Figure 1.
Principle of in vivo integrated PA-PT nano-theranostics of bacteria in blood and distant infected sites.
Figure 2.
Label free PA diagnosis and PT therapy of S. aureus.
(a) Linear absorption (yellow curve) and nonlinear PT spectra of bacteria in solution (106 CFU/mL) in 1 cm optical pathway at two energy fluence levels: 1.8 J/cm2 (blue curve) and 4.8 J/cm2 (red curve). The average standard deviation in PT data for each wavelength at automated laser spectral scanning was 21%. (b) High-resolution optical image (top, left) and linear PT signal (top, right) from single intact S. aureus at laser wavelength of 740 nm and low energy fluence (0.9 J/cm2), and high-resolution optical image of photodamaged S. aureus (bottom, left) and nonlinear PT signal associated with laser-induced nanobubbles at high laser fluence of 10 J/cm2 (bottom, right). The intact bacteria had round shapes compared to irregular shapes of the photodamaged bacterium. (c) In vivo label-free PA detection of bacteria in mouse ear (left) showing increase of PA signal (right, top) compared to control measurement without bacteria (right, bottom). The infected site was modeled by injection of 20-µL bacteria suspension in concentration of 104 CFC/mL in mouse ear. Laser parameter: wavelength, 741 nm; energy fluence, 1 J/cm2. (d) Laser irradiation of ∼300-µm abdominal blood vessel (callout) in skin-fold mouse model followed by detection of acoustic waves with the ultrasound transducer closely attached to laser beam.
Figure 3.
Schematic of label-free and targeted theranostics of S. aureus.
(a) Absorption spectra of blood (red), skin (orange), S. aureus (yellow), and different NPs: GNTs, GNRs (green), and siMNPs (blue). Dashed and solid lines indicate averaged linear and nonlinear PA spectra, respectively. (b) Multiplex targeting S. aureus surface biomarker protein A (Spa) and lipoprotein (Lpp) by siMNPs, GNRs and GNTs functionalized with either anti-Spa or anti-Lpp Abs. (c) Atomic force microscopy (AFM) images of GNTs on the surface of an S. aureus cell (top) and transmission electronic microscopy (TEM) images of single and clustered siMNPs with a ∼30-nm magnetic core and ∼10-nm silica layer (bottom, left and right, respectively). Scale bars, 10 nm.
Figure 4.
Assessment of systemic toxicity with Ab-conjugated GNRs in kidney and liver.
The 1× and 10× designations refer to approximately 1011 and 1012 Ab-conjugated GNR NPs, respectively, injected into the bloodstream of mice via the tail vein. Samples from liver and kidney at 24 h after NP injections were assayed for evidence of apoptotic and/or necrotic damage using the TUNEL assay (green). Nuclei were counterstained with DAPI (blue). Cisplatin (20 mg/kg) was used as a positive control for kidney damage, while acetaminophen (300 mg/kg) was used as a positive control for liver damage.
Figure 5.
Molecular targeting of S. aureus with fluorescent dyes and siMNPs functionalized with anti-Spa and anti-Lpp Abs.
(a–c) Optical (a) and fluorescence images of bacteria targeted with FITC-labeled anti-Spa Abs (green, b) or phycoerythrin (PE)-labeled anti-Lpp (SA0486) Abs (red, c). (d) Overlaid image that confirms co-localization of Abs. Green and red arrows indicate Spa-positive and Lpp-positive bacteria, respectively, and the white arrow indicates a rare unlabeled bacterium. Scale bars, 5 µm.
Table 1.
Labeling efficiency (%) of S. aureus (1 h, 37°C) in static condition in vitro obtained with fluorescent and PA (in brackets) techniques.
Figure 6.
Giant nonlinear signal amplification in single siMNPs and magnet-induced siMNP clusters.
(a) Nonlinear PA signal amplification from siMNPs (red curve) compared to MNPs without a silica layer (blue curve) at 639 nm. (b) Nonlinear PA spectra of siMNPs (blue and red curves) at 0.3 and 5 J/cm2 energy fluences. Black curve indicates the linear absorption spectrum. PA spectra were normalized on absorption spectra at 800 nm. The error bars represent the standard deviations in five measurements. (c–h) Optical (c, d) and fluorescent (f, d) images of many (c, f) and two (d, g) S. aureus labeled by 30-nm siMNPs with FITC, and PT signals from two labeled bacteria before (e) and after (h) magnet exposure for 3 minutes. Laser parameters: wavelength, 640 nm; energy fluence, 200 mJ/cm2; pulse rate, 10 Hz.
Figure 7.
In vivo detection of CBCs with Ab-conjugated NPs and magnet-induced CBC capturing and signal amplification.
(a) In vivo PA monitoring of CBCs labeled with Ab-functionalized NPs in vitro prior to injection. (b) PA monitoring of CBCs labeled in vivo with a cocktail of functionalized NPs consisting of GNRs690 with anti-Lpp Ab and GNRs900 with anti-Spa Ab in a 50%∶50% proportion in 20 µL of phosphate-buffered saline (n = 3). The inset shows typical PA signal traces. Dashed line indicates PA monitoring observed when the same cocktail was used to label S. aureus cells in vitro prior to injection (similar to Fig. 7a). The average standard deviation for each time point in (a) and (b) is 18% (c) Verification of magnetic amplification of PA signals from bacteria labeled with 30-nm MNPs with anti-Spa Ab in rat mesentery. The inset shows typical PA signal traces. (d) In vivo real-time monitoring of PA signal dynamic from bacteria labeled in vivo by siMNPs with anti-Spa Ab before, during, and after magnetic field action in mouse ear. Laser parameters: wavelengths, 671 nm and 820 nm (a, b), 639 nm (c), 671 nm (d); energy fluence, 0.1 J/cm2.
Figure 8.
In vivo monitoring of labeled bacteria in tissue.
(a) In vivo monitoring of bacterial extravasation from a mouse ear vein. (b) Arrest of circulating bacteria in mouse tibial bone labeled in vitro with 30-nm MNPs functionalized with FITC-conjugated Abs (left). The presence of bacteria in bone was confirmed by the appearance of PA signals after fluorescence-guided laser irradiation of green spots (right, top) compared to background signals from bone alone (right, bottom). (c) PT map of a 120-µm slide containing blood spiked with bacteria labeled with anti-Spa-Ab-conjugated GNTs900. Black peaks are associated with single bacteria as verified by optical imaging (callout). Laser parameters: wavelengths, 639 nm (a, b, c); energy fluence, 0.1 J/cm2.
Figure 9.
Molecular diagnosis and targeted eradication of S. aureus in blood with real-time monitoring of therapeutic efficacy.
(a) Two-color (639 and 850 nm) monitoring of PT (left) and PA (right) linear (top) and nonlinear (bottom) signals from CBCs labeled with anti-Spa-conjugated siMNPs. (b) In vivo real-time monitoring of the efficacy of PT nanotherapy of targeted CBCs. The average standard deviation in PA data for each time point at automated data collection was 19%. (c) Numbers of viable bacteria in blood samples was determined by plate count as a function of laser irradiation.
Figure 10.
Molecular diagnosis and targeted eradication of S. aureus in tissue with real-time monitoring of therapeutic efficacy.
(a) PA control of targeted PT purging of an infected area in mouse ear (left) by comparison of signals before (right, top) and after (right, bottom) PT therapy at 850 nm (0.8 J/cm2). (b) Targeted PT therapy of a secondary infection in the liver.