and zeta potential (red) of (A) γFe2O3@HMBP-COOH and (B) γFe2O3@HMBP-CCH. Magnetization curve and second derivative of magnetization in the inset of (C) γFe2O3@HMBP-COOH and (D) γFe2O3@HMBP-CCH.

Table 1. Average Number of Dyes per NPs before and after Molecule Desorption, Quenching Factor, Yield Coupling, and Colloidal Behavior (Hydrodynamic Diameter and Surface Charge) Obtained by Click and Carbodiimide Chemistry for Different Ratio R grafted molecule number carbodiimide click

R

before and after desorption

2 20 2 20

1 6 7 36

± ± ± ±

1 1 3 5

4 40 25 165

± ± ± ±

quenching

1 4 5 11

4 7 4 5

% 0.7 7 5.4 38

± ± ± ±

0.2 1 0.5 3

Dh (nm)

PDI

zeta (mV)

13.2 58 20 26

0.3 0.3 0.4 0.5

−37 −43 −37 −45

Table 2. Average Number of PEG per Nanoparticle, Yield Coupling, and Colloidal Behavior (Hydrodynamic Diameter and Surface Charge) Obtained by Click and Carbodiimide Chemistry for Different Ratio R and Different UV-Irradiation Time for Thiol−Yne Reaction R carbodiimide

click

■

time (h)

2 5 20 50 5 5 5 50

0.5 1.5 16 1.5

grafted molecule number 50 50 63 87 204 221 144 192

± ± ± ± ± ± ± ±

2 9 3 13 20 22 15 20

% 9 9 11 16 51 55 36 48

± ± ± ± ± ± ± ±

1 2 1 2 5 5 4 5

Dh (nm)

PDI

zeta (mV)

16 16 18 18 22 19 22 20

0.3 0.3 0.3 0.3 0.2 0.2 0.2 0.3

−35 −38 −34 −38 −44 −40 −41 −43

sphosphonate (HMBP) anchors bearing either alkyne19 or carboxylate terminal groups.27 Several techniques can be used for the ligand quantification. Thermogravimetric analysis20 is the more conventional and versatile method though it requires

RESULTS AND DISCUSSION

γFe2O3@HMBP Nanoplatforms. We used two nanoplatforms, previously reported, consisting of γFe2O3 nanoparticles (crystalline diameter 9 nm) with hydroxylmethylene bi14642

dx.doi.org/10.1021/la403245h | Langmuir 2013, 29, 14639−14647

Langmuir

Article

Table 3. Average Number of cRGD or cRGD-PEG per Nanoparticle, Yield Coupling, and Colloidal Behavior (Hydrodynamic Diameter and Surface Charge) Obtained by Click and Carbodiimide Chemistry for a Ratio R = 2 RGD carbodiimide click

cRGDfK cRGDfK-PEG-NH2 cRGDfK(N3) cRGDfK-PEG-N3

grafted molecule number 6 12 36 50

± ± ± ±

1 1 4 5

% 1 2 8 11

± ± ± ±

0.2 0.2 1 1

Dh (nm)

PDI

zeta (mV)

10 16 15 19

0.3 0.2 0.3 0.3

−36 −39 −39 −43

Table 4. PEG-cRGD Number and % per Multimodal Nanoplatform for R = 2, Fluorescence Imaging, Quenching, and Dynamic Light Scattering Properties (Hydrodynamic Size, Polydispersity Index, and Zeta Potential) before and after Coupling PEGcRGD

(Figure S6). The average number of dyes per NPs was determined both on NPs solutions and after forced desorption to evaluate quenching phenomena. The average number of dyes per NPs, quenching factor, grafting yield, and colloidal behaviorhydrodynamic diameter and surface chargeare reported in Table 1. The grafting yield express in percentage corresponds to the ratio between the number of conjugated molecule over the number of HMBP ligand (see Figure S8 for infrared characterization). Hence, increasing ratio R allows to increase the average number of dye per NPs, and the emission is decreased by a factor around 5 (Table 1). The grafting yield and stability are slightly better using click chemistry. This behavior could be related to the presence of a spacer between azide and dye aromatic functions, reducing steric hindrance (Figure S4). Comparison of PEG Coupling. Thiol−yne and carbodiimide reaction were also evaluated for PEG coupling onto the two nanoplatforms. The grafting of the HS-PEG-COOH and H2NPEG-COOH was evaluated quantitatively by EDX analysis, measuring the ratio of iron to phosphorus and sulfur signals, and the fluoraldehyde reagent (OPA) method quantifying amine functions after alkaline decomposition of the peptide bond. Indeed, the limitation of EDX analysis for small atom quantification, such as carbon or nitrogen, requires the use of an other method for H2N-PEG-COOH detection on nanoparticle surface. The OPA method involves the destruction of low quantity of nanoparticles (less than 1 mg) and before a calibration curve establishment. Therefore, this method is appropriate and advantageous for biological molecule in view of the presence of amine or amide functions and the cost of molecule. Several molar ratios for the PEG versus COOH/C CH functions were tested (ratio R), and the UV-irradiation time for thiol−yne reaction was also explored (Table 2). Thiol−yne reaction clearly appears as far more efficient than carbodiimide chemistry even with microwave assistance, leading to a 50% coupling yield. Thiol−yne reaction is completed after 1 h 30 min, and the increase in the ratio R did not improve the grafting yield. Indeed, after 16 h of reaction, the grafting rate is lower (about 36%) than those observed at shorter time (about 50%), and increasing the ratio R by a factor of 10 induces similar coverage yield. The hydrodynamic size and zeta potential are quite constant and similar for each nanoplatform.

important sample quantity. Other methods are more dependent on the molecule structure: FTIR,32 NMR,25 pH titration,33 energy-dispersive X-ray analysis,19 specific colorimetric test.34 In our case, the number of HMBP per particles was deduced by energy-dispersive X-ray measurements and thermogravimetric analysis and was found to be 440 ± 40 HMBP-CCH and 550 ± 30 HMBP-COOH per particles which corresponds to 2.2 and 1.7 ligands/nm2, values in agreement with the literature35 (see Supporting Information for TEM images, size distribution, FT-IR spectra, and quantification of HMBPs per nanoparticle). The γFe2O3@HMBP nanoplatforms formed highly stable ferrofluids in a large range of pH, from pH 4 to pH 11, and in different salt concentration solutions (see Supporting Information). At physiological pH, the hydrodynamical diameter and zeta potential are 11 nm, −56 mV and 13 nm, −55 mV for alkyne and carboxylate nanoplatforms, respectively, suggesting very low aggregation, (Figure 1A,B). Concerning magnetic measurements, the magnetization curve and second derivative of magnetization, measured with MIAplex detector, are characteristic of superparamagnetic behavior (Figure 1C,D). γFe2O3@COOH and γFe2O3@CCH present equivalent saturate magnetization of 43 and 47 emu g−1, respectively. Once these nanoplatforms are well characterized, the carboxylic acid functions or the alkynes ones at the outer surface of the nanoparticles were used for the covalent coupling of different ligands (dye, PEG, or RGD derivatives) using carbodiimide or click chemistry. Coupling Efficiency Study by Carbodiimide and Click Chemistry. Comparison of Dye Coupling. The two nanoplatforms were first modified with rhodamine (Rh) derivatives with microwave-assisted procedure: azido-functionalized rhodamine B (Rh-N3) when using CuAAC methodology and 123rhodamine (Rh-123) when using carbodiimide chemistry (Figure S4). We recently shown that in the case of triazole/ amide bond formation the functionalization yield could be increased by using microwave energy.28,29 (See Supporting Information for comparison conventional versus microwaves covalent coupling, Table S2). Several molar ratios for the dye versus CCH/COOH functions were tested (ratio R). After coupling of both Rh derivatives, fluorescence emission spectra of the fluorescent γFe2O3 nanoplatforms gave respectively a maximum wavelength at 518 and 575 nm slightly shifted compared to free Rh-123 (522 nm) and Rh-N3 (573 nm) 14643

dx.doi.org/10.1021/la403245h | Langmuir 2013, 29, 14639−14647

Langmuir

Article

Table 5. IC50, Ki, and Beta Factor toward αvβ3, Obtained by Competition Binding Assay, of γFe2O3@HMBP-cRGD and γFe2O3@HMBP-(PEG-cRGD) Nanoplatforms for a Ratio R = 0 and R = 2, and Multimodal Nanoplatforms Obtained by Carbodiimide and Click Chemistry RGD carbodiimide

click

Figure 2. Binding of 125I-echistatin was added to a final concentration of 0.05 nM in the presence of the various competing ligands at the indicated concentrations. The ligands studied were (A) γFe2O3@ HMBP-COOH (black), cRGDfK (green), cRGDfK-PEG-NH 2 (purple), with the red and blue curves corresponding to their coupling respectively on γFe2O3@HMBP-COOH by carbodiimide chemistry for a ratio R = 2. (B) γFe2O3@HMBP-CCH (black), cRGDfKN3(green), and cRGDfK-PEG-N3 (purple) with the red and blue curves corresponding to their coupling respectively on γFe2O3@ HMBP-CCH by click chemistry for a ratio R = 2. (C) Multimodal nanoplatforms obtained by carbodiimide chemistry (red) and click chemistry (green) and their blanks (black) and (blue), respectively.

cRGDfK γFe2O3@ cRGDfK cRGDfK-PEGNH2 γFe2O3@ cRGDfKPEG-NH2 multimodal cRGDfK(N3) γFe2O3@ cRGDfK(N3) cRGDfK-PEGN3 γFe2O3@ cRGDfKPEG-N3 multimodal

grafted RGD number

%

6±1

1 ± 0.2

IC50 (nM)

Ki (nM)

93 99

76 81

32

26

12 ± 1

2 ± 0.2

21

17

11 ± 2

2 ± 0.4

36 ± 4

8±1

27 54 23

22 44 19

77

63

10

8

50 ± 5

11 ± 1

48 ± 3

11 ± 1

5.3

4.3

Figure 3. T2-weighted MR images of (A) γFe2O3@HMBP-COOH and (B) γFe2O3@HMBP-CCH at CFe = 0.061, 0.125, 0.188, and 0.25 mM (TR = 2000 ms, TE = 20 ms). Transverse relaxivities r2 measured on a 1.5 T clinical MRI (C) of the nanoplatform (a) γFe2O3@HMBP-COOH functionalized with (b) PEG (R = 50), (c) cRGD-PEG (R = 2), and (d) the multimodal nanoplatform obtained by carbodiimide chemistry; the nanoplatform (a′) γFe2O3@HMBPCCH functionalized with (b′) PEG (R = 5; 1 h 30 min), (c′) cRGD-PEG (R = 2), and (d′) the multimodal nanoplatform obtained by click chemistry.

Comparison of RGD Derivatives Coupling. In order to introduce αvβ3 targeting, the NPs were coupled to a cRGD motif. Ongoing with our investigation on the coupling methodology, we choose to link the peptide c(RGDfK) onto the carboxylated nanoplatform via the lysine residue and to link an azido cRGD peptide on the alkyne nanoplatform using CuAAC (Figure S11). The influence of the introduction of PEG linker between the peptide and its functionality on the grafting efficiency was also investigated. The average number of peptides grafted on each nanoplatform was evaluated using OPA reactant (Table 3).

As observed for dye and PEG coupling, click chemistry allowed better coupling yield. When grafting RGD, Lu et al.21 obtained similar yields with higher peptide molar ratio. In both methodologies, the addition of PEG linker is correlated to an increase in grafting efficiency resulting from a decrease in steric hindrance between peptides in accordance with results already obtained by Lin et al.36 As expected, the hydrodynamic 14644

dx.doi.org/10.1021/la403245h | Langmuir 2013, 29, 14639−14647

Langmuir

Article

Figure 4. Release of (A) γFe2O3@HMBP-COOH-Rh123 (R = 2) and γFe2O3@HMBP-CCH-RhN3 (R = 2) in 10% (black) and 50% (red) FBS.

As shown in Figure 3A,B, T2-weighted images obtained with a 1.5 T clinical MRI change drastically in signal intensity with an increasing amount of nanoparticles, indicating that assynthesized nanoparticles generated MR contrast on transverse (T2) proton relaxation times weighted sequences. The relaxation relaxivities r2 are 127 and 179 mM−1 s−1 for γFe2O3@COOH and γFe2O3@CCH, respectively (see Figure S14). The difference in r2 values could be due to the nature of terminal function (alkyne−carboxylate) and/or to the coverage density. For each grafted nanoplatform the transverse relaxivity r2 was nearly constant (around 140 mM−1 s−1) and was independent of the grafted molecules on NPs surface (Figure 3C). The transverse relaxivities, r2, of the different nanoplatforms are similar to those of commercial MRI contrast agents such as Endorem from Guerbet (98 mM−1 s−1) and Resovist from Bayer Healthcare (151 mM−1 s−1)37 and suggest that the assynthesized nanoplatforms could be useful as MRI contrast agents. Biological Stability. NP stability in serum is an important criterion for their utility as targeting nanosystems in vivo. Using the change in fluorescence intensity in the presence of plasma as a surrogate for protein adsorption, we studied stability in 10% and 50% FBS (fetal bovine serum) of γFe2O3@HMBPCOOH-Rh123 (R = 2) and γFe2O3@HMBP-CCH-RhN3 (R = 2). After 24 h, less than 15% of release is reached (Figure 4), showing the biostability of these two nanoplatforms through the high HMBP anchoring on iron oxide surface and the covalent coupling through amide or triazole bonds.

diameter increased slightly when adding PEG chains. One must note that when comparing the increase in grafting yield from carbodiimide to click chemistry with really similar molecules, it is perfectly consistent with what was observed for rhodamine grafting (approximately a 5-fold grafting increase). So we can conclude for rhodamine coupling though derivatives were not the same that the grafting increase is mostly due to the chemical methodology used and not the steric hindrance between both derivatives. Finally, we elaborated multimodal nanoplatforms by coupling in a first step the rhodamine derivatives and then the RGD derivatives with a ratio R = 2. PEG-cRGD derivatives were used in order to reduce the potential steric hindrance between Rh and RGD and to maximize the grafting efficiency. The two functionalization steps induced an increase of hydrodynamic diameters and consequently an increase in Rh quenching,27 but it was still sufficient for fluorescence imaging (Table 4). This effect was more drastic for the γFe2O3@COOH nanoplatform compared to the γFe2O3@CCH nanoplatform. Furthermore, the grafting yield for PEG-cRGD derivatives was not influenced by the presence of Rh for both nanoplatforms (compare Tables 3 and 4). Comparative Study toward αvβ3 Integrins Targeting. The affinities of cRGD derivatives and γFe2O3-labeled nanoplatforms to αvβ3 integrins were investigated using radioactive 125Ilabeled echistatin solid phase binding assay (Supporting Information Ki calculations). We first tested γFe2O3@COOH and γFe2O3@CCH nanoplatforms for nonspecific binding to αvβ3, and no binding was observed (Figure S13). Ki values were markedly decreased compared to the corresponding free ligand for the click nanoplatform, whereas no significant enhancement was obtained with the carbodiimide nanoplatform (Figure 2 and Table 5). This latter result as to be related to the lower number of RGD derivatives grafted on the nanoplatform so shows the crucial importance of being able to control the number of peptides at the surface. Thus, the binding to the target when using click is improved (2−5-fold), which is in good agreement with results obtained by Thorek and coworkers.24 MRI Analysis. Finally, the potential of these nanoplatforms as contrast agent was studied using a 1.5 T clinical MRI. The transverse relaxation times, T2, were measured with a TR of 2000 ms and increasing TEs of 20, 40, 60, and 80 ms at room temperature.

■

CONCLUSION In summary, the design of multifunctional nanoparticles is a challenging task as for biological applications it is of crucial importance to be able to control the grafting of molecules onto the surface without imparting NPs and/or surface ligands properties. We here wanted to compare various chemistries for the elaboration of a multimodal nanoplatform: the well-known carbodiimide chemistry and CuAAC click chemistry as well as the thiol−yne chemistry recently described onto NPs surface. We studied the grafting of a fluorophore rhodamine, PEG chains, and the αvβ3 integrin targeting peptide cRGD. We showed that whatever the grafted molecules and in addition to their chemoselective natures click reactions are far more efficient for the nanoparticle functionalization than the carbodiimide coupling even under microwave assistance. 14645

dx.doi.org/10.1021/la403245h | Langmuir 2013, 29, 14639−14647

Langmuir

Article

azide-alkyne “click” chemistry. Adv. Drug Delivery Rev. 2008, 60, 958− 970. (13) Erathodiyil, N.; Ying, J. Y. Functionalization of inorganic nanoparticles for bioimaging applications. Acc. Chem. Res. 2011, 44, 925−935. (14) Papst, S.; Cheong, S.; Banholzer, M. J.; Brimble, M. A.; Williams, D. E.; Tilley, R. D. One-pot synthesis of water soluble iron nanoparticles using rationally-designed peptides and ligand release. Chem. Commun. 2013, 49, 4540−4542. (15) Lutz, J.-F. 1,3-dipolar cycloadditions of azides and alkynes: A universal ligation tool in polymer and materials science. Angew. Chem., Int. Ed. 2007, 46, 1018−1025. (16) Cutler, E. C.; Lundin, E.; Garabato, B. D.; Choi, D.; Shon, Y.-S. Dendritic functionalization of monolayer-protected gold nanoparticles. Mater. Res. Bull. 2007, 42, 1178−1185. (17) Korthals, B.; Morant-Miñana, M. C.; Schmid, M.; Mecking, S. Functionalization of polymer nanoparticles by thiol-ene addition. Macromolecules 2010, 43, 8071−8078. (18) Tucker-Schwartz, A. K.; Farrell, R. A.; Garrell, R. L. Thiol-ene click reaction as a general route to functional trialkoxysilanes for surface coating applications. J. Am. Chem. Soc. 2011, 133, 11026− 11029. (19) Demay-Drouhard, P.; Nehlig, E.; Hardouin, J.; Motte, L.; Guénin, E. Nanoparticles under the light: Click functionalization by photochemical thiol-yne reaction, towards double click functionalization. Chem.Eur. J. 2013, 19, 8388−8392. (20) Hayashi, K.; Moriya, M.; Sakamoto, W.; Yogo, T. Chemoselective synthesis of folic acid functionalized magnetite nanoparticles via click chemistry for magnetic hyperthermia. Chem. Mater. 2009, 21, 1318−1325. (21) Lu, J.; Shi, M.; Shoichet, M. S. Click chemistry functionalized polymeric nanoparticles target corneal epithelial cells through RGDcell surface receptors. Bioconjugate Chem. 2008, 20, 87−94. (22) Polito, L.; Monti, D.; Caneva, E.; Delnevo, E.; Russo, G.; Prosperi, D. One-step bioengineering of magnetic nanoparticles via a surface diazo transfer/azide-alkyne click reaction sequence. Chem. Commun. 2008, 621−623. (23) Schätz, A.; Hager, M.; Reiser, O. Cu(II)-azabis(oxazoline)complexes immobilized on superparamagnetic magnetite@silica-nanoparticles: A highly selective and recyclable catalyst for the kinetic resolution of 1,2-diols. Adv. Funct. Mater. 2009, 19, 2109−2115. (24) Thorek, D.; Elias, D.; Tsourkas, A. Comparative analysis of nanoparticle-antibody conjugations: Carbodiimide versus click chemistry. Mol. Imaging 2009, 8, 221−9. (25) Benyettou, F.; Lalatonne, Y.; Chebbi, I.; Benedetto, M. D.; Serfaty, J.-M.; Lecouvey, M.; Motte, L. A multimodal magnetic resonance imaging nanoplatform for cancer theranostics. Phys. Chem. Chem. Phys. 2011, 13, 10020−10027. (26) de Montferrand, C.; Lalatonne, Y.; Bonnin, D.; Lièvre, N.; Lecouvey, M.; Monod, P.; Russier, V.; Motte, L. Size-dependent nonlinear weak-field magnetic behavior of maghemite nanoparticles. Small 2012, 8, 1945−1956. (27) Lalatonne, Y.; Paris, C.; Serfaty, J. M.; Weinmann, P.; Lecouvey, M.; Motte, L. Bis-phosphonates-ultra small superparamagnetic iron oxide nanoparticles: a platform towards diagnosis and therapy. Chem. Commun. 2008, 22, 2553−2555. (28) Guénin, E.; Hardouin, J.; Lalatonne, Y.; Motte, L. Bivalent alkyne-bisphosphonate as clickable and solid anchor to elaborate multifunctional iron oxide nanoparticles with microwave enhancement. J. Nanopart. Res. 2012, 14, 1−10. (29) Benyettou, F.; Guenin, E.; Lalatonne, Y.; Motte, L. Microwave assisted nanoparticle surface functionalization. Nanotechnology 2011, 22, 055102. (30) Orlando, R. A.; Cheresh, D. A. Arginine-glycine-aspartic acid binding leading to molecular stabilization between integrin alpha v beta 3 and its ligand. J. Biol. Chem. 1991, 266, 19543−50. (31) Kumar, C. C.; Rogers, H.-N. C. P.; Malkowski, M.; Maxwell, E.; Catino, J. J.; Armstrong, L. Biochemical characterization of the binding

Moreover, the grafting increase was shown to enhance targeting affinity to integrin. Finally, we proved biostability and the multimodality: fluorescence, MRI properties, and targeting of our nanoplatform.

■

ASSOCIATED CONTENT

S Supporting Information *

[TEM images, size distribution, MRI curves, and FTIR spectra of γFe2O3@HMBP-COOH and γFe2O3@HMBP-CCH, quantification of HMBP ligands, chemical structures, comparison of conventional and microwaves coupling, calibration curves for OPA method, biological stability determination, solid phase binding assay, determination of echistatin Kd. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Fax +33 1 41 08 85 28 (L.M.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by ANR 2010-BLAN-1007-1 fundings. We are grateful to Magnisense Corporation for providing the MIAplex reader.

■

REFERENCES

(1) Catherine, C. B.; Adam, S. G. C. Functionalisation of magnetic nanoparticles for applications in biomedicine. J. Phys. D: Appl. Phys. 2003, 36, R198. (2) Hu, J.; Qian, Y.; Wang, X.; Liu, T.; Liu, S. Drug-loaded and superparamagnetic iron oxide nanoparticle surface-embedded amphiphilic block copolymer micelles for integrated chemotherapeutic drug delivery and MR imaging. Langmuir 2012, 28, 2073−2082. (3) Tanaka, K.; Narita, A.; Kitamura, N.; Uchiyama, W.; Morita, M.; Inubushi, T.; Chujo, Y. Preparation for highly sensitive mri contrast agents using core/shell type nanoparticles consisting of multiple SPIO cores with thin silica coating. Langmuir 2010, 26, 11759−11762. (4) Frimpong, R. A.; Hilt, J. Z. Magnetic nanoparticles in biomedicine: synthesis, functionalization and applications. Nanomedicine 2010, 5, 1401−1414. (5) Pankhurst, Q. A.; Thanh, N. T. K.; Jones, S. K.; Dobson, J. Progress in applications of magnetic nanoparticles in biomedicine. J. Phys. D: Appl. Phys. 2009, 42, 224001. (6) Catherine, C. B. Progress in functionalization of magnetic nanoparticles for applications in biomedicine. J. Phys. D: Appl. Phys. 2009, 42, 224003. (7) Wang, Y.; Li, B.; Zhang, L.; Song, H. Multifunctional mesoporous nanocomposites with magnetic, optical, and sensing features: Synthesis, characterization, and their oxygen-sensing performance. Langmuir 2013, 29, 1273−1279. (8) Shen, J.; Zhu, Y.; Yang, X.; Zong, J.; Li, C. Multifunctional Fe3O4@Ag/SiO2/Au core-shell microspheres as a novel SERS-activity label via long-range plasmon coupling. Langmuir 2013, 29, 690−695. (9) Perrier, T.; Saulnier, P.; Benoît, J.-P. Methods for the functionalisation of nanoparticles: new insights and perspectives. Chem.Eur. J. 2010, 16, 11516−11529. (10) He, H.; Gao, C. Click chemistry on nano-surfaces. Curr. Org. Chem. 2011, 15, 3667−3691. (11) Li, N.; Binder, W. H. Click-chemistry for nanoparticlemodification. J. Mater. Chem. 2011, 21, 16717−16734. (12) Lutz, J.-F. o.; Zarafshani, Z. Efficient construction of therapeutics, bioconjugates, biomaterials and bioactive surfaces using 14646

dx.doi.org/10.1021/la403245h | Langmuir 2013, 29, 14639−14647

Langmuir

Article

of echistatin to integrin αvβ3 receptor. J. Pharmacol. Exp. Ther. 1997, 283, 843−853. (32) Martin, A.; Hickey, J.; Ablack, A.; Lewis, J.; Luyt, L.; Gillies, E. Synthesis of bombesin-functionalized iron oxide nanoparticles and their specific uptake in prostate cancer cells. J. Nanopart. Res. 2010, 12, 1599−1608. (33) Charron, G.; Hühn, D.; Perrier, A.; Cordier, L.; Pickett, C. J.; Nann, T.; Parak, W. J. On the use of pH titration to quantitatively characterize colloidal nanoparticles. Langmuir 2012, 28, 15141−15149. (34) Bolley, J.; Lalatonne, Y.; Haddad, O.; Letourneur, D.; Soussan, M.; Perard-Viret, J.; Motte, L. Optimized multimodal nanoplatforms for targeting αvβ3 integrins. Nanoscale 2013, DOI: 10.1039/ C3NR03763K. (35) Ghobril, C.; Popa, G.; Parat, A.; Billotey, C.; Taleb, J.; Bonazza, P.; Begin-Colin, S.; Felder-Flesch, D. A bisphosphonate tweezers and clickable PEGylated PAMAM dendrons for the preparation of functional iron oxide nanoparticles displaying renal and hepatobiliary elimination. Chem. Commun. 2013, 49, 9158−9160. (36) Lin, P.-C.; Ueng, S.-H.; Yu, S.-C.; Jan, M.-D.; Adak, A. K.; Yu, C.-C.; Lin, C.-C. Surface modification of magnetic nanoparticle via Cu(I)-catalyzed alkyne-azide [2 + 3] cycloaddition. Org. Lett. 2007, 9, 2131−2134. (37) Wang, Y.-X. J. Superparamagnetic iron oxide based MRI contrast agents: Current status of clinical application. Quant. Imaging Med. Surg. 2011, 1, 35−40.

14647

dx.doi.org/10.1021/la403245h | Langmuir 2013, 29, 14639−14647