Supplementary Figures

Supplementary Figure 1. Transmission electron microscopy (TEM) images of quantum dots (QDs)

and magnetic nanoparticles (MNPs). TEM image of CdSe-CdS core-shell QDs with an average

diameter of 9.0 ± 0.4 nm and a standard deviation of ~ 4.4% (a) and Fe3O4 MNPs with an average

diameter of 5.9 ± 0.3 nm and a standard deviation of ~ 5.0% (b).

Supplementary Figure 2. Scanning electron microscopy (SEM) measurement of core-shell

supernanoparticles (CS-SPs). A SEM image of multilayered CS-SPs with an average diameter of ~120

nm.

Supplementary Figure 3. Hydrodynamic (HD) size measurement. Dynamic light scaterring (DLS)

measurement of PVP-coated CS-SPs shown in Figure 1 of the main text. The inorganic diameter of the

sample obtained by TEM measurement is ~120 nm. The increased size is due to the hydration layer

formed by the surface PVP bulky polymer in ethonal solution.

Supplementary Figure 4. Core-shell superstructure revealed by high-resolution TEM (HR-TEM)

measurement. A TEM image of one CS-SP (a) and a HR-TEM image of the area indicated by bule line

in panel a (b). The displayed lattice distance of ~2.53 Å in yellow dashed circles and 3.62 Å in red

dashed circles correspond to the (311) lattice spacing of the cubic Fe3O4 MNPs and the (100) lattice

spacing of the wurtzite CdSe-CdS QDs, respectively.

Supplementary Figure 5. TEM images of reverse CS-SPs. TEM images show that CS-SPs (QDs as

shell and MNPs as core) are formed using small QDs and large MNPs. TEM images of CdSe-CdS QDs

(~ 6.0 ± 0.3 nm) (a), Fe3O4 MNPs (9.6 nm ± 0.4 nm) (b), CS-SPs (c) and one zoom-in CS-SP (d). Red

and yellow dash-circles are indicating the positions of QDs (on the surface) and MNPs (stay inside),

respectively.

Supplementary Figure 6. Elemental mapping of silica coated CS-SPs (silica-CS-SPs). Images of

dark-field scanning TEM (STEM) and elemental mapping from the silica-CS-SPs sample. The results

show that a thin layer of silica shell is uniformly coated on the surface of CS-SPs, while the core-shell

superstructure (MNPs as “core” and QDs as “shell”) is preserved.

Supplementary Figure 7. HD size measurements of CS-SPs before and after silica shell growth.

DLS measurement of PVP-coated CS-SPs (PVP-CS-SPs, blue line) and silica-coated CS-SPs (silica-CS-

SPs, red line). The results display that after silica shell growth, the HD diameter of CS-SPs decreases

from ~ 130 nm to ~100 nm, suggesting removal of the PVP bulky polymer layer.

Supplementary Figure 8. Temperature dependence of magnetization (M/Ms). Zero field cool (ZFC,

red) and field cool (FC, blue) curves for silica-coated CS-SPs (silica-CS-SPs). The blocking temperature

is ~ 35 K, showing the superparamagnetism of silica-CS-SPs at room temperature (~300K).

Supplementary Figure 9. Magnetic characterization of free MNPs. Superconducting quantum

interference device (SQUID) measurement shows that free Fe3O4 MNPs (~5.9 nm in diameter) at 300K

exhibit a saturation magnetization of 63.7 emu/g [Fe3O4].

Supplementary Figure 10. Elemental analysis of silica-CS-SPs. Energy-dispersive X-ray (EDX)

spectrum of ~100nm silica-CS-SPs (a) and atomic percentages of Fe (29.4%), Cd (10.5%) and Si

(60.1%) atoms (b).

Supplementary Figure 11. Photoluminescence (PL) measurements of CS-SPs. Relative (Relat.) PL

intensities of the CS-SPs before (PVP-CS-SPs, blue line) and after silica shell growth (silica-CS-SPs).

Supplementary Figure 12. HD size measurements of silica-CS-SPs before and after mPEG

functionalization. DLS measurement of silica-coated CS-SPs (Silica-CS-SPs, red line) and mPEG-

silane functionalized silica-CS-SPs (mPEG func. Silica-CS-SPs, green line). The results demonstrate

that after mPEG functionalization, the hydrodynamic (HD) diameter of silica-CS-SPs increases from ~

102 nm to ~120 nm, suggesting a successfull PEGylation process.

Supplementary Figure 13. Surface charge measurements of silica-CS-SPs before and after mPEG

functionalization. Zeta potential (ζ) measurement of silica-CS-SPs (Silica-CS-SPs, blue line) and

mPEG-silane (MW5000) functionalized silica-CS-SPs (mPEG func. Silica-CS-SPs, red line). The

results show that after mPEG-silane surface functionalization, the surface charge of silica-CS-SPs

changed from -27.2 ± 3.1 mV to -5.1 ± 2.3 mV.

Supplementary Figure 14. In vitro cytotoxicity study of mPEG functionalized silica-CS-SPs. We

studied whether these nanoparticles had any deleterious biological properties. Cytotoxicity of silica-CS-

SPs was examined in three cell lines: Hs27 (a), U937 (b), HMVEC (c), representing stromal, immune,

and vascular cells respectively. We found that mPEG functionalized silica-CS-SPs are biologically

nontoxic in our cytotoxicity studies.

Supplementary Figure 15. Serum protein adsorption study. Representative correlation functions

from fluorescence correlation spectroscopy (blue line) and their respective fits (red line) for particles

incubated in PBS (a) and FBS (b). The average diffusion times and their corresponding HD diameters

shown in panel c. The results show that there is no difference in diffusion times for particles incubated

in PBS and FBS, indicating no significant protein adsorption for the mPEG functionalized silica-CS-SPs.

Error bars represent standard error over the 10 measurements.

Supplementary Figure 16. In vitro magnetic resonence imaging (MRI) measurements. In vitro

transverse relaxivity coefficients (r2) of silica-CS-SPs (red spheres) and free Fe3O4 MNPs (blue squares).

T2-weighted MR images (right) of silica-CS-SPs at various dilutions. A 7.2-fold increase in r2 is

observed for silica-CS-SPs due to synergisitic MR enhancement effect of highly aggregated Fe3O4

MNPs inside silica-CS-SPs.

Supplementary Tables

Supplementary Table 1: Calculated lattice constant (a) based on the SAXS data.

hkl d (nm) a (nm) 111 6.0 10.4 200 5.3 10.6 220 3.6 10.2 311 3.2 10.6 222 3.0 10.4 331 2.4 10.5 422 2.3 10.3 333 2.1 10.8 442 1.7 10.2

Based on the d spacing measured from the SAXS data, the calculated lattice constant (a) is 10.4 ± 0.2 nm.

Supplementary Table 2: Size-controlled synthesis of CS-SPs.

QDs (mg) MNPs (mg) DTAB (mg) Weight ratio (QDs+MNPs)/DTAB

Diameter of CS-SPs (nm)

5.2 4.8 20 0.5 80 ± 9 4.0 6.0 20 0.5 120 ± 13 4.0 13.5 20 0.875 235 ± 30 5.0 23.0 20 1.4 360 ± 60

Supplementary Table 3: Calculation of the magnetization per single silica-CS-SP (~100nm).

Diameter of the magnetic “core” (nm) 60.8

Volume of the magnetic “core“ (nm3) 1.18 × 105

MNPs inorganic volume packing density (%) 39

Volume of MNPs per silica-CS-SP (nm3) 4.6 × 104

Mass of MNPs per silica-CS-SP (g) 2.3 × 10-16

Mass of single silica-CS-SP (g) 9.3 × 10-16

Number of silica-CS-SPs per gram of sample 1.1 × 1015

Magnetization per single silica-CS-SP (emu/particle) 1.4 × 10-14

The diameter of the magnetic “core” is calculated based on the diameter of the silica-CS-SPs (100nm),

the silica shell thickness (10.6nm) and the diameter of the QDs (9.0 nm).

The MNPs inorganic packing density is obtained based on the face-centered-cubic packing nature (close

packing density of 74%) of the MNPs inside magnetic “core” and the inter-particle distance of 1.4nm

determined by small angle x-ray scattering (SAXS) measurement.

The mass of single silica-CS-SP is calculated based on the mass (2.3 × 10-16g) and mass percentage

(24.8%) of MNPs per silica-CS-SP.

The magnetization per single silica-CS-SP is calculated based on the saturation magnetization of silica-

CS-SPs per gram (15.2 emu/g measured by superconducting quantum interference device) and the

number of silica-CS-SPs in a gram of sample.

Supplementary Methods

Chemicals

1-octadecene (ODE, 90%), 1-tetradecene (TDE, 92%), 1-octanethiol (> 98.5%), trioctylphosphine oxide

(TOPO 99%), trioctylphosphine (TOP, 97%), oleic acid (OLA, 90%), oleylamine (OAm, 70%),

polyvinylpyrrolidone (PVP, 55k MW), fluorescein isothiocyanate–dextran (FITC-Dextran), ethylene

glycol (spectrophotometric grade, ≥ 99%), ammonia hydroxide (28% NH3·H2O) and tetraethyl

orthosilicate (99.999%) were obtained from Aldrich. Cadmium oxide (CdO, 99.998%), selenium powder

(99.999%), dodecyltrimethylammonium bromide (DTAB, 99%) and octadecylphosphonic acid (ODPA)

were purchased from Alfa Aesar. Iron chloride hexahydrate (FeCl3·6H2O 99+%) was purchased from

Acros. Sodium oleate (95%) was purchased from TCL. Methoxy-polyethylene-glycol silane (mPEG-

Silane, 5000 MW) was obtained from Creative PEGWorks. Nanopure water was made by Barnstead

Nanopure Diamond system.

Synthesis of CdSe-CdS quantum dots (QDs)

CdSe-CdS core-shell QDs were synthesized using a previously published method1.

For CdSe core synthesis: briefly, 60 mg CdO, 280 mg ODPA and 3 g TOPO were added to a 50 mL

flask. The mixture was heated to 150 °C and degassed under vacuum for 1 hour. Under nitrogen flow,

the reaction mixture was heated to 320 °C to form a colorless clear solution. After adding 1.0 mL TOP

to the solution, the temperature was brought up to 380 °C, at which point Se/TOP (60 mg Se in 0.5 mL

TOP) solution was swiftly injected into the flask. When the CdSe core nanocrystals reached the desired

size, the reaction was terminated by removing the heat. The resulting CdSe particles were precipitated

by adding acetone and dispersed in hexane for further uses.

For CdS shell growth: a hexane solution containing 300 nmol of CdSe QDs was loaded in a mixture of 6

mL ODE and 6 mL OAm. The reaction solution was degassed under vacuum at room temperature for 1

hour and 120 °C for 10 min to completely remove the hexane, water and oxygen inside the reaction

solution. After that the reaction solution was heated up to the growth temperature under nitrogen flow

and magnetic stirring. When the reaction solution had reached 240 °C, a desired amount of cadmium (II)

oleate (Cd-oleate, diluted in 6 mL ODE) and 1.2 equivalent amount of octanethiol (diluted in 6 mL ODE)

were injected dropwise into the growth solution at a rate of 3 mL/hr using a syringe pump. The reaction

temperature was further raised to 310 °C and maintained for the rest of the reaction. After finishing the

precursor infusion, 1 ml oleic acid was quickly injected and the solution was further annealed at 310 °C

for 30 min. The resulting CdSe-CdS core-shell QDs were precipitated by adding acetone, and then

redispersed in chloroform as a stock solution.

Synthesis of Fe3O4 magnetic nanoparticles (MNPs)

Fe3O4 MNPs were prepared according to a modified literature method2. Briefly, 900 mg pre-made iron-

oleate, 190 µL OLA, and 5 mL of ODE or ODE/TDE mixture were loaded in a 50 mL flask. Under

Nitrogen flow, the mixture was gradually heated to solution boiling temperature and kept boiling for one

hour. The reaction was stopped by removing the heat. The resulting Fe3O4 MNPs were precipitated by

adding acetone and dispersed in chloroform as a stock solution.

Synthesis of “core-shell” superparticles (CS-SPs) with a random magnetic “core”

For a typical synthesis, 1 mL chloroform solution containing QDs (9.0 ± 0.4 nm) and MNPs (5.9 ± 0.3

nm) was injected into 1 mL DTAB aqueous solution (20 mg/mL in Nanopure water). The solution was

thoroughly mixed by vortex for 5 second. After removing the chloroform from the mixture by blowing

Ar at room temperature, a clear QD-MNP-micelle aqueous solution was obtained. This QD-MNP-

micelle solution was swiftly injected into 5mL poly(vinylpyrrolidone) (PVP, MW 55000) ethylene

glycol (EG) solution (2 mM) under vigorous stirring with a stirring speed of 700 rpm for 30 min at room

temperature. The resulting CS-SPs were isolated by centrifugation, redispersed in ethanol and stored at

~4 °C as a stock solution.

Synthesis of CS-SPs with a supercrystalline magnetic “core”

The synthesis procedure is the same as the synthesis of CS-SPs described above, except that an

additional thermal annealing process was carried out after the CS-SPs synthesis. In this thermal

annealing process, the resulting CS-SPs in PVP/EG solution was heated to 80 °C with a slow stirring

speed (300 rpm) and kept for 24 hours before cooling to room temperature. The resulting

supercrystalline CS-SPs were isolated by centrifugation, redispersed in ethanol and stored at ~ 4 °C as a

stock solution.

Synthesis of silica-coated CS-SPs (silica-CS-SPs)

For a typical synthesis, 2 mg CS-SPs was dissolved in 20 mL ethanol. Under vigorous stirring, 3 mL

deionized-water (DI-water) and 1 mL NH3·H2O solution was added dropwise in the reaction solution,

followed by the addition of 50 µL TEOS. The final solution was kept stirring at room temperature for 20

min. The resulting silica-coated CS-SPs were isolated by centrifugation, washed three times with

deionized-water (DI-water) and finally dissolved in 2 mL DI-water and stored at ~ 4 °C as a stock

solution.

Synthesis of methoxy-polyethylene-glycol silane (mPEG-silane, MW5000) functionalized silica-CS-

SPs

2 mL silica-CS-SPs were diluted in 10 mL DI-water. Under magnetic stirring, 50 µL NH3·H2O solution

and 20 µL TEOS were added into the solution, sequentially. After the mixture solution was heated to

70 °C, 1 mL mPEG-silane (MW 5000) aqueous solution (0.02 M) was dropped in. The reaction solution

was kept stirring at 70 °C for 3 hrs and then stirred at room temperature for another 15 hrs. The resulting

mPEG-silane functionalized silica-CS-SPs were isolated by centrifugation, washed three times with

nanopure water and finally dissolved in 1 mL phosphate buffer solution (PBS, 1X) and stored at ~4 °C

fridge.

Synchrotron-based X-ray scattering measurement

Wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) were performed inside a

diamond anvil cell (DAC) in which two well-aligned diamond anvils with a culet size of 0.5 mm are

used. A pre-indented stainless-steel gasket with a ~ 200 µm hole was used to hold the sample. A purified

CS-SPs sample (~ 1 mg) was dried and loaded in the gasket hole. The X-ray scattering measurements

were performed at room temperature at B2 station, Cornell High Energy Synchrotron Source (CHESS).

Double Ge crystals cut along (111) collimate the white beam into monochromatic beam. The

monochromatic X-ray with a wavelength of 0.48595 Å was used for WAXS and SAXS measurements.

A single tube collimator reduces the x-ray beam down to 100µm. The x-ray scattering signals from the

samples were collected using a large area MAR345 detector. Several parameters including the tilt and

rotating angles of the detector and the sample-to-detector distance were calibrated by using both CeO2

and silver behenate standard. The SAXS and WAXS pattern were collected at the sample-to-detector

distance of 859 mm. The obtained images were integrated using the Fit2D software3. The lattice

constant (a) was calculated by the d spacing between superlattice planes with Miller indices {hkl} using

the formula: 𝑎 = 𝑑!!" ℎ! + 𝑑! + 𝑙!.

Tomography 3D-TEM measurement

For the tomograms shown in 3D-TEM tomography Movies, 241 images were recorded every 0.5° from

+60° to −60° using a FEI Tecnai G20 (Netherlands) transmission electron microscope operated at 200

kV. Image acquisition was undertaken using the FEI software package Xplore3D. Images were then

aligned sequentially using IMOD/etomo4. Reconstructions were performed using either weighted back-

projection (WBP) routines or an iterative routine (SIRT). Projections were constructed in IMOD/etomo

and Imaris.

Photoluminescence (PL) intensity trace measurement for single silica-CS-SPs

The silica-CS-SPs water solution was spun cast onto glass coverslips (Electron Microscopy Sciences).

Individual silica-CS-SPs were studied by confocal fluorescence microscopy using an oil immersion

microscope objective (100X, 1.40 NA, Plan Apochromat). The silica-CS-SPs were excited with a

continuous wave laser at 532 nm with a power density of 22 W/cm2. PL intensity time traces were

recorded using pulse counters (National Instruments) and a correlator card (Timeharp 200, PicoQuant).

All measurements were performed at room temperature.

Calculation of the mass percentage of magnetic component inside silica-CS-SPs

The mass percentage of magnetic component (𝑓 𝑀 ) can be expressed as:

𝑓 𝑀 = !(!"#)!(!"!#$)

(1)

= !(!"#)! !"# !! !" !! !"#"$% !!(!"#$%&')

(2)

Where 𝑚 𝑖 is the mass of each component inside silica-CS-SPs. For QD component (CdSe/CdS core-

shell structure), there are more than 95% of CdS molecules. Therefore, to simplify the calculation, we

assume that the QDs contain only CdS molecules. Therefore, Equation (2) can be further expressed as:

𝑓 𝑀 =   [!"!!!]×!"(!"!!!)!"!!! ×!" !"!!! ! !"# ×!" !"# ! !"!! ×!" !"!! !!(!"#$%&')

(3)

=   !" /!×!"(!"!!!)!" /!×!" !"!!! ! !" ×!" !"# ! !" ×!" !"!! !!(!"#$%&')

(4)

Where [𝑖] are the molecular percentages of Fe3O4, CdS and SiO2 molecules inside silica-CS-SPs, and

𝑀𝑊(𝑖) is the molecular weight.

As an example, energy-dispersive X-ray spectrum measurement for ~100nm silica-CS-SPs

(Supplementary Fig. 10) shows that atomic percentages of [Fe], [Cd] and [Si] are 29.4%, 10.5% and

60.1%, respectively. After applying the molecular weight of each molecule and the mass percentage of

organic component (𝑚(𝑜𝑟𝑔𝑎𝑛𝑖𝑐) ) in silica-CS-SPs (19.1% of the total mass of silica-CS-SPs,

determined by thermogravimetric analysis) in Equation (4), the mass percentage of the magnetic

component (𝑓 𝑀 ) inside silica-CS-SPs is calculated to be 24.8%.

Calculation of the magnetization per single silica-CS-SP

The magnetization per single silica-CS-SP can be calculated by the saturation magnetization of silica-

CS-SPs per gram divided by the number of silica-CS-SPs in a gram of sample.

As an example, for which we detail our analysis, ~100 nm silica-CS-SPs has a saturation magnetization

of 15.2 emu/g measured by superconducting quantum interference device (SQUID). The calculation is

summarized in Supplementary Table 3. Based on the calculation (Supplementary Table 3), the saturation

magnetization for ~100nm silica-CS-SPs is 1.4 × 10-14 emu/particle.

Serum protein adsorption measurement

Protein adsorption was measured after incubating the mPEG-silane functionalized silica-CS-SPs

solution (50 µL, 2 mg/mL) with 150µL of Fetal Bovine Serum for 4 hours at 37 °C. The sample

incubated in PBS (1X, pH 7.4) was used as control. After incubation, the samples were diluted in PBS

(1X, pH 7.4) and their diffusion times were measured by fluorescence correlation spectroscopy (FCS)

using a setup described previously5. Samples were excited with 1.8 µW (power in front of the

microscope objective), and ten measurements with an acquisition time of 60s for each measurement

were performed for each sample. Correlation functions were fit in Matlab (The Mathworks) with the

isotropic 2D translational diffusion model to extract the average particle diffusion time. The diffusion

times of the samples incubated with FBS and PBS were compared.

Cytotoxicity study

Cell culture: Human dermal fibroblast cell Hs27, human macrophage cell U937 and human dermal

microvascular endothelial cell HMVEC were obtained from American Type Culture Collection (ATCC,

Manassas, VA) and cultured in RPMI-1640 (Hs27 and U937) and EGM™-2MV BulletKit™ medium

(HMVEC) with supplements, respectively. All cells were maintained at 37°C in a humidified incubator

with 5% CO2.

In vitro cytotoxicity: In vitro cytotoxicity of mPEG-silane functionalized silica-CS-SPs was evaluated

using a cell viability assay. 5000 cells (Hs27, U937 and HMVEC) were seeded in each well of a 96 well

plate and incubated for 24 h. Then cells were treated with mPEG-silane functionalized silica-CS-SPs at

different particle concentrations of 0, 12.5 25, 50, 100 and 200 µg/mL for 24 hours. Cell viability was

determined by a Dojindo cell counting kit using the protocol from the manufacturer (Rockville, MD).

Force measurements on single silica-CS-SPs

Force calibration on the single silica-CS-SPs was performed by dipping the magnetic tip in a viscous

solution made of 80% glycerol and 20% water (w/w), in which the silica-CS-SPs were dispersed.

Concentration was kept low to avoid any hydrodynamic flux whereas the particles were dragged inside

the viscous solution. Movies were acquired at 25 Hz while the CS-SPs were attracted toward the magnet.

Particle positions were then determined by means of a tracking software6, and the particle velocities

along the tangential direction of the tip were calculated. ~500 individual SPs were tracked for the force

calibration measurement. The associated forces were finally determined as a function of the distance to

tip using Stoke's law: 𝐹 = 6𝜋𝜂𝑟𝑣, where the force 𝐹 is related to particle's velocity 𝑣 through medium

viscosity 𝜂 and the particle's radius 𝑟. We determined the spatial precision of individual SPs tracking by

absorbing SPs on a coverslip and imaging the individual SPs for 250 consecutive frames. Standard

deviation of the positions gave a pointing accuracy of 15 nm.

Magnetic manipulation of the silica-CS-SPs at the single particle level in water droplet

Silica-CS-SPs were observed with a wide field Olympus IX70 inverted microscope, equipped with a

100X, 1.45 NA objective, and coupled to a 488 nm laser line in an epifluorescence configuration. The

emitted light was filtered out from the excitation laser by a dichroic filter with a 495 nm cut off and a

band-pass emission filter centered at 625nm, before being collected on a QUANTEM 512SC EMCCD

camera (Photometrics).

To demonstrate the magnetic manipulation of the silica-CS-SPs at the single particle level, we filled the

sample holder of the microscope with mineral oil and injected inside a mixture of 80% glycerol and 20%

water (w/w) containing silica-CS-SPs at very low concentration (in the sub-nanomolar range). We were

thus able to create an aqueous droplet of around 50µm diameter, containing the silica-CS-SPs inside the

oil phase. Injection was performed with a micro-injector system (Femtojet, Eppendorf) and home-made

micropipettes of 1µm tip, pulled from borosilicates capillaries using the P-97 puller (Sutter Instrument).

Silica-CS-SPs were finally magnetically manipulated inside the confined environnement of the droplet

using a simple magnetic tweezer (see Supplementary Movie S6) made from a paramagnetic tip put on

top of a small permanent magnet of Neodymium Iron Boron whose shape was a parallelepiped of

4×1×0.5 mm (supermagnet). To create the tip, a piano string in steel (0.1 mm diameter) was approached

to the flame of a Bunsen burner while pulling on both string sides. The string was slowly broken,

resulting in two sharp extremities of around 20 µm that were used as paramagnetic tips on top of the

permanent magnet. This magnetic tweezer was attached to the arm of the injection system

micromanipulator in order to bring the tweezer with high precision and stability close to the droplet

where particle manipulation was imaged at a frame rate of 2 Hz.

Magnetic manipulation of the silica-CS-SPs at the single particle level in living cells

For manipulation in living cells, HeLa cells and Cos7 cell lines were cultured at 37°C in 5% CO2 in

Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum. For imaging

and manipulation, cells were grown on 25 mm glass coverslips. Experiments were performed at 37 °C in

a heating chamber placed on the inverted microscope. Microinjection was performed with the same

borosilicate needles as described above and filled with a concentrated solution of SPs. After

microinjection, the cells were left for 20 minutes recovery. In order to bring the tip close to the sample

with the orientation adapted to each unique cell morphology, the magnetic tip was attached to a circular

teflon holder, allowing us to rotate the tip in respect to the optical axis of the microscope, as described in

Etoc et al.7. Tracking of the manipulated SPs was performed semi-manually using the Manual Tracking

plugin of ImageJ.

In vivo multiphoton (MP) imaging study

Animal models: Imaging studies were carried out using a MCaIV cranial window model in C3H mice8.

To prepare the window, the mouse head is fixed by a stereotactic apparatus. The skin on top of the

frontal and parietal regions of the skull is cleaned with antimicrobial solution. A longitudinal incision of

the skin is made between the occiput and forehead. Then the skin is cut in a circular manner on top of

the skull, and the periosteum underneath is scraped off to the temporal crests. A 6-mm circle is drawn

over the frontal and parietal regions of the skull bilaterally. Using a high-speed air-turbine drill with a

burr-tip 0.5 mm in diameter, a groove is made on the margin of the drawn circle. This groove is made

thinner by cautious and continuous drilling of the groove until the bone flap becomes loose. Cold saline

is applied during the drilling process to avoid thermal injury of the cortical regions. After removal of the

bone flap, gel foam is placed on the cutting edge and the dura mater is continuously kept moist with

physiologicalsaline. The dura and arachnoid membranes are cut completely from the surface of brain

surface. The window is sealed with a 6-mm cover glass, which is glued to the bone with histocompatible

cyanoacrylate glue. The cranial window is removed 10 days after initial surgery and MCaIV tumor cell

pellet (~ 2.5×105 cells) is injected into the exposed parenchymal space at about 0.1 mm depth. In vivo

imaging is performed on established tumors on day 9 after pellet implantation.

All animal procedures were carried out following the Public Health Service Policy on Humane Care of

Laboratory Animals and approved by the Institutional Animal Care and Use Committee of the

Massachusetts General Hospital.

In vivo imaging: Fluorescein isothiocyanate–dextran/phosphate buffered saline solution (FITC-Dextran)

was prepared for in vivo vessel tracing in a concentration of 10 mg/mL. Following tail vain injection of

100 µl of FITC-Dextran, a baseline vessel image was acquired using multiphoton imaging described

previously9 on a custom-built multiphoton laser-scanning microscope using confocal laser-scanning

microscope body (Olympus 300; Optical Analysis Corp.) and a broadband femtosecond laser source

(High Performance MaiTai, Spectra-Physics). After acquisition of a baseline image, mPEG-silane

functionalized silica-CS-SPs solution (200µL, 2mg/mL) were intravenously injected via tail vein. Image

slices were taken with 400 mW at a wavelength of 820 nm in three ROIs in tumor area and one in the

healthy contra-lateral hemisphere. Images were taken with depths from 0 – 150 µm. Imaging studies

were performed with a 20X magnification, 0.95NA water immersion objective (Olympus XLUMPlanFl,

1-UB965, Optical Analysis). Image analysis was carried out using ImageJ.

In vitro T2-relaxivity measurement

Linear dilutions of silica-CS-SPs were prepared in PBS (1X, pH 7.4) added to the center wells of a 384-

well microtiter plate (BD Falcon) at concentrations 0.018-0.09 mM [Fe]. Surrounding wells were filled

with PBS to reduce susceptibility-based image artifacts and improve shimming efficacy. T2 relaxation

measurements were carried out at ambient temperature on a Bruker Avance III 7 T system with a 20 cm

bore and using a Bruker Biospin MRI GmbH coil. A 2 mm thick horizontal slice with a 5.0 × 5.0 field of

view was imaged through the center of the filled wells using a T2-weighted multiecho spin echo pulse

sequence with a 10 ms inter-echo spacing time and a 3 s repetition time. Thirty 2D Carr-Purcell-

Meiboom-Gill (CPMG) echo images were collected as 256 × 256-point matrices and subsequently

analyzed using custom Matlab (Mathworks, Natick, MA) routines. The echo time vs. integrated intensity

from imaged wells was fit to an exponential decay function to obtain a transverse relaxation rates, R2,

for each sample. The reported relaxivity, r2, was computed from the slope of the relationship between

relaxation rate and iron concentration for each sample dilution.

In vivo magnetic resonance (MR) imaging study

All experiments were performed on a 9.4 Tesla magnet (Magnex Scientific Ltd, Oxford, UK) equipped

with a 60 mm inner diameter gradient coil (Resonance Research, Billerica, MA) and interfaced with a

Bruker MRI console (Bruker Biospin, Billerica, MA). The gradient coil has a maximum strength of

1500 mT/m and a rise time of 100 µs. Images were acquired using a home built mouse head bird-cage

coil. Mice were positioned on a custom made mouse cradle and anesthetized with 1.5% isoflurane in

50/50 O2/medical air mixture with total flow rate of 1200 ml/min.

T2-weighted RARE MRI: T2-weighted Rapid Acquisition with Refocused Echoes (RARE) images were

acquired with the following acquisition parameters: TE = 10, RARE factor = 8, TR = 2500 ms, NA = 4,

15 image slices, 0.5 mm slice thickness, 125 µm in-plane resolution. Tumor volume was determined

from the T2 hyperintense tumor region of the brain.

T2 Maps: T2 maps were generated from multi-echo spin-echo and multi-echo gradient-echo images,

respectively, using a custom written MATLAB program for voxel-wise fitting of the T2 relaxation times.

Multi-echo spin-echo image acquisition parameters were: TE = 10 ms, 15 echoes with 10 ms increment,

TR = 2.5 s, 2 averages, FOV = 1.6 cm, matrix = 128 × 128 (in-plane resolution 125 µm), slice thickness

= 0.5 mm, 15 image slices. Multi-echo gradient-echo image acquisition parameters were: TE = 2.5 ms,

10 echoes with 2.5 ms increment, TR = 1.5 s, 4 averages, FOV = 1.6 cm, matrix = 96 × 96, slice

thickness = 0.5 mm, 15 image slices.

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