Chemical nature and structure of organic coating of quantum dots is crucial for their application in imaging diagnostics

Figures & data

Figure 1 A flow cytometric histogram of cell fluorescence with quadrant markers, drawn to distinguished viable (nonlabeled) from dead (propidium iodide-labeled) cells. Quadrant B shows viable cells and quadrant D shows dead cells.

Figure 2 Model structure of quantum dot probes, consisting of CdSe/ZnS quantum dots coated with non-crosslinked PAMAM dendrimers or encapsulated in crosslinked PAMAM dendrimers.

Abbreviations: PAMAM, polyamidoamine; EDC, 1-ethyl-3(3-dimethylamino propyl)-carbodiimide; QD, quantum dot.

Figure 3 Two-photon excited fluorescent images of brain vasculature in horizontal planes (at 150 μm depth from brain surface) after intravenous injection of QD⁵²⁵ (coated with non-crosslinked PAMAM dendrimers [A] or capsulated in crosslinked dendrimers [B] in the anesthetized rat).

Notes: All images were obtained at λ_ex = 810 nm and a mean laser power of about 2.7 W. Detectors: filter/band-pass 525/50 nm (via 560/10 nm beam splitter).

Abbreviations: PAMAM, polyamidoamine; QD, quantum dot.

Table 1 Physiological characteristics of rats before and after intravenous injection of QDs (coated with noncrosslinked PAMAM dendrimers)

Parameters	Before injection	About 20 minutes after injection	P value
Mean arterial blood pressure (mmHg)	102.5 ± 2.5	75.5 ± 7.1	<0.05
Heart rate (beats per minute)	378 ± 29	310 ± 15	<0.05
Mean diameter of detected arteries (μm)	28 ± 9	35 ± 10 (vasodilatation)	<0.05
Mean diameter of detected veins (μm)	42 ± 23	45 ± 18 (slight vasodilatation)	NS

Notes: Means ± standard deviations from six independent experiments. QD concentration 0.3 nmol/kg bodyweight.

Abbreviations: QD, quantum dot; NS, not significant; PAMAM, polyamidoamine.

Table 2 Physiological characteristics of rats before and after intravenous injection of QDs (encapsulated in crosslinked PAMAM dendrimers)

Parameters	Before injection	About 20 minutes after injection	P value
Mean arterial blood pressure (mmHg)	96.8 ± 4.9	100.5 ± 8.4	NS
Heart rate (beats per minute)	340 ± 21	336 ± 23	NS
Mean diameter of detected arteries (μm)	23 ± 10	Constant	NS
Mean diameter of detected veins (μm)	41 ± 20	Constant	NS

Notes: Means ± standard deviations from six independent experiments. QD concentration 0.3 nmol/kg bodyweight.

Abbreviations: QD, quantum dot; NS, not significant; PAMAM, polyamidoamine.

Table 3 Effect of CdSe/ZnS QD⁴⁸⁵ (coated with noncrosslinked PAMAM dendrimers) on cell viability in vitro (% from control)

Cell line	10 nM QD	50 nM QD	100 nM QD	200 nM QD	500 nM QD
Jurkat	99 ± 7	97 ± 5	62 ± 5	30 ± 7	21 ± 4
K562	101 ± 6	98 ± 9	62 ± 9	37 ± 5	16 ± 3
A549	103 ± 9	101 ± 5	65 ± 7	39 ± 5	28 ± 5
HeLa	99 ± 3	100 ± 5	60 ± 8	32 ± 3	20 ± 4

Notes: The cell suspension (5 × 10⁵ cells/mL) was incubated with QDs over 24 hours in a humidified atmosphere, and cell viability was analyzed by flow cytometry. Cell viability in the absence of QD was considered 100% (control). The data are mean ± standard deviation from six independent experiments.

Abbreviations: QD, quantum dot; PAMAM, polyamidoamine.

Figure 4 (A) Dynamics of tetramethylrhodamine ethyl ester (TMRE) fluorescence of Jurkat cells in the absence (control) or presence of dendrimer-coated QD⁴⁸⁵ 100 nM. (B) Hypothetical mechanism of cytotoxicity of QD, coated with non-crosslinked polyamidoamine dendrimers.

Abbreviations: Apaf-1, apoptotic peptidase-activating factor 1; QD, quantum dot.

Figure 5 Model structure of QD probe, consisting of silica-shelled QD conjugated with PEG.⁴⁸

Note: Reprinted with permission from Zhelev Z, Ohba H, Bakalova R. Single quantum dot-micelles coated with silica shell as potentially non-cytotoxic fluorescent cell tracers. J Am Chem Soc. 2006;128:6324–6325. Copyright 2006 American Chemical Society.

Abbreviations: QD, quantum dot; PEG, polyethylene glycol.

Figure 6 Electron micrographs of nasal mucosa one day after intranasal inoculation of amino-functionalized silica-shelled quantum dots (dissolved in distilled water, 30 drops per animal, single dose).

Abbreviation: QD, quantum dot.

Table 4 Physiological characteristics of rats before and after intravenous injection of PEG1100-grafted silica-shelled QDs

Parameters	Before injection	About 20 minutes after injection	P value
Mean arterial blood pressure (mmHg)	96.2 ± 5.4	95.3 ± 12.8	NS
Heart rate (beats per minute)	320 ± 55	345 ± 51	NS
Mean diameter of detected arteries (μm)	24 ± 9	Constant	NS
Mean diameter of detected veins (μm)	41 ± 22	Constant	NS

Notes: Means ± standard deviations from six independent experiments. QD concentration 0.4 nmol/kg bodyweight.

Abbreviations: QD, quantum dot; NS, not significant; PEG, polyethylene glycol.

Table 5 Effect of silica-shelled CdSe/ZnS QDs (conjugated with PEG1100) on cell viability in vitro (% from control)

Cell line	10 nM QD	50 nM QD	100 nM QD	200 nM QD	500 nM QD
Jurkat	101 ± 9	99 ± 7	100 ± 7	103 ± 10	98 ± 7
K562	100 ± 5	102 ± 8	98 ± 5	96 ± 8	98 ± 7
A549	98 ± 4	101 ± 5	98 ± 7	98 ± 9	96 ± 10
HeLa	102 ± 6	103 ± 4	99 ± 9	98 ± 5	96 ± 8

Notes: The cell suspension (5 × 10⁵ cells/mL) was incubated with QDs for 24 hours in a humidified atmosphere, and cell viability was analyzed by flow cytometry. Cell viability in the absence of QDs was considered to be 100% (control). The data are means ± standard deviations from six independent experiments.

Abbreviations: QD, quantum dot; PEG, polyethylene glycol.

Figure 7 Two-photon excited fluorescent images of brain vasculature in horizontal planes (at 150 μm depth from the brain surface) after intravenous injection of PEG1100-grafted silica-shelled QD⁵²⁵ (2.5 nmol/kg bodyweight) in the anesthetized rat. The images were obtained at λ_ex = 810 nm and a mean laser power of about 2.7 W. Detectors: filter/band-pass: 525/50 nm (via a 560/10 nm beam splitter).

Abbreviations: QD, quantum dot; PEG, polyethylene glycol.

Figure 8 Model structure of PEGylated multimodal silica-shelled quantum dots.⁴⁸

Note: Reprinted with permission from Zhelev Z, Ohba H, Bakalova R. Single quantum dot-micelles coated with silica shell as potentially non-cytotoxic fluorescent cell tracers. J Am Chem Soc. 2006;128:6324–6325. Copyright 2006 American Chemical Society.

Abbreviations: PEG, polyethylene glycol; DOTA, tetraazacyclododecanetetraacetic acid.

Table 6 Spectral characteristics of PEG1100-grafted multimodal silica-shelled QDs

Spectral characteristic	Value
QY in 10 mM PBS, pH 7.4 (%)	35–48
QY in 100 mM PBS, pH 7.4 (%)	32–45
Longitudinal relaxation time, T1 (msec)	293.4 ± 23.5
Transverse relaxation time, T2 (msec)	128.5 ± 5.3

Notes: T₁ and T₂ data are means ± standard deviations from six independent experiments. T₁ and T₂ data were calculated at a QD concentration of 500 nM. The magnetic resonance imaging measurements were performed in a 4.7 T magnet (General Electric, Fairfield, CT) interfaced to a Bruker Avance console (Bruker Medical GmbH, Germany). A 30 mm diameter Litz coil (Doty Scientific Inc, Columbia, SC) was used for measurement of the samples. The sample temperature was maintained at room temperature (approximately 22–24°C). The measurements were performed in the following order: T₁-weighted imaging using conventional spin echo sequence; multiecho spin echo imaging for T₂ calculations; and inversion recovery spin echo imaging for T₁ calculations. For calculation of fluorescence quantum yield, the spectrally integrated emission of particle dispersion in different aqueous solutions was compared with the integrated emission of an ethanol solution of rhodamine G (Fluka) of identical optical density (<0.015) at an excitation wavelength of 365 nm.

Abbreviations: QD, quantum dot; PEG, polyethylene glycol; PBS, phosphate-buffered saline; QY, quantum yield.

Figure 9 (A) Fluorescent imaging of tumor by angiogenesis in the anesthetized mouse injected intravenously with PEG1100-grafted multimodal QD⁶⁵⁵ (1.6 nmol/kg bodyweight, single dose). Dashed lines indicate liver area and tumor area (including angiogenic network). The images were obtained 15 minutes after injection using an IVIS^® imaging system, with excitation at 450 ± 30 nm and emission at 650 nm (DsRed filter). The images were obtained on day 10 after inoculation (1 × 10⁵ cells in 10 μL). (B) Fluorescence intensity in tumor area, liver area, and other parts of the body (background fluorescence) after intravenous injection of PEG1100-grafted multimodal QD⁶⁵⁵ (1.6 nmol/kg bodyweight, single dose). Data were calculated from the images in A.

Abbreviations: QD, quantum dot; PEG, polyethylene glycol.

Figure 10 T₁-weighted magnetic resonance images of colon cancer in the anesthetized mouse injected intravenously with PEG1100-grafted multimodal QD⁶⁵⁵ embedded with gadolinium (1 μmol/kg bodyweight, single dose). Dashed lines indicate tumor area (including angiogenic vasculature). The images were obtained immediately after injection, using 7.0 Tesla magnetic resonance imaging. Imaging parameters: spin echo sequence with fat suppression preparation pulse; repetition time 400 msec; echo time 9.574 msec; field of view 32 × 32 mm; matrix size 256 × 256; slice thickness 1.0 mm; number of averages 4.

Abbreviations: QD, quantum dot; PEG, polyethylene glycol.

Figure S1 Experimental scheme of imaging of two-photon excited fluorescence in rats.

Abbreviation: QD, quantum dot.

Table S1 Physiological characteristics of rats before and after intravenous injection of polyamidoamine C12 dendrimer 0.1 nmol/kg bodyweight

Parameters	Before injection	About 20 minutes after injection	P value
Mean arterial blood pressure (mmHg)	114.0 ± 5.5	68.3 ± 8.5	<0.01
Heart rate (beats per minute)	392 ± 24	317 ± 18	<0.05
Mean diameter of detected arteries (μm)	29 ± 7	39 ± 11 (vasodilatation)	<0.05
Mean diameter of detected veins (μm)	42 ± 21	47 ± 14 (vasodilatation)	<0.05

Notes: Data are means ± standard deviations from five independent experiments. The experimental conditions are the same as in Table 1.

Table S2 Effect of PAMAM C12 dendrimers on cell viability in vitro (% from control)

Cell line	50 nM dendrimer	100 nM dendrimer	500 nM dendrimer
Jurkat	65 ± 5	37 ± 4	8 ± 4
K562	57 ± 9	31 ± 5	3 ± 1
A549	68 ± 9	40 ± 8	5 ± 1
HeLa	70 ± 8	42 ± 4	12 ± 4

Notes: The cell suspension (5 × 10⁵ cells/mL) was incubated with dendrimers over 24 hours in a humidified atmosphere, and cell viability was analyzed by flow cytometry. Cell viability in the absence of dendrimer was considered 100% (control). The data are means ± standards deviation from six independent experiments.

Table S3 Effect of water-soluble amino-functionalized CdSe/ZnS QD⁴⁸⁵ without additional organic shell on cell viability in vitro (% from control)

Cell line	10 nM QD	50 nM QD	100 nM QD	200 nM QD	500 nM QD
Jurkat	104 ± 5	98 ± 8	100 ± 2	97 ± 6	96 ± 8
K562	100 ± 3	101 ± 5	103 ± 4	97 ± 5	95 ± 5
A549	100 ± 6	103 ± 5	99 ± 8	100 ± 4	97 ± 4
HeLa	102 ± 4	98 ± 7	97 ± 6	98 ± 5	94 ± 9

Notes: The cell suspension (5 × 10⁵ cells/mL) was incubated with QDs within 24 hours at humidified atmosphere, and cell viability was analyzed by flow cytometry. The cell viability in absence of QD was considered 100% (control). Data are means ± standard deviations from six independent experiments.

Abbreviation: QD, quantum dot.

ZhelevZOhbaHBakalovaRSingle quantum dot-micelles coated with silica shell as potentially non-cytotoxic fluorescent cell tracersJ Am Chem Soc20061286324632516683790

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