Clothilde Comby-Zerbino,a Rodolphe Antoine, a Philippe Dugourd,a
Frederic Boschetti,c Franck Denat, d Cedric Louis,b Stephane Roux,g
Tristan Doussineau,h Olivier Tillementa and François Lux *a
Ultrasmall silica nanoparticles (NPs), having hydrodynamic diameters under 10 nm are promising
inorganic platforms for imaging and therapeutic applications in medicine. Herein is described a new way
for synthesizing such kind of NPs in a one-pot scalable protocol. These NPs bear DOTA (1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid) ligands on their surface that can chelate different
metals suitable for a wide variety of biomedical applications. By varying the ratio of the precursors, the
hydrodynamic diameters of the particles can be controlled over the range of 3 to 15 nm. The resulting
NPs have been characterized extensively by complementary techniques like dynamic light scattering
(DLS), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), mass
spectrometry (MS), phosphorescence titration, photophysical measurements, relaxometry and elemental
analysis to elucidate their structures. Chelation of gadolinium (Gd) allowed its use as an effective
intravenous contrast agent in MRI and was illustrated in mice bearing colorectal CT26 tumors. The new
particle appears to sufficiently accumulate in the tumors and efficiently clear out of animal bodies
through kidneys. This new synthesis is an original, time/material-saving and very flexible process that
can be applied for creating versatile ultrasmall multifunctional nanomedicines.
Introduction
After the success of DOXILs, a liposome formulation ofdoxorubicin, at the end of the 90s, the field of nanomedicine hasexploded exponentially with a tremendous amount of researchesdedicated to a wide variety of materials and applications.1,2
Among them, multifunctional NPs for theranostic applicationsattract a lot of attention. These NPs can combine variousimaging and therapeutic modalities in a single object. Forimproving medical imaging, NPs can be designed as imagingagents for X-ray computed tomography (CT), magnetic reso-nance imaging (MRI), ultrasonography, scintigraphy or opticalimaging (OI). On the other hand, for therapeutic purposes, theycan act as either drug carriers, heat emitters in hyperthermiatherapy, or sensitizers for photodynamic, neutron/radiotherapy etc.3–5
However, one of the most challenging requirements for NPsdesigned for clinical translation lies in their biocompatibility,toxicity and pharmacokinetic profile. For the latter, NPs need tobe able to evade mononuclear phagocytic system (MPS) to avoidbeing trapped in liver, spleen or lung. This requires them tohave a hydrodynamic diameter (DH) less than 50 nm and/or astealth coating e.g. by polyethylene glycol (PEG).2 Even if manyparticles showed longer circulation time in blood by satisfyingthese criteria, they still eventually ended up mostly in the liverand relied fully or partly on hepatic clearance. This excretionmechanism is a complicated process and implies possiblelong term side effects due to prolonged exposure to toxiccompounds as well as interference with other diagnoses and
a Institut Lumiere Matiere, Universite Claude Bernard Lyon 1, CNRS UMR 5306,
69622 Villeurbanne, France. E-mail: franç[email protected] Nano-H S.A.S., 2 Place de l’ Europe, 38070 Saint Quentin Fallavier, Francec CheMatech, 2 rue Pauline Kergomard, 21000 Dijon, Franced Institut de Chimie Moleculaire de l’Universite de Bourgogne (ICMUB),
UMR CNRS 6302, Universite de Bourgogne Franche-Comte, 9 avenue Alain Savary,
21078 Dijon, Francee Centre Commun de RMN, Universite Claude Bernard Lyon 1,
3 rue Victor Grignard, 69616 Villeurbanne, Francef Chimie ParisTech, CNRS UMR8258, INSERM U1022, Universite Paris Descartes,
Unite de Technologies Chimiques et Biologiques pour la Sante, 11 rue Pierre &
Marie Curie, 75005 Paris, Franceg Institut UTINAM, Universite de Bourgogne Franche-Comte, CNRS UMR 6213,
16 route de Gray, 25030 Besançon, Franceh NHTherAguix S.A.S., 43 Boulevard du 11 Novembre 1918, 69100 Villeurbanne,
France
† Electronic supplementary information (ESI) available: Synthesis of chelatingsilane precursor and experimental details. See DOI: 10.1039/c8tb00195b
treatments. To overcome this pitfall, NPs can be designed to berenal excretable.6,7 Taking into account the porosity of glomerularbasement membrane in the kidney, some studies have shown thatan upper limit of DH for renal clearance should be approximately10 nm. Similarly, for larger nanosystems, elimination by thekidneys can be achieved by the capacity to be biodegradable insmall fragments.8–11 If NPs are not biodegradable but containtoxic and persistent compounds such as some metals, they shouldbe preferably ultrasmall (DH o 10 nm). Moreover, as effectiveimaging agents, theranostic NPs should accumulate rapidly andspecifically in a target site and be cleared rapidly from non-targeted tissues. These features facilitate a shorter acquisitiontime and higher contrast for imaging. Therefore, it is moredesirable for the particle to have a small size with greater tissuepenetrability and to be eliminated through the renal clearancewhich is the most efficient excretion pathway.11 Despite highdemand, few nanosystems in the literature can satisfy thissize limit while maintaining their intact structures and multi-functionality since the latter require relatively high stability andthe addition of different molecules or layers that possibly makethe overall size exceed the limit.
Of different types of materials, silanes appear to be aninteresting source to fabricate ultrasmall theranostic NPs.In fact, silica NPs have been widely accepted as having a verylow toxicity and are therefore recognized as safe for biologicalapplications.12 In addition, these materials are quite cheapcompared to other organic or precious metals counterparts.There are two principal methods for synthesizing these silicastructures in the literature: Stober method and reverse micro-emulsion. The latter can produce NPs with a wide range of sizedown to 10 nm with a very good monodispersity.13–15 However,this process suffers from a long and complicated purificationstep which lowers the final yield to remove all the involvedsurfactants. Stober method, in which the hydrolysis and con-densation of silane precursors are catalyzed by a weak base inalcohol–water mixture, is far more practical and easy to bescaled up for big syntheses.16 However, DH of NPs achieved bythis method cannot stay below 10 nm.17,18 Some authorshave modified this process using amino acids as catalyzers.Nevertheless, the size limit stays in the order of 10 nm.19–21
Recently, Wiesner et al. have developed a Stober-like synthesismethod in water using PEG-silane to stop the growth of silicaNPs and prevent the recognition of mononuclear phagocyticsystem. They reported to obtain NPs with DH from 2.5 to7.5 nm.22 However, in this particle, the ratio of active com-pounds i.e. drugs, imaging agents, targeting ligands over non-active excipients, i.e. polysiloxane, organic polymer, is relativelylow. Our team has previously proposed a top-down method toproduce ultrasmall (DH B 3–5 nm) silica NPs functionalizedwith Gd chelates (see Fig. S1, ESI†): AGuIX.23 Shortly, a gado-linium oxide core is formed in diethylene glycol then this coreis coated by a polysiloxane shell before grafting of the chelates.During the transfer to the water, gadolinium oxide core dissolvedand gadolinium ions are chelated by the ligands, then thepolysiloxane shell collapsed leading to the final ultrasmallnanoparticles. The ratio between active compound, Gd chelates,
over non-active excipients is relatively high (B15% of gadolinium).This NP can be used as a safe and effective multimodal imagingand radiosensitizing agent in cancer diagnosis and treatment23–26
and is now implicated in a phase Ib clinical trial for the treatmentof brain metastases in association with whole brain radiationtherapy (NANO-RAD; ClinicalTrials.gov Identifier: NCT02820454).A second phase I clinical trial (NANO-COL; ClinicalTrials.govIdentifier NCT03308604) has recently begun for the treatment oflocally advanced cervical cancer by radiotherapy in associationwith AGuIX nanoparticles. Nevertheless, the described synthesisprocess was quite complicated and included multiple steps.Moreover, the metals chelated by the nanoparticle are dependenton the metal oxide core we used as a precursor. Herein, we reportan original strategy to obtain directly such kind of ultrasmall silicaNPs having high colloidal stability with a one-pot protocol basedon silane chemistry. This method also offers more flexibility interms of size and compositions (nature of the metals present atthe surface of the NPs is not limited anymore) tailoring. By varyingthe amount of silane precursors, we can tune finely the DH ofthe particles between 3 and 15 nm. On the other hand, differentmetallic ions can be easily complexed on the particles post-synthetically implying a wide range of applications in medicalimaging and therapy. Furthermore, given the stringent regula-tions of medical authority for drugs used in human, the productswere thoroughly investigated using different analytical techniquesi.e. DLS, HPLC, NMR, MS, phosphorescence titration, relaxometryand elemental analysis to reveal their structures. To illustrate theirpotential as imaging agents, first MRI experiment on tumor-bearing mice were also performed with the new Gd complexedparticles in order to assess their in vivo behavior after intravenousadministration.
Results and discussion
In this study, ultrasmall silica nanoparticles (USNP), weresynthesized directly from molecular silane precursors i.e. TEOS(tetraethyl orthosilicate), APTES (aminopropyl triethoxysilane)and macrocyclic chelator DOTAGA anhydride (1,4,7,10-tetra-azacyclododecane-1-glutaric anhydride-4,7,10-triacetic acid).To realize this synthesis, DOTAGA anhydride needed to befirst coupled with APTES to produce a bulky chelating silaneAPTES–DOTAGA (see Fig. 1B for the structures of silanes usedin this study). Two different strategies were applied: (i) APTES–DOTAGA was prepared as a ready-to-use powder and thenmixed with other precursors (see Fig. 1) or (ii) APTES–DOTAGAwas formed in situ during the synthesis of NP in a singlecontinuous process (see Fig. 3). The particles synthesized bythese two strategies will be referred to as USNPr and USNPirespectively.
1. Synthesis of USNP with ready-to-use chelatingsilane (USNPr)
1.1. Synthesis of USNPr. APTES–DOTAGA was synthesizedand isolated before being mixed with other silanes. The synthesisand isolation of APTES–DOTAGA were described in ESI,† S2.
Briefly, APTES–DOTAGA can be synthesized from two methods(i) the reaction between APTES and t-butyl protected DOTAGA((t-bu)4DOTAGA) or (ii) the reaction between APTES andDOTAGA anhydride. Fig. 1A shows the reaction where (t-bu)4-DOTAGA has been used. The peptide bond was formed by usingHBTU ((2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa-fluorophosphate)) as coupling agent. After purification, APTES–(t-bu)4DOTAGA was deprotected by concentrated hydrochloric acidto obtain final product. The excess of acid was removed byevaporation. Solution was lyophilized for storage. In the secondmethod, APTES and an excess of DOTAGA anhydride can bemixed in DMSO at 75 1C under argon (Ar) atmosphere. Thesolution was precipitated by adding acetone. The solid waswashed with acetone and dried overnight at 40 1C. The productsfrom two methods can be used interchangeably as APTES–DOTAGA sources.
The synthesis of USNPr was presented schematically inFig. 1B. APTES–DOTAGA was dissolved in water at pH 9 andstirred for 1 h to hydrolyze preformed siloxane bonds betweenAPTES–DOTAGA and homogenize the solution. APTES andTEOS were added to achieve at the end the concentration ofAPTES–DOTAGA, APTES and TEOS as 10 mM, 10 mM and 20mM respectively. In this condition including slightly basic pH,relatively low concentrations of silanes and high concentrationof water, the hydrolysis of silane precursors happens veryrapidly. Meanwhile the condensation, which produces wateras a byproduct took place very slowly. This allows the silanes tobe in the forms of monomers or small oligomers and distributedevenly.22 HCl was added gradually under vigorous stirring to bringthe pH to 4.5. This step which led to the protonation of silanolgroups and of a part of carboxylate groups on DOTA reduces theelectrostatic repulsion between the precursors and facilitates theirassembling. At this stage, probably, most of precursors especiallyorganosilane precursors i.e. APTES–DOTAGA and APTES were heldtogether by hydrogen bonds between silanol groups. Then the
solution was heated at 80 1C overnight to complete the condensa-tion i.e. the formation of covalent bonds between precursors.27
This process also assured the thermodynamic equilibriumbetween silanes and particles was reached. The particlesolution was concentrated to twenty times smaller volume.pH of solution was brought to 2 to protonate DOTAGA, therebyreducing the electrostatic interaction between the free chelatorsand amine groups on the surface of the newly formed particles.Particle solution was purified at this concentration and pHby tangential filtration through Vivaspin (MWCO = 3 kDa) toremove all unreacted precursors, filtered through 0.2 mmmembrane and then lyophilized for long term storage.
Theoretically, there should be some unwanted productscreated in this process as shown in Fig. 1B such as naked poly-siloxane particles from only TEOS precursors or functionalizedparticles from only APTES and APTES–DOTAGA precursors.However, these structures are not sufficiently stable and willbe removed during the purification. The naked polysiloxaneparticles or the ones poorly covered by an insufficient ratio oforganosilanes i.e. APTES–DOTAGA and APTES in this range ofsize would not exist. They will either dissolve in the condition ofpurification i.e. aqueous milieu at pH 2 and be removed bytangential filtration or aggregate into microparticles by Oswaldripening and be removed by filtration.28 On the other hand,structures made from only organosilanes will be fragile. Due tosteric hindrance, they could not form an extensive network.Hence, they have a high surface curvature that more readilyexposes the siloxane bonds to hydrolysis by water. This highcurvature also makes it difficult for all three siloxane bondsper each organosilane to form.29 This further weakens thehydrolysis stability of such particles. This phenomenon isfurther demonstrated by the results in 2.2. Therefore, only thestructures containing a matrix core at a certain size protected by asufficient ratio of organosilanes were expected to stably form andpossibly remain after the purification.
Fig. 1 The synthesis of USNP with ready-to-use chelating silane (USNPr): (A) synthesis of the chelating silane, APTES–DOTAGA, from t-butyl protectedDOTAGA. (B) Synthesis and nomenclature of USNPr.
1.2. Characterization of USNPr. Fig. 2A shows the sizedistribution of the particles after purification measured byDLS with average DH = 4.6 � 1.6 nm. The zeta potential of theparticle was �27.1 mV at pH 7.4. Fig. 2B shows the chromato-gram at 295 nm of the purified product. Among compoundspresent in the synthesis mixture, APTES–DOTAGA and DOTAGA(remaining reactant) absorb more significantly in UV due totheir bulkier structures. Meanwhile, APTES and TEOS areknown for their UV transparency. The first eluted group ofpeaks with retention time (tR) from 2 to 5 min might beassigned to the unreacted precursors and the second widerpeak (tR B 13 min) can be assigned to the produced NPsas previously shown for similar NPs.30 The results show thatby concentrating and purifying, we were able to obtain ahomogeneous population of NPs with high purity (490%).Chromatograms with detection at 700 nm of the NPs aftercomplexed with CuSO4 were also recorded to visualize specifi-cally the eluates containing DOTAGA (Fig. S11F, ESI†). Theevolution during each stage of the synthesis was illustrated inmore details by chromatograms in Fig. S11 (ESI†).
To evaluate the diameter of USNPr, the presence and theratio of APTES–DOTAGA and APTES on its surface, 1H NMR and
NMR DOSY spectra were collected from USNPr at 127 g l�1.Fig. 2D shows a 2D NMR-DOSY spectrum of USNPr in D2O.Most of protons seem to have the same diffusion coefficient (D)at 54.4 mm2 s�1. The result indicates that expected organicgroups i.e. APTES–DOTAGA and APTES were grafted on thesame particles. Besides, there are some free hydrolyzed silaneswhich have much faster coefficient (194.5 mm2 s�1). Hydro-dynamic diameter of the main particles can be calculated fromEinstein equation which is around 7.0 � 2.5 nm. The viscosityof the solution was unknown and might be considerably higherthan pure D2O at this concentration. Therefore, the calculatedDH might be a bit overestimated compared to the value mea-sured from DLS (4.6 � 1.6 nm). Nevertheless, it stayed less than10 nm. Fig. 2E shows the peak integration of 1H spectrum of theUSNPr. Most of 1H peaks were superimposed on each otherdue to the complex 1H spectrum of DOTAGA that could beconfirmed elsewhere.31 Fortunately, DOTAGA has no 1H peakat higher field than 1 ppm where the peak of 1H of carbonat position 1 (see Fig. 2E), closest to Si of APTES and APTES–DOTAGA, can be found (0.63 ppm).32 The area of this peakis the sum of contributions from 2 protons of APTES and2 protons of APTES–DOTAGA, whereas the total area of all of
Fig. 2 Characterization of USNPr: (A) DLS diagram; (B) chromatogram at 295 nm; (C) ESI-MS spectrum, inset: deconvoluted spectrum; (D) NMR-DOSYspectrum; (E) the positions of H1, H2 and H3 on the APTES and APTES–DOTAGA functional groups on the particle and 1H NMR spectrum;(F) chromatograms at 700 nm, pH 3 of USNPr at 7.14 g l�1 complexed with Cu2+ at 10 mM (solid line), CuSO4 solution at 16 mM (dotted line) andDOTAGA(Cu2+) complex solution at 1.76 mM (dashed line) and (G) Eu titration curve at 594 nm (circle) and 616 nm (square), lex = 395 nm.
peaks is the sum of 6 protons of APTES and 33 protons ofAPTES–DOTAGA (only non-exchangeable protons were counted).Presumably, NP solution was free from other organic impurities.Solving this two-variable linear equation system will give the ratiobetween the amount of APTES over the amount of APTES–DOTAGA in the sample. The result was found as 1.35.
Elemental analysis revealed the contents of USNPr in Si,C and N (see Table S8, ESI†). From the results, we cancalculate the contribution of each species i.e. APTES–DOTAGA,APTES and TEOS if we assume that after the condensation,each molecule of APTES–DOTAGA, APTES and TEOS impliesthe molecular structure as SiO1.5(CH2)3NH–DOTAGA(2�),SiO1.5(CH2)3NH3
+ and SiO2 respectively. As the results suggest,the ratio of APTES–DOTAGA : APTES : TEOS in USNPr is 1 : 1 : 5.1.
To obtain the average chemical formula of a single USNPr,the mass of the particle should be determined. For this purpose,ESI-MS was performed taking its advantage as a soft ionizationmethod which is suitable for studying nano assemblies.33 Fig. 2Cshows the MS spectrum of USNPr averaged from 5 differentspectra acquired in different ESI-MS conditions to collect allcharge states of this NP. As reported previously, this type of NPusually gives a complex and unresolved spectrum due to thepresence of multiple ionizable functional groups i.e. carboxylicacids, amines, silanols.23 Despite this complex appearance, theposition of the peaks in the spectrum is stable, although therelative abundance of the different peaks is found to be slightlydependent on the ESI-MS conditions. This spectrum can bedeconvoluted by using a multiplicative correlation algorithm(MCA) adapted to estimate the mass spectra from the mass-to-charge spectra produced by ESI-MS as described by Shen et al.34
The deconvoluted spectrum was shown in the inset. A main peakat around 12.5 kDa and two other peaks other at around 10.9 kDaand 14.3 kDa were observed. Combining with the ratio of speciesdeduced from elemental analysis, this result corresponds with thechemical formula APTES–DOTAGA12.5APTES12.5TEOS64 for themain peak and APTES–DOTAGA11APTES11TEOS56; APTES–DOTA-GA14.5APTES14.5TEOS74 for the two other peaks.
Quantification of DOTAGA on USNPr. The content of DOTAGAon the particle was also determined by two methods: (1) HPLCanalysis with copper (Cu2+) and (2) titration with europium(Eu3+) phosphorescence.
HPLC analysis with Cu2+. This method has been describedpreviously for quantifying free chelators on silica NP.35 A smallamount of USNPr was complexed with an excess of CuSO4 andanalyzed by HPLC (Fig. 2F). The chromatograms of the solutionat 700 nm was superimposed with the ones of CuSO4 andDOTAGA(Cu2+). The first peak, the second peak and the thirdpeak correspond to free copper ions, (APTES)–DOTAGA(Cu2+)and USNPr(Cu2+) respectively. The shape of NPs peak shows ahomogenous distribution after the complexation. The calibra-tion curves and the detailed results can be found in Fig. S12and Table S4 (ESI†). From the results, we can find out the totalconcentration of DOTAGA in the sample and deduce its contentwhich was about 0.72 mmol mg�1.
Phosphorescence titration with Eu3+. A series of samplescontaining a fixed amount of USNPr and an increasing amountof EuCl3 was prepared. Eu3+ has two specific phosphorescenceemission peaks at 594 nm (5D0 -
7F1) and 616 nm (5D0 -7F2)
when being excited at 395 nm (see Fig. S9A, ESI†).36,37 Theintensity of these two peaks can be strongly enhanced whenEu3+ was complexed due to the protective effect of DOTAGAligand from the quenching effect of O–H oscillator of wateron Eu3+ phosphorescence. Upon the addition of increasingamount of Eu3+, the phosphorescence intensity increasessharply until no DOTAGA is available for chelation. After thispoint, the intensity reaches a plateau or increases slowly dueonly to the phosphorescence of free Eu3+.38 Fig. 2G shows thetitration curve of the sample with the equivalent point ataround 0.79 mmol mg�1.
In addition, it is also possible to come up with the contentsof each species based on the result of the elemental analysis.The content of APTES–DOTAGA was calculated as 0.95 mmol mg�1
(Table S8, ESI†). HPLC analysis using Cu2+ is limited by a low levelof signal/noise at 700 nm with our detector, the small volume ofsample taken and the possible lack of access of some chelatorson the particle in a highly concentrated solution. Meanwhile,the result from elemental analysis was based on several indirectcalculations that imply accumulating subtle errors. These leftEu3+ titration as probably the most precise method. Thus, itsresult (0.79 mmol mg�1) will be used for the next steps.
2. Synthesis of USNP using in situ formed chelatingsilane (USNPi)
In order to propose an even simpler protocol, we combined thereaction of APTES and DOTAGA anhydride with the hydrolysisand condensation to produce the NPs into a single continuousprocess while reducing cost and experimental time. In addition, bychanging the ratio of silane precursors in the formula, we cancontrol the size of the particles (see Fig. 3). The starting ratios ofcomponents and characterization results are summarized in TableS10 (ESI†).
2.1. Synthesis of USNPi. For the first series of samples(USNPi-1, 2, 3, 4), DOTAGA anhydride and APTES were mixedand heated at 75 1C in DMSO anhydrous under Ar atmosphereovernight. DMSO was chosen since it is an aprotic solvent andcannot hydrolyse anhydride. This will assure a high yield ofthis coupling reaction. Then an amount of water was added todilute DMSO to less than 5% in order not to dissolve thetangential filtration membrane used in the next step. The restof the protocol remains very similar to the above protocol.The pH of the solution was adjusted to 9 to hydrolyse pre-formed siloxane bonds between APTES–DOTAGA and allowthem disperse evenly in the solution. Then the solution wasseparated in 4 volumes. Increasing amounts of TEOS and waterwere added to each volume to obtain increasing concentrationsof TEOS and keep total silane concentrations in all samples at20 mM. These solutions were stirred overnight to completelyhydrolyse TEOS. pHs of 4 solutions were readjusted to 4.5before heating at 40 1C was applied overnight. These solutions
were concentrated and purified at pH 2 by tangential filtrationusing Vivaspin (MWCO = 3 kDa).
To demonstrate the possibility of scaling up this process,larger batch (ca. 5.4 g vs. normal ca. 0.7 g batch) has beensynthesized (USNPi-5). DEG, previously used in our top-downsynthesis, was used as a replacement for DMSO. DEG as otherethylene glycols can be added up to 10% in the aqueousmixture without damaging the filtration membrane used inthe purification step. Subsequently, the total silane concen-tration could be raised to ca. 60 mM. The equilibrium betweensiloxane bond formation and dissociation depends very muchon the concentration.39–42 So by increasing moderately the totalsilane concentration, the yield in DOTAGA of the reaction wasexpected to significantly increase. A predicted difficulty of thisprotocol was the solubility of DOTAGA anhydride in DEG whichcould hamper the coupling reaction with APTES. Unlike thereaction in DMSO, heating, which could increase the solubilityof DOTAGA anhydride, was not applied in this case. Thisprevented the possibility of uncontrolled reaction of DOTAGAanhydride with alcohol groups of DEG which can probablyreduce the yield of the reaction with amine groups. Instead, themixture was kept stirring at room temperature for a long periodof time (5 days). The starting ratio of APTES and DOTAGAanhydride was increased to 3 : 2 to obtain a more balanced ratioof APTES and APTES–DOTAGA at the end. After the addition ofTEOS, pH during the hydrolysis process was maintained at 4instead of being adjusted to 9. Since pH was kept at 4, twoprocesses of hydrolysis and heating to complete the condensa-tion were combined into a single one. The purification processwas done similarly at pH 2 by tangential filtration with Vivaflowsetup for larger scale.
2.2. Characterization of USNPi. Different samples ofUSNPi-1 (after the reaction between APTES and DOTAGA anhy-dride and after being exposed at pH 9 overnight) and USNPi-5(after the reaction between APTES and DOTAGA anhydride)were taken to quantify the efficacy of the coupling by amide
bond between DOTAGA anhydride and APTES (see S2.2.2.3 andS2.2.2.4 for the detailed principle, ESI†).
The results for USNPi-1 show that the exposure to pH 9affects neither the DOTAGA structure nor the amide bond ofAPTES–DOTAGA (Fig. S14 and Table S5 (USNPi-1), ESI†).According to the result, around 70% of DOTAGA anhydridehas reacted with APTES.
On the other hand, sample of USNPi-5 was first diluted10 times in water to obtain a clear solution instead of theinitial suspension before being analyzed. Unexpectedly, despiteappearing initially as an insoluble suspension after the synthesis,the produced mixture showed a reaction yield effectively equivalentto the one carried out in DMSO (70%) (Fig. S15 and Table S5(USNPi-5), ESI†).
Fig. 4A shows the diameter diagrams in DLS of the firstseries (USNPi-1, 2, 3, 4) after being redispersed at pH 7. Theresults showed a clear dependence of NP size on the addedamount of TEOS as expected. DH of USNPi-1, 2, 3, 4 were 2.8,4.3, 7.6 and 14.4 nm respectively. Chromatography was alsoperformed with these four particles and confirmed the DLSresults (see Fig. 4B). The more TEOS added in the formula, theslower the retention time of NPs. This indirectly shows thedependence of NP size on the amount of TEOS since a highertR usually implies a bigger NP.
Another indirect method to evaluate the size of the particlesis to look at the relaxivities after being complexed with Gd.The bigger the particles are, the lower their rotational correla-tion times and so the higher their relaxivities are.43 Forthis experiment, Gd3+ was complexed with a small sample ofUSNPi-2, 3, 4 with an excess of DOTAGA (molar ratio Gd :DOTAGA = 1 : 10) to have a quick and complete complexationwithout the need of purification for studying their size evolution.Their relaxation times were measured by relaxometry andconcentrations of Gd3+ in each sample were verified by ICP-OES. Their relaxivities were calculated and shown in Fig. 5C.As expected, r1 (and also r2/r1) increases in the order of USNPi-2
Fig. 3 The synthesis of USNP using in situ formed chelating silane (USNPi) and the complexation with different metals (Mn+ = Gd3+, Lu3+, Eu3+, Tb3+,Ho3+, Cu2+, Bi3+).
(r1 = 16.9 mM�1 s�1, r2/r1 = 1.53) o USNPi-3 (r1 = 19.2 mM�1 s�1,r2/r1 = 1.80) o USNPi-4 (r1 = 19.8 mM�1 s�1, r2/r1 = 2.04). (Due toinsufficient reaction yield, USNPi-1 could not be extensivelycharacterized.)
In short, these results show that by varying the starting ratioof siloxane network creating precursor, TEOS, and organo-silanes, APTES–DOTAGA and APTES, the size of the NPs canbe tuned.
Elemental analyses also showed an increase of the molarratio of Si over N or C in the order of USNPi-2 o USNPi-3 oUSNPi-4 in accordance with the amount of added TEOS (seeTable S8, ESI†).
For the scaled-up batch (USNPi-5), DLS diagrams at differentsteps during the synthesis show that before water was added,no stable particle was created yet as indicated by small valuesof DH (B1 nm). After the hydrolysis and condensation ofsilanes, stable particles started to form. The final particle afterlyophilization displayed DH of 5.2 � 2.0 nm (see Fig. S18B andTable S6, ESI†). Chromatography was also performed to verifythe purity of the particle after the purification process (seeFig. S19 and Table S6, ESI†). Its chromatogram showed asymmetrical peak demonstrating a homogenous NP distribution.Zeta potential of the particle at pH 7.3 was �32.6 mV. Theisoelectric point (pI) of the particle was around 4.24 (see Fig. S20and Table S7, ESI†) which seems reasonable given that at this pHDOTA predominantly has minus one or two charge (second andthird protonation constant, pKH2L and pKH3L, of DOTAGA are 9.67and 4.68 respectively44) and the starting ratio of APTES–DOTAGA/APTES was almost 1 : 1.
USNPi-5 lyophilized powder was redispersed in D2O forNMR analysis. DOSY spectrum shows that the diffusion coeffi-cient of USNPi-5 was around 55 mm2 s�1 (see Fig. S22B, ESI†).
DH of USNPi-5 calculated from Einstein equation was 7.0 �2.5 nm. This result was very similar to the value of USNPr.Except two multiplets at 3.52 and 3.62 indicating a smallremaining amount of DEG, the 1H spectrum of USNPi-5 wasalso very similar to that of USNPr (see Fig. S22C, ESI† vs.Fig. 2E). The integration of 1H peaks excluding the ones fromDEG showed that the ratio APTES/APTES–DOTAGA in thiscase was 1.26. The ratio of DEG/APTES–DOTAGA can also becalculated by this technique as 0.33.
From elemental analysis, the ratio of APTES–DOTAGA :APTES : TEOS in USNPi-5 was calculated as 1.0 : 1.0 : 4.7 takinginto account the amount of DEG calculated from NMR experi-ment (Table S8, ESI†).
Finally, Fig. 4C shows the original MS spectrum and thedeconvoluted spectrum of USNPi-5. They were very similar toUSNPr with a main peak at around 11.5 kDa and two minorpeaks at around 10.2 kDa and 13.5 kDa. This result wascombined with the ratio of species inferred from elementalanalysis to give the chemical formula of the main peakas APTES–DOTAGA12APTES12TEOS56 and the ones of twominor peaks as APTES–DOTAGA10.5APTES10.5TEOS50 and APTES–DOTAGA14APTES14TEOS66.
Quantification of DOTAGA on USNPi. The DOTAGA contentof each sample was determined by Eu titration. For the firstseries of samples, USNPi-1, 2, 3, 4, we see clearly a decreaseof DOTAGA content from 1.1 mmol mg�1 to 0.7 mmol mg�1
when TEOS was increased in the formula as expected (seeFig. 4D and Fig. S17, ESI†). Combining this result with thetotal weight of produced particle, the yield of the process can bedetermined. Interestingly, the more TEOS we put, the higherthe yield of APTES–DOTAGA at the end from 1.15% to 15.41%
Fig. 4 Characterization of USNPi: (A) DLS diagram of USNPi-1 (black), USNPi-2 (blue), USNPi-3 (green), USNPi-4 (red) redispersed at pH 7;(B) chromatograms at 295 nm (normalized to the same height) of the same samples with respective color code (A–D: APTES–DOTAGA, A: APTES,T: TEOS); (C) ESI-MS spectrum of USNPi-5, inset: deconvoluted spectrum; (D) Eu titration curves of USNPi-1 (left), USNPi-2 (middle), USNPi-3 (right) at594 nm (circle) and 616 nm (square), lex = 395 nm.
(Table S10, ESI†). Presumably, the siloxane bond between silicicacid and organosilanol might be stronger than the siloxanebond between the organosilanols themselves. In addition,aminosilanes, especially the ones with short carbon chainsuch as APTES, are known as having quite low hydrolyticstability.41,45,46 Another possible explanation might be due tothe presence of flatter surface curvature on bigger particleswhich allow the formation of all three siloxane bonds per eachorganosilane i.e. APTES–DOTAGA and APTES. This featuremakes organosilanes stick more stably on the surface of theparticles and have higher resistance to water hydrolysis. On thecontrary, smaller particles might have higher surface curvaturewhich imposes more steric constraint on the formation ofall three siloxane bonds per each organosilane as previouslyshown by Mei et al. in the case of thiol ligands functionalizedon gold NPs.29 It seems the thermodynamic equilibrium wasdriven more and more towards the creation and maintaining ofthe particle when more TEOS was added, while the mixture ofAPTES and APTES–DOTAGA alone cannot make up stableparticles. This is the reason why pH was not necessarilyadjusted to 9 before adding TEOS anymore in the case ofUSNPi-5 to simplify the protocol. In fact, pH 4 was shown aswhere the condensation happens the most slowly.47 Hence, thiswould actually facilitate the equilibrium towards keeping theNPs at ultrasmall size.
The DOTAGA content of USNPi-5 was determined by Eutitration as B0.8 mmol mg�1 (see Fig. S21, ESI†). The yield was24.7% compared to the introduced quantity of DOTAGA. Thisresult was 3 times higher than the yield obtained in DMSO/H2Osystem for the NP with an equivalent DH (e.g. 7.9% for USNPi-3).
3. USNP as a flexible platform for complexing different metals
3.1. Complexation of different metals on USNPr/i. Differentmetals were used to complex with USNPr or USNPi to demonstratetheir flexibility and help study their nanostructures. First of all,as we have seen above, Eu3+ was introduced on every particle andCu2+ was complexed with USNPr to quantify the amount of freeDOTAGA. This implies the possibility of using USNP for opticalimaging while exploiting luminescence property of Eu3+ and/orscintigraphy using radioactivity of 64Cu isotope.48,49 On the otherhand, samples were complexed with Gd3+ to exploit its magneticproperties as positive contrast agent in MRI. For example, USNPrand USNPi-5 were complexed with Gd3+ to produce USNPr@Gdand USNPi-5@Gd respectively. USNPr was also complexed witha diamagnetic lanthanide ion i.e. Lu3+ (USNPr@Lu) for NMRexperiments. Besides, USNPi-5 was complexed with Tb3+, Ho3+
and Bi3+ to give respective complexed particles i.e. USNPi-5@Tb,USNPi-5@Ho and USNPi-5@Bi. These metals have potentials fordifferent biomedical applications. Tb3+ as Eu3+ has been usedfrequently as a luminescent probe.50 Natural isotope of Bi3+ hasbeen reported recently along with our previously developed USNPas a promising radiosensitizing agent.51 On the other hand, thechelation of Lu, Tb, Ho and Bi here can imply the potential ofUSNP as a nanocarrier for 177Lu, 161Tb, 166Ho radioisotopes asbeta-particle-emitters and/or 213Bi, 212Bi as alpha-particle-emittersfor therapeutic applications.52 In all cases, the amounts of metals
were kept at 90 to 95% compared to the amount of DOTAGA tomake sure no free metal was left in the solution.
3.2. Characterization of complexed USNPr/i. Hydrodynamicdiameters measured by DLS of USNPr@Gd, USNPi-5@Gd/Tb/Ho/Bi were 5.7 nm, 6.3 nm, 6.1 nm, 5.8 nm and 6.0 nm respec-tively (Fig. 5A, Fig. S11G and Table S8, ESI†). These results showa high level of colloidal stability of USNP platform and therepetitiveness of the protocol. Zeta potentials of these samplesat pH 7.4 were �8.2, �21.8, �19.3, �19.8, �3.4 mV respectively.This indicates the presence of metals in the chelators whichreduces the negative charges of chelators at neutral pH. Thepresence of metals was further confirmed by their infrared (IR)spectra (see Fig. S25, ESI†). The disappearance/decrease ofthe peak at 1720 cm�1 (CQO stretching vibration band offree carboxylic acid) indicated the complexation of metals inDOTA.40,41 Chromatograms of these samples indicated homo-geneous populations of particles with high purities (490%).All samples have a very similar tR at around 16 min (Fig. 5B andFig. S11H and I, ESI†).
The relaxivities of USNPr@Gd and USNPi-5@Gd were shownin Fig. 5C. High longitudinal relaxivity (r1) (21.4 mM�1 s�1 and23.2 mM�1 s�1) combining with a low ratio r2/r1 (1.59 and 1.65)indicating their potential as good positive contrast agentsfor MRI.
NMR-DOSY spectrum of USNPr@Lu is shown in Fig. 5D.Lu was chosen for NMR studies because of its diamagneticnature and its similarities in terms of chemical properties toother lanthanides. Other paramagnetic lanthanides i.e. Gd, Eu,Tb, Ho will detrimentally broaden the peaks in NMR spectraand unable the signal acquisition process of DOSY technique.Bi as a diamagnetic element can also be used for NMRexperiments. However, since this study focuses more onlanthanides, Lu represents as the best candidate. In the future,different diamagnetic metals can also be tested to verifytheir structural differences. The spectrum shows a similarresult to that of USNPr. The main products still had D around56 mm2 s�1. They coexisted with some other smaller specieswhich have faster D (410 and 214 mm2 s�1). DH of the mainparticles calculated from Einstein equation was 6.8 � 2.4 nm.1H spectrum of USNPr@Lu was not exactly similar to the one ofUSNPr (see Fig. S22A, ESI† vs. Fig. 2E) since the complexationchanged significantly the configuration of DOTAGA.42 However,still no 1H of DOTAGA showed a peak at the region smaller than1 ppm. So the integration method described above for USNPrcan still be applied and give the ratio APTES/APTES–DOTAGAas 0.87.
To determine specifically the presence and the contents ofmetals in USNPr@Gd and USNPi-5@Gd/Tb/Ho/Bi, ICP-OESmeasurements were conducted. The results were 0.604, 0.654,0.558, 0.625 and 0.442 mmol mg�1 for USNPr@Gd, and USNPi-5@Gd/Tb/Ho/Bi respectively (see Table S11, ESI†). The ratio ofeach species in USNPr@Gd was calculated from the elementalratio of Gd, Si, C and N as (Gd0.8APTES–DOTAGA1APTES1.2-TEOS6)x (see Table S7, ESI†) which is in accordance with non-complexed USNPr. Si, C and N in metal-complexed USNPi-5were not analyzed. However, if we assume, after the complexation
and purification, the ratio of elements remain the same, DEGhave been washed completely and each molecule of APTES–DOTAGA, APTES and TEOS still implies the molecular structureas SiO1.5(CH2)3NH–DOTAGA(2�), SiO1.5(CH2)3NH3
+ and SiO2
respectively, then we can propose the molecular structures ofUSNPi-5@Gd/Ho/Tb/Bi as (Gd0.70APTES–DOTAGA1.00APTES0.95-TEOS4.75)x, (Tb0.59APTES–DOTAGA1.00APTES0.95TEOS4.75)x, (Ho0.67-APTES–DOTAGA1.00APTES0.95TEOS4.75)x and (Bi0.47APTES–DOTA-GA1.00APTES0.95TEOS4.75)x respectively.
To complement the results of ICP-OES, UV-vis and phos-phorescence spectra of metals complexed USNP were recordedto show the photophysical imprints of respective metals. Fig. 5Eshows the excitation and emission spectra of USNPi-5@Gd withtypical excitation peak and emission peak for Gd at 273 nm and313 nm respectively.53 Fig. 5F shows those spectra of USNPi-5@Tbwith typical emission peaks at 489, 546, 586, 622 nm for Tb.50 TheUV-vis spectra of USNPr and different metals-complexed particleswere recorded. Sample of USNPi-5@Bi showed an intense peak at309 nm which is typical for DOTA(Bi3+) complex (see Fig. S24A,ESI†).54 This result was also demonstrated by a more intense NPpeak of the chromatogram of USNPi-5@Bi (see Fig. S23, ESI†).Meanwhile, UV-vis spectrum of USNPi-5@Ho shows severalabsorption peaks of Ho3+ (see Fig. S24B, ESI†).
4. USNP produced from chelated APTES–DOTAGA
We have also noticed that complexed APTES–DOTAGA canaffect slightly differently the silane chemistry compared touncomplexed APTES–DOTAGA and produce smaller USNP.For this experiment, another synthesis, where APTES–DOTAGAwas first complexed with Gd3+ before being used in the con-densation process, was conducted. The product is referred toas USNPr@Gd* (see ESI,† S3.1). Briefly, GdCl3 solution wasadded in APTES–DOTAGA solution in molar ratio Gd : APTES–DOTAGA = 0.9 : 1.0. The mixture was kept at pH 5 and incu-bated at 80 1C for 48 h to ensure the reaction was complete. pHof the solution was increased to 9 in 1 h before TEOS andAPTES were introduced to achieve at the end the concentrationof APTES–DOTAGA(Gd), APTES–DOTAGA, APTES and TEOS as9 mM, 1 mM, 10 mM and 20 mM respectively. The next stepsof synthesis and purification were repeated similarly as in thecase of USNPr.
USNPr@Gd* was also characterized by DLS, HPLC, relaxo-metry, Eu titration and elemental analysis (Table S9, ESI†). Thechromatograms of this particle were very similar to those ofUSNPr@Gd with similar tR (15.2 min for USNPr@Gd* vs.15.7 min for USNPr@Gd) (see Fig. S13A–F, ESI†). HoweverDH measured in DLS was slightly smaller (2.8 � 0.7 nm)
Fig. 5 Characterization of USNP(r/i) after complexed with metals: (A) DLS diagrams of USNPi-5 complexed with: Gd3+ (black), Tb3+ (green), Ho3+ (blue),Bi3+ (red); (B) chromatograms at 295 nm (normalized to the same height) of the same samples with respective color code; (C) relaxivities of USNPi-2, 3and 4 after complexed with 10% Gd3+ and USNPi-5@Gd; (D) NMR-DOSY spectrum of USNPr@Lu; (E) excitation spectrum (blue, lem = 313 nm)and emission spectrum (red, lex = 273 nm) of USNPi-5@Gd at 0.06 g l�1; (F) excitation spectrum (blue, lem = 546 nm) and emission spectrum(red, lex = 221 nm) of USNPi-5@Tb at 0.06 g l�1.
(see Fig. S13G, ESI†). The zeta potential of USNPr@Gd was�35.6 mV at pH 7.4 which is lower than the one of USNPr@Gd(see Table S9, ESI†). According to the elemental analysis, theratio of Gd : APTES–DOTAGA : APTES : TEOS can be estimated as0.7 : 1 : 0.3 : 5.1 (see Table S8, ESI†). USNPr@Gd* has less amine(condensed APTES) and slightly more free DOTAGA in itsstructure which explains a more negative zeta potential com-pared to USNPr@Gd. The reason for the difference between thetwo particles is not totally understood. Probably, the complexedchelating silane (APTES–DOTAGA(Gd3+)) somehow has slightlydifferent effect on the condensation and silane distributioncompared to the free chelating silane (APTES–DOTAGA). Thismight be because the former has a fixed charge �1 and a morecompact configuration whereas the latter has varied negativecharges depending on the pH of the solution and a moreflexible configuration. Nevertheless, this subtle difference didnot compromise the potential of USNPr@Gd* as an efficientMRI contrast agent demonstrated by a high r1 (18.5 mM�1 s�1)and low r2/r1 (1.55) similar to other Gd complexed USNP(see Fig. 5C).
5. In vivo MRI experiment with USNPr@Gd*
As an illustration of the potential for biomedical applications ofthese ultrasmall NPs, USNPr@Gd* was chosen for studying thecontrast-enhancing property in MRI of new Gd complexedparticles. The solution of particle was intravenously injectedin BALB/c mice bearing ectopically induced colorectal CT26tumors on both flanks. The images were obtained usingadapted T1 weighted motion free artifacts with intragate acqui-sition, dynamic MRI sequences at 7 T.
Fig. 6A shows MRI cross-sections where the tumor regions arehighlighted as expected. Comparison of the pre- and post-contrastimages clearly reveals the higher brightness at the tumor regionscaused by the particle. Contrast enhancement was expressed aspercentage of enhancement compared to the pre-contrast image.In the tumor tissue, USNPr@Gd* showed an intake phase with amaximal enhancement 30 minutes post injection (35% of signalincrease) and a prolonged clearance phase, with a half-time of 3hours, demonstrating the EPR (Enhanced Permeability and Reten-tion) effect (see Fig. 6B). In comparison, commercial molecularcontrast agent (DOTAREMs) shows a half-life of dozens minutes(see Fig. 6C). In the liver, peak of enhancement (90% increase ofsignal) was observed at 6 minutes post-injection of the particlefollowed by a clearance phase (see Fig. S26, ESI†). After 40 minpost-injection, the signal was at half of maximal intensity, indicat-ing a hepatic half-time around 30 minutes. After bloodstreamcirculation with a complementary transitory visualization throughthe vascular network in the liver, particles were excreted from thekidney cortex to the bladder as shown by a 150% increase of thesignal and a kidney half-life superior to 90 min, as previouslyshown with ultrasmall nanoparticles.55
This imaging study evidences that USNPr@Gd* displayscontrast enhancement in both the tumors, kidneys and hepatictissues over the full observation period, without the typical liveraccumulation observed for macromolecular agents.56 Thus,they improve the imaging properties without undesired liveruptake. Compared to commercial agent DOTAREMs, the clear-ance phase of the tumor with the USNPr@Gd* is slower asillustrated in the Fig. 6C. Meanwhile, the relatively long reten-tion time in the tumors opens the perspective for vectorizationtowards tumor tissues.
Fig. 6 In vivo MRI contrast enhancement of USNPr@Gd*: (A) MRI cross-sections of the tumor tissues (white arrows) pre- (left) and post-injection (right,up to 6 h) of USNPr@Gd*; (B) dynamic MRI signal enhancement in tumor tissues after injection of the particle. (C) Zoom of the 45 min period showing thefaster clearance rate (black arrow) in tumor for DOTAREM compared to USNPr@Gd*.
In this study, we have shown original and simple one-potprotocols for synthesizing thermodynamically stable ultrasmallsilica NPs functionalized with DOTAGA chelates using bio-compatible starting materials. The products were thoroughlycharacterized with different physicochemical analyses. Theseprotocols lead to well-defined monodisperse NPs with DH
ranging from 3 to 15 nm. The obtained NPs display freeDOTAGA ligands on their surface that can chelate a large varietyof metals depending on the biological application. In particular,longitudinal and transverse relaxivities of Gd complexed particlesshow that they behave as efficient positive contrast agents for MRI(r1 = from 17 to 23 mM�1 s�1 and r2/r1 = from 1.5 to 2). In vivo MRIexperiment was performed using one of these new Gd particles todemonstrate their efficacy in accumulating in the tumor, avoidingliver entrapment and rapidly escaping from the bodies ofthe animals through kidneys. These NPs might be applied asmultimodal imaging or radiosensitizing agents for theranosticpurposes in medical applications.
Experimental section1. Synthesis of USNPr and USNPr@Gd* with ready-to-usechelating silane
200 mL of water were added to an amount of powder containing2.228 mmol of APTES–DOTAGA. The pH of the solution isadjusted to 9. The solution was stirred for 1–2 h. Then TEOS(1015 mL, 4.457 mmol) and APTES (527 mL, 2.228 mmol) wereadded one by one to the above solution. Water was added toobtain the final concentrations of APTES–DOTAGA, TEOS andAPTES around 10 mM, 20 mM and 10 mM respectively. Themixture was stirred at 25 1C for 18 h to let the solution becomehomogeneous. The solution was adjusted to pH 4.5 and heatedat 80 1C for 18 h.
1 mL of solution was filtered through 0.2 mm membrane andanalyzed by DLS and HPLC (see ESI,† S3.1).
The solution was concentrated to 10 mL by tangentialultrafiltration through Vivaspint (MWCO = 3 kDa). Again,similar HPLC analyses were repeated.
Then, the solution was purified further with tangentialultrafiltration at pH 2 for 1024 purification factor (purificationfactor = starting volume/end volume). Then, the solution wasfiltered through 0.2 mm membrane to remove dust and largeimpurities. Finally, the solution was freeze dried for long termstorage. 706 mg of lyophilized powder of USNPr were obtained.
Another similar protocol was repeated for USNPr@Gd*.200 mL of water were added to an amount of powder containing2.333 mmol of APTES–DOTAGA. The pH of the solution isadjusted to 4. 1.938 mL of GdCl3 solution at 2.188 M (molarratio (APTES–DOTAGA + DOTAGA) : Gd = 1 : 0.9) were added in3 times. Between each time, pH was carefully increased to 4–5.This solution was incubated at 80 1C in 48 h. Then, pH of the abovesolution was adjusted to 9 before TEOS (1063 mL, 4.666 mmol)and APTES (551 mL, 2.333 mmol) were added. Water was addedto obtain the final concentrations of APTES–DOTAGA(Gd3+),
APTES–DOTAGA, TEOS and APTES at 9 mM, 1 mM, 20 mM and10 mM respectively. The rest of the protocol followed the samesteps as described for USNPr. 716 mg of powder of USNPr@Gd*were obtained. Similarly, samples were also taken for DLS andHPLC analyses during the synthesis.
2. Synthesis of USNPi-1, 2, 3, 4 using in situ formed chelatingsilane in DMSO/H2O
8 g of DOTAGA anhydride (13.96 mmol) were put in a flask towhich 31.6 mL of DMSO anhydrous and 3.300 mL of APTES(13.96 mmol) were quickly added. The reaction was stirredunder argon atmosphere and heated to 75 1C for 18 h. Theproduct was a golden solution. The mixture was let to cooldown to room temperature before 663 mL of ultrapure waterwere added. A small amount of sample was taken to quantifythe amount of produced APTES–DOTAGA by the combinationof Eu titration and HPLC probed by Gd3+.
The pH of the solution was adjusted to 9. Then the solutionwas separated in 4 volumes. 0, 1.814, 3.466, 5.199 mmol ofTEOS and 0.819, 91, 173, 260 mL of water were added to eachvolume. The samples were named as USNPi-1, 2, 3, 4 accordingly.The concentration of each component is indicated in Table S3(ESI†). These solutions were stirred at 25 1C overnight.
With USNPi-1, a small amount of sample was taken to verifythe amount of APTES–DOTAGA after being exposed to basicpH overnight.
The day after, 4 solutions were readjusted to pH 4.5 andheated to 40 1C overnight. These solutions were analysedby DLS.
These solutions were concentrated by Vivaspin (MWCO =3 kDa) to appropriate volumes in which the theoretical concen-tration of APTES–DOTAGA reaches 200 mM. These solutionswere purified at pH 2 by Vivaspin for 64 purification factor.Samples were taken for HPLC analyses (see ESI,† S3.2).
After the purification, the solutions were filtered through0.2 mm membrane and freeze-dried for long term storage.
Samples were redispersed and analysed by DLS (see S3.2, ESI†)and titrated with EuCl3 using common method shown below.
Samples of USNPi-2, 3, 4 were prepared for relaxationmeasurement (see S3.2, ESI†).
3. Synthesis of USNPi-5 using in situ formed chelating silanesin DEG/H2O
6.187 mL of APTES (26.17 mmol) were added in a 200 mL glassbottle containing 90 mL of diethylene glycol (DEG). Thesolution was stirred at RT for 1 h before 10 g of DOTAGAanhydride (17.45 mmol) were added. The mixture was stirred atRT for 5 days to allow a complete reaction. The product was afine suspension. Sample was prepared for DLS measurement(see S3.3, ESI†). 7.952 mL of TEOS (34.90 mmol) were added tothe suspension. This mixture was stirred for 1 h.
Then 900 mL of ultrapure water were added. The mixturewas heated to 50 1C and kept stirring for 18 h to allow acomplete hydrolysis of TEOS. A small amount of sample wastaken to analyze by DLS and HPLC. The solution was concen-trated by Vivaflow cassette (MWCO = 5 kDa) to 200 mL. Solution
was purified at pH 2 by Vivaflow for 50 purification factor(200 mL–1 L–200 mL–1 L–100 mL). After the purification, thesolution was neutralized to pH 7.4, filtered through 0.2 mmmembrane and freeze-dried for long term storage. 5.4 g oflyophilized powder of USNPi-5 were obtained. Sample wasprepared for DLS or HPLC analyses (see S3.3, ESI†). TheDOTAGA content of the particle was determined by Eu titration.
4. Complexation of metals in USNP
4.1. Complexation of Gd and Lu on USNPr to createUSNPr@Gd and USNPr@Lu. 333 mg of USNP lyophilizedpowder were redispersed in ultrapure water to obtain a solutionof 200 g L�1. 36 mL of GdCl3 2.2 M solution at pH 4 (molar ratioDOTAGA : Gd = 1 : 0.9) were added slowly to the solution ofUSNP in three times. The pH of the solution was adjusted toaround 4 during each addition of GdCl3 solution. The solutionwas incubated at 80 1C. pH was readjusted frequently to around5. After 48 h, pH of the solution was stable at around 5. Waterwas added to obtain a final concentration of 127 g L�1 or100 mM in DOTAGA of USNPr and 90 mM in Gd3+. The solutionwas further purified through Vivaspint 3 kDa for 32 purifica-tion factor in case some free Gd3+ might remain. Sample wasprepared for HPLC analysis (see S4.1, ESI†).
4.2. Complexation of USNPi-5 with different metals (Gd,Ho, Tb and Bi). 283 mg of lyophilized powder of USNPi-5containing 227 mmol DOTAGA, were redispersed in water tohave a concentration around 200 mM of DOTAGA. pH of thesolution was adjusted to 5.5 by adding NaOH solutions.To produce Gd particle, 98.5 mL of GdCl3 solution at 2.188 M(molar ratio DOTAGA : Gd = 1 : 0.95) were added slowly in3 times while the solution was heated and stirred on a heatplate at 70 1C. After each addition, pH was carefully increasedto 5.5. After 3 additions, water was added to obtain a concen-tration of 100 mM of DOTAGA. This solution was stirred at80 1C for 18 h. This solution was purified by tangential filtra-tion (MWCO = 3 kDa) with 16 purification factor to get rid ofany free Gd3+. Finally, the solution was neutralized to pH 7 andfiltered through 0.2 mm membrane and freeze-dried for longterm storage. Sample of purified solution was analyzed by DLS.
Similar protocols were applied using 431 mL of HoCl3 orTbCl3 solutions at 500 mM instead to produce Ho and Tbparticles respectively.
For Bi particles, 817 mL of BiCl3 solutions at 250 mM in HCl6 M was used. The nanoparticles solution had to be heated at70 1C before any addition was carried out. NaOH solutionat 10 M was needed to neutralize the solution to pH 5.5 andsolution was heated at 80 1C for 1 h between each step ofaddition. The molar ratio DOTAGA : Bi = 1 : 0.9 was used. Therest of the protocol was similar. Samples were prepared forHPLC analysis (see S4.2, ESI†).
5. Analytical methods (see S5, ESI† for more details)
5.1. Dynamic light scattering (DLS) and zeta potential.Hydrodynamic diameter distribution and zeta potential of thenanoparticles were measured in a Zetasizer Nano-S (633 nmHe-Ne laser) from Malvern Instruments (USA).
5.2. High performance liquid chromatography (HPLC).Gradient HPLC analysis was done by using the ShimadzuProminence series UFLC system with a CBM-20A controllerbus module, a LC-20AD pump, a CTO-20A column oven, aSPD-20A UV-vis detector and a FR-20A Fluorescence detector.
5.3. Phosphorescence spectroscopy. Phosphorescencemeasurements were carried out using a Varian Cary Eclipsefluorescence spectrophotometer, in the resolved time mode.
5.4. 1H nuclear magnetic resonance (NMR) and diffusionordered spectroscopy (DOSY). For experiments conductedon USNP, all experiments were performed at 298 K, withoutspinning, on a Bruker Avance III 500 MHz spectrometerequipped with 5 mm PABBI probe.
For 1H NMR diffusion experiments, the standard ledbpgp2ssequence from Bruker was used. The diffusion delay d20 wasset to 100 ms, and the bipolar pulses p30 were adjusted toobtain a 95% attenuation at full strength, typically in the rangeof 2 to 4 ms. 64 points of 32 scans each were acquired in thediffusion dimension, with 5 s relaxation delay allowingfull relaxation of protons, giving a total experiment time of3 h. The data were processed with NMRnotebook software fromNMRTec, (France).
The reported hydrodynamic diameters (DH) are simplyderived from the diffusion coefficients (D) with the well-knownStokes–Einstein formula: DH = kBT/3pZD, in which kB is theBoltzmann constant, T the absolute temperature, and Z theviscosity of the solvent (1.13 cP for D2O at 298 K).
5.5. Relaxivity measurement. Relaxivity measurementswere performed on a Brukers minispec mq60NMR analyzer(Bruker, USA) at 37 1C at 1.4 T (60 MHz).
5.6. Elemental analysis. For experiments conducted onUSNP, the determination of the accurate concentration ofmetals in the nanoparticle was performed by inductivelycoupled plasma-optical emission spectrometry (ICP-OES) (witha Varian 710-ES spectrometer, USA). Otherwise, elementalanalysis was also conducted for some samples by FILAB SAS,Dijon, France and enabled the determination of the Gd, C, Nand Si contents of the powder samples.
5.7. Mass spectrometry. For experiments conducted onUSNP, full scan mass experiments were recorded by electrosprayionization (ESI) on a commercial quadrupole time-of-flight(micro-qTOF, Bruker-Daltonics, Bremen, Germany). Externalcalibration was realized with a commercial peptides set.
5.8. In vivo magnetic resonance imaging (MRI) experiment.Three BALB/c mice were inoculated subcutaneously with coloncarcinoma (CT26) cells on both flanks.
USNPr@Gd* lyophilized powder was dispersed in physio-logical serum at 100 mM (in Gd). This concentrated solution wasdiluted to 20 mM in serum before being injected intravenously tothe mice at the dose of 200 mmol (in Gd) per kg.
Images were acquired before (pre-contrast) and after injection(post-contrast) using a 7 T MRI system 300WB micro imagingspectrometer, with a 1H 40 mm coil, Paravision 5.11 software(Bruker, Germany). The respiratory rate was continuously moni-tored by adjusting isoflurane concentration (1.5%) dynamic con-trast enhanced (DCE) sequence was recorded using Intragate Flash
multislices for motion free artifacts with TR = 100 ms, TE = 4 ms,flip angle = 801. The repetition number was set to 15 and a numberof time frames to reconstruct was 1. A field-of-view (FOV) of 3 cm�3 cm and a matrix of 256 � 256, 4 slices with a thickness of 1 mmwere chosen, giving a spatial resolution of 117 mm � 117 mm inplane. The total scan time was in the order of 3 min 14 s. Finally,an elongated version of the Intragate Flash multislices sequencewas used for the dynamic follow-up to obtain the same temporalresolution in a scan time of 40 min. 2–3 min scans were performed3–6 hours post-contrast as follow up.
Several regions of interest (ROI) in tumors and liver weremonitored and the MRI intensities of ROIs were plotted pre-and post-injection of the particle. Tissue enhancement level ofthe signal in each tissue area was calculated as (St � S0)/S0,where St was the signal intensity measured at each time pointafter injection, and S0 was the signal intensity before injection.
Of three mice, only one showed clear enough images forassessing hepatic accumulation and clearance due to thedifficulty in positioning the animals. In fact, they were positionedrather to reveal better the tumors than the other organs.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
V. L. T. thanks Mr Bernard Fenet (Centre Commun de RMN,UCBL I) and Kevin Sanders (ENS Lyon) for conducting pre-liminary NMR experiments and explaining DOSY principles;Arthur Marais, Eloıse Thomas, Matteo Martini, Chayma Nouajaaand Mathilde Maillard (ILM, UCBL I) for helpful discussions andtechnical assistance during the synthesis and characterization ofthe precursors and NPs. The authors gratefully acknowledgeEuropean Union FP7-PEOPLE Initial Training Network ARGENT(Advanced Radiotherapy, Generated by Exploiting Nanoprocessesand Technologies) Project ID 608163 for the financial support.The in vivo bioimaging experiments were performed at the UTCBSChimie Paristech Bioimaging facility (LIOPA from PIV ParisDescartes facility).
Notes and references
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