Near-Infrared Fluorescence Imaging Sensor with Laser Diffuser for Visualizing Photoimmunotherapy Effects under Endoscopy.

The drug efficacy evaluation of tumor-selective photosensitive substances was expected to be enabled by imaging the fluorescence intensity in the tumor area. However, fluorescence observation is difficult during treatments that are performed during gastrointestinal endoscopy because of the challenges associated with including the fluorescence filter in the camera part. To address this issue, this study developed a device that integrates a narrow camera and a laser diffuser to enable fluorescence imaging through a forceps port. This device was employed to demonstrate that a laser diffuser with an NIR fluorescence imaging sensor could be delivered through a 3.2 mm diameter port. In addition, fluorescence images of Cetuximab-IR700 were successfully observed in two mice, and the fluorescence intensity confirmed that the fluorescence decayed within 330 s. This device is expected to have practical application as a tool to identify the optimal irradiation dose for tumor-selective photosensitive substances under endoscopy.

PET/NIR Fluorescence Bimodal Imaging for Targeted Tumor Detection.

Cancer is one of the greatest threats to human health due to late diagnosis and incomplete resection. The bimodal probe combines positron emission tomography (PET) imaging for noninvasive whole-body scanning with intraoperative near-infrared fluorescence (NIRF) surgical guidance for preoperative tumor detection, tumor resection during surgery, and postoperative monitoring. We developed a new PET/NIRF bimodal imaging agent, [68Ga]Ga-DOTA-NPC, covalently coupled to DCDSTCY and DOTA via ethylenediamine and radiolabeled with gallium-68, and investigated it in vitro and in vivo. The probe was found to be preferential for colon cancer cells due to the organic anion-transporting polypeptide1B3 (OATP1B3). PET/NIRF imaging allowed us to confirm [68Ga]Ga-DOTA-NPC as a promising probe for tumor detection, as it provides good biosafety and high-contrast tumor accumulation. Orthotopic and subcutaneous colon tumors were successfully resected under real-time NIRF guidance. [68Ga]Ga-DOTA-NPC provides highly sensitive and unlimited tissue-penetrating PET/NIRF imaging, helping to visualize and differentiate tumors from adjacent tissue.

Bioorthogonal Lanthanide Molecular Probes for Near-Infrared Fluorescence and Mass Spectrometry Imaging.

We report here the development of clickable and highly near-infrared (NIR) fluorescent lanthanide (Ln) complexes for bioorthogonal labeling of biomolecules. These azide- or alkyne-functionalized Ln complexes are hydrophilic and fluorogenic, exhibiting a strong increase of NIR fluorescence upon conjugation with biomolecules. Metabolic labeling of biomolecules with azide or alkyne, followed by click labeling with the Ln complexes, enables NIR fluorescence (NIRF) imaging of DNA, RNA, proteins, and glycans in cells. Furthermore, multicolor imaging is performed by combining click-labeling with the Ln complexes and immunostaining. In addition, the Ln complexes is compatible with click-expansion microscopy (click-ExM), which enables high-resolution NIRF imaging of cellular glycoproteins. Finally, the Ln complexes can be used for time-of-flight secondary-ion mass spectrometry (ToF-SIMS) imaging, thus achieving the first example of dual-modal imaging combining NIRF and SIMS microscopies.

Efficacy of bisphosphonates in detection of early enamel caries using NIR fluorescence imaging and inhibition of caries progression.

NIR fluorescence imaging using bisphosphonate-Indocyanine green has been indicated for early interproximal caries detection. This study assessed diagnostic accuracy of caries detection by NIR fluorescence imaging with OsteoSense 750® (OS750) in vitro and ex vivo, and to analyze the therapeutic efficacy of a bisphosphonate (Etidronate) in inhibiting enamel caries progression in vitro. Methods: Four experiments were conducted using extracted human teeth; 1) to calculate the infiltration rate of OS750 into interproximal white spot lesions using fluorescence microscope, 2) to assess diagnostic accuracy of interproximal natural white spot lesions using desktop NIR fluorescence imaging device in vitro setting, 3) to assess diagnostic accuracy of artificially created deeper enamel carious lesion (0.5 mm~1.0 mm) using NIR fluorescence image through the head-mount display in ex vivo setting, 4) to compare the progression on the enamel caries lesions treated by Etidronate, NaF and distilled-water. Diagnostic accuracy was analyzed using sensitivity, specificity and receiver operating curves (ROC). The caries progression was calculated with micro-CT and was statistically analyzed using a two-way ANOVA and the Tukey HDS post-hoc test. Results: 1) The infiltration rate of OS750 was 101.83% ± 8.66 (Min: 90.10%, Max: 133.94%). 2) The average of sensitivity and specificity in vitro setting experiments were 86.7% ± 4.4% and 70% ± 11%, respectively. The average of area under the ROC curves (AUC) was 0.883 ± 0.059 indicating excellent performance. 3) The mean sensitivity and specificity in ex vivo setting was 82.97% ± 15% and 76.78% ± 13.27% respectively. 4) The carious lesion volume treated by Etidronate was significantly smaller at post treatment-1 (p<0.05) and treatment-2 (p<0.01) than the control. There was no significant difference in lesion volume in the Etidronate and NaF group at the time point of post treatment-1. Conclusion: This study suggests that bisphosphonates contribute to both early diagnosis of enamel caries and inhibition of caries progression.

Cyanine-Dyad Molecular Probe for the Simultaneous Profiling of the Evolution of Multiple Radical Species During Bacterial Infections.

Real-time monitoring of the evolution of bacterial infection-associated multiple radical species is critical to accurately profile the pathogenesis and host-defense mechanisms. Here, we present a unique dual wavelength near-infrared (NIR) cyanine-dyad molecular probe (HCy5-Cy7) for simultaneous monitoring of reactive oxygen and nitrogen species (RONS) variations both in vitro and in vivo. HCy5-Cy7 specifically turns on its fluorescence at 660 nm via superoxide or hydroxyl radical (O2.- , . OH)-mediated oxidation of reduced HCy5 moiety to Cy5, while peroxynitrite or hypochlorous species (ONOO- , ClO- )-induced Cy7 structural degradation causes the emission turn-off at 800 nm. Such multispectral but reverse signal responses allow multiplex manifestation of in situ oxidative and nitrosative stress events during the pathogenic and defensive processes in both bacteria-infected macrophage cells and living mice. Most importantly, this study may also provide new perspectives for understanding the bacterial pathogenesis and advancing the precision medicine against infectious diseases.

Real-Time Fluorescence Imaging Using Indocyanine Green to Assess Therapeutic Effects of Near-Infrared Photoimmunotherapy in Tumor Model Mice.

BACKGROUND: Near-infrared photoimmunotherapy (NIR-PIT) is a cancer therapy that causes an increase in tumor perfusion, a phenomenon termed the super-enhanced permeability and retention effect. Currently, in vivo treatment efficacy of NIR-PIT is observable days after treatment, but monitoring would be improved by more acute detection of intratumor change. Fluorescence imaging may detect increased tumor perfusion immediately after treatment. METHODS: In the first experiment, athymic nude mouse models bearing unilateral subcutaneous flank tumors were treated with either NIR-PIT or laser therapy only. In the second experiment, mice bearing bilateral flank tumors were treated with NIR-PIT only on the left-sided tumor. In both groups, immediately after treatment, indocyanine green was injected at different doses intravenously, and mice were monitored with the Shimadzu LIGHTVISION fluorescence imaging system for 1 hour. RESULTS: Tumor-to-background ratio of fluorescence intensity increased over the 60 minutes of monitoring in treated mice but did not vary significantly in control mice. Tumor-to-background ratio was highest in the 1 mg kg-1 and 0.3 mg kg-1 doses. In mice with bilateral tumors, tumor-to-untreated tumor ratio increased similarly. CONCLUSIONS: Acute changes in tumor perfusion after NIR-PIT can be detected by real-time fluorescence imaging.

Enzyme-Triggered Disassembly of Perylene Monoimide-based Nanoclusters for Activatable and Deep Photodynamic Therapy.

Photodynamic therapy (PDT) exhibits great potential for cancer therapy, but still suffers from nonspecific photosensitivity and poor penetration of photosensitizer. Herein, a smart perylene monoimide-based nanocluster capable of enzyme-triggered disassembly is reported as an activatable and deeply penetrable photosensitizer. A novel carboxylesterase (CE)-responsive tetrachloroperylene monoimide (P1) was synthesized and assembled with folate-decorated albumins into a nanocluster (FHP) with a diameter of circa 100 nm. Once P1 is hydrolyzed by the tumor-specific CE, FHP disassembles into ultrasmall nanoparticles (ca. 10 nm), facilitating the deep tumor penetration of FHP. Furthermore, such enzyme-triggered disassembly of FHP leads to enhanced fluorescence intensity (ca. 8-fold) and elevated singlet oxygen generation ability (ca. 4-fold), enabling in situ near-infrared fluorescence imaging and promoted PDT. FHP permits remarkable tumor inhibition in vivo with minimal side effects through imaging-guided, activatable, and deep PDT. This work confirms that this cascaded multifunctional control through enzyme-triggered molecular disassembly is an effective strategy for precise cancer theranostics.

Intraoperative fluorescence imaging in thoracic surgery.

Intraoperative fluorescence imaging (IFI) can improve real-time identification of cancer cells during an operation. Phase I clinical trials in thoracic surgery have demonstrated that IFI with second window indocyanine green (TumorGlow® ) can identify subcentimeter pulmonary nodules, anterior mediastinal masses, and mesothelioma, while the use of a folate receptor-targeted near-infrared agent, OTL38, can improve the specificity for diagnosing tumors with folate receptor expression. Here, we review the existing preclinical and clinical data on IFI in thoracic surgery.

A Near-infrared Fluorescence and Positron Emission Tomography Bimodal Probe for In Vivo Imaging of Amyloid-ß Species.

Noninvasive imaging of amyloid-ß (Aß) species in vivo is important for the early diagnosis of Alzheimer's disease (AD). In this paper, we report a near-infrared (NIR) fluorescence (FL) and positron emission tomography (PET) bimodal probe (NIR-[68Ga]) for in vivo imaging of both soluble and insoluble Aß species. NIR-[68Ga] holds a high binding affinity, high selectivity and high sensitivity toward Aß42 monomers, oligomers, and aggregates in vitro. In vivo imaging results show that NIR-[68Ga] can cross the blood-brain-barrier (BBB), and produce significantly higher PET and NIR FL bimodal signals in the brains of APP/PS1 transgenic AD mice relative to that of age-matched wild-type mice, which are also validated by the ex vivo autoradiography and histological staining images. Our results demonstrate that NIR-[68Ga] is an efficient NIR FL and PET bimodal probe for the sensitive imaging of soluble and insoluble Aß species in AD mice.

2nd Window NIR Imaging of Radiation Injury Mitigation Provided by Reduced Notch-Dll4 Expression on Vasculature.

PURPOSE: Vascular endothelium plays a central role in the pathogenesis of acute and chronic radiation injuries, yet the mechanisms which promote sustained endothelial dysfunction and contribute to late responding organ failure are unclear. We employed 2nd window (> 1100 nm emission) Near-Infrared (NIR) imaging using indocyanine green (ICG) to track and define the role of the notch ligand Delta-like ligand 4 (Dll4) in mediating vascular injury in two late-responding radiosensitive organs: the lung and kidney. PROCEDURES: Consomic strains of female Salt Sensitive or SS (Dll4-high) and SS with 3rd chromosome inherited from Brown Norway, SS.BN3 (Dll4-low) rats at ages 11-12 weeks were used to demonstrate the impact of reduced Dll4 expression on long-term vascular integrity, renal function, and survival following high-dose 13 Gy partial body irradiation at 42- and 90 days post-radiation. 2nd window dynamic NIR fluorescence imaging with ICG was analyzed with physiology-based pharmacokinetic modeling and confirmed with assays of endothelial Dll4 expression to assess the role of endogenous Dll4 expression on radiation injury protection. RESULTS: We show that SS.BN3 (Dll4-low) rats are relatively protected from vascular permeability disruption compared to the SS (Dll4-high) strain. We further demonstrated that SS.BN3 (Dll4-low) rats have reduced radiation induced loss of CD31+ vascular endothelial cells, and increased Dll4 vascular expression is correlated with vascular dysfunction. CONCLUSIONS: Together, these data suggest Dll4 plays a key role in pathogenesis of radiation-induced vascular injury to the lung and kidney.

Real-Time Objective Evaluation of the Ischemic Stroke through pH-Responsive Fluorescence Imaging.

A rapid and comprehensive assessment of ischemic stroke (IS) is critical for clinicians to take the most appropriate treatment. Currently, IS assessment is mainly carried out by computed tomography and magnetic resonance imaging in combination with observing the clinical symptoms and inquiring about contraindications. However, they cannot diagnose pathological conditions and judge the microenvironment in real-time. Near-infrared fluorescence imaging has advantages for IS imaging, such as high sensitivity, high spatiotemporal resolution, and straightforward real-time operation. Herein, a pH-responsive fluorescent liposomal probe (BOD@Lip) is prepared for in vivo real-time visualization of the degree of IS based on the different acid microenvironments in the progression of the disease. The fluorescence imaging with BOD@Lip shows the degree of IS, and the correlation between fluorescence signals and the neurological deficit scores is established for the first time. This work provides a new method to objectively evaluate the degree of IS through a visualized route and a new insight into the relationship between the acidic microenvironment and the progression of IS.

Inhibition of Pro-Survival Autophagy Induced by Rare-Earth Nanocomposites for Promoting Photothermal Therapy of Visualized Tumors.

Manipulation of autophagic processes has emerged as a promising strategy for synergizing nanoagent-mediated photothermal therapy (PTT). Most of the current studies focus on improving PTT efficacy by inhibiting pro-survival autophagy induced by the heat generated from the photothermal process. However, autophagy induced by the nanoagents is usually ignored, which may weaken the effect of autophagy-mediated efficacy improvement in PTT if induced autophagy is pro-death. Therefore, this work aims at developing a nanoagent that is able to induce heat-synergetic pro-survival autophagy to optimize the efficacy of PTT. An approach is developed to coat carbon layer, polyethylenimine (PEI), and folic acid (FA) on NaYF4 :Er,Yb,Nd@NaNdF4 (DCNPs@C@PEI@FA, DCPF) nanoparticles successively, giving access to the nanoagent to induce pro-survival autophagy. The synthetic imaging-guided photothermal nanoagent displays outstanding targeting ability and biocompatibility based on the surface modification of PEI and FA. By using an autophagy inhibitor chloroquine, a conspicuously synergistic effect on DCPF-mediated PTT in vitro and in vivo tumor models (HeLa) is achieved. A promising strategy is presented here to enhance the efficacy of imaging-guided PTT by modulating the autophagy induced by the nanoagent.

Near-infrared Fluorescence Imaging for Detecting Pancreatic Liver Metastasis in an Orthotopic Athymic Mouse Model.

BACKGROUND/AIM: Evidence of metastatic disease precludes oncological resection of pancreatic cancer. Near-infrared (NIR) fluorescent labels, such as indocyanine green (ICG), assist in the intraoperative detection of occult and micrometastatic liver disease. The present study aimed to analyse the role of NIR fluorescence imaging using ICG for pancreatic liver disease as proof of concept in an orthotopic athymic mouse model. MATERIALS AND METHODS: Pancreatic ductal adenocarcinoma was induced by injecting L3.6pl human pancreatic tumour cells into the pancreatic tail of seven athymic mice. After four weeks of tumour growth, ICG was injected into the tail vein and NIR fluorescence imaging was performed at harvest to determine tumour-to-liver ratios (TLR) using Quest Spectrum® Fluorescence Imaging Platform. RESULTS: Pancreatic tumour growth and liver metastasis could be visually confirmed for all seven animals. None of the hepatic metastases showed any detectable ICG-uptake. ICG-staining failed to visualize the liver metastases or to increase fluorescence intensity of the rim around the hepatic lesions. CONCLUSION: ICG-staining fails to visualize liver metastases induced by L3.6pl pancreatic tumour cells in athymic nude mice by NIR fluorescence imaging. Further studies are necessary to delineate the underlying mechanism for insufficient ICG uptake in these pancreatic liver metastases and for the lack of a fluorescent rim around the liver lesions.

Near-infrared fluorophores methylene blue for targeted imaging of the stomach in intraoperative navigation.

Near-infrared (NIR) fluorescence imaging-guided surgery is increasingly concerned in gastrointestinal surgery because it can potentially improve clinical outcomes. This new technique can provide intraoperative image guidance for surgical margin evaluation and help surgeons examine residual lesions and small tumors during surgery. NIR fluorophores methylene blue (MB) is a promising fluorescent probe because of its safety and intraoperative imaging in the clinic. However, whether MB possesses the potential to perform intraoperative navigation of the stomach and gastric tumors needs to be further explored. Therefore, the current study mainly validated MB's usefulness in animal models' intraoperative imaging of stomach and gastric tumors. NIR fluorophores MB can exhibit specific uptake by the gastric epithelial cells and cancer cells. It is primarily found that MB can directly target the stomach in mice. Interestingly, MB was applied for the NIR imaging of gastric cancer cell xenografts, suggesting that MB cannot specifically target subcutaneous and orthotopic gastric tumors in xenograft models. Thus, it can be concluded that MB has no inherent specificity for gastric tumors but specificity for gastric tissues. Apparently, MB-positive and negative NIR imaging are meaningful in targeting gastric tissues and tumors. MB is expected to represent a helpful NIR agent to secure precise resection margins during the gastrectomy and resection of gastric tumors.

Novel selenium-containing photosensitizers for near-infrared fluorescence imaging-guided photodynamic therapy.

Benzopyran nitrile dyes cannot be used as qualified photosensitizers due to the low quantum yield of triplet state. The benzopyran derivatives containing selenium instead of oxygen atom based on the heavy atom effect are expected to become potential agents for photodynamic therapy. In this paper, a series of selenium-containing photosensitizers (PSX) were prepared according to this strategy. PSX can effectively produce both singlet oxygen and superoxide anions upon laser irradiation. PSX exhibited the emission wavelength at 500-800 nm and near-infrared (NIR) fluorescence imaging in HeLa cells. Excellent biocompatibility and phototoxicity further indicated that PSX could be used as efficient photosensitizers for NIR fluorescence imaging and photodynamic therapy.

Hexa-BODIPY-cyclotriphosphazene based nanoparticle for NIR fluorescence/photoacoustic dual-modal imaging and photothermal cancer therapy.

Theranostic, which integrates the diagnosis and tumor treatment in tandem, is an emerging strategy in cancer treatment. Here, we report a novel and unique theranostic nanoparticle, HBCP NP, based on hexa-BODIPY cyclophosphazene (HBCP). Due to the unique bulky molecular structure of HBCP, this nanoparticle can simultaneously perform near-infrared (NIR) fluorescence imaging and photoacoustic imaging (PAI). Interestingly, since reactive oxygen species (ROS) generation of HBCP NPs is completely inhibited, 'safe' fluorescence imaging is possible without the risk of cell damage even under laser irradiation. Finally, NIR fluorescence imaging and PAI in 4T1 tumor-bearing mice demonstrated selective accumulation of HBCP NPs at tumor sites. In addition, HBCP NPs exhibited excellent photothermal effects under high-power laser irradiation, achieving effective tumor growth inhibition.

Development and Preclinical Evaluation of a Near-Infrared Fluorescence Probe Based on Tailored Hepatitis B Core Particles for Imaging-Guided Surgery in Breast Cancer.

Purpose: Tumor-free surgical margin is crucial but challenging in breast-conserving surgery (BCS). Fluorescence imaging is a promising strategy for surgical navigation that can reliably assist the surgeon with visualization Of the tumor in real-time. Notably, finding an optimized fluorescent probe has been a challenging research topic. Herein, we developed a novel near-infrared (NIR) fluorescent probe based on tailored Hepatitis B Core virus-like protein (HBc VLP) and presented the preclinical imaging-guided surgery. Methods: The RGD-HBc160 VLP was synthesized by genetic engineering followed encapsulation of ICG via disassembly-reassembly. The applicability of the probe was tested for cell and tissue binding capacities through cell-based plate assays, xenograft mice model, and MMTV-PyVT mammary tumor transgenic mice. Subsequently, the efficacy of RGD-HBc160/ICG-guided surgery was evaluated in an infiltrative tumor-bearing mouse model. The protein-induced body's immune response was further assessed. Results: The prepared RGD-HBc160/ICG showed outstanding integrin αvß3 targeting ability in vitro and in vivo. After intravenous administration of probe, the fluorescence guidance facilitated more complete tumor resection and improved overall survival Of the infiltrative tumor-bearing mice. The probe also showed the excellent capability to differentiate between benign and malignant breast tissues in the mammary tumor transgenic mice. Interestingly, the ingenious tailoring of HBc VLP could not only endow its tumor-targeting ability towards integrin αvß3 but also significantly reduce the humoral and cellular immune response. Conclusion: The RGD-HBc160/ICG holds promise as an effective tool to delineate tumor margin. These results have translational potential to achieve margin-negative resection and improve the stratification of patients for a potentially curative.

A NIR-II emissive polymer AIEgen for imaging-guided photothermal elimination of bacterial infection.

The development of antibiotics resistance has made multidrug-resistant (MDR) bacterial infection one of the most serious global health issues. Photothermal therapy (PTT) is an emerging therapeutic mode which can be applied to bacterial infection without inducing resistance. Moreover, enhanced therapeutic efficacy and less tissue damage can be realized with NIR-II fluorescence imaging (FLI) guided PTT. Herein, a polymeric luminogen with aggregation-induced emission (AIEgens) characteristics, poly(dithieno[3,2-b:2',3'-d]pyrrole-benzo[1,2-c:4,5-c']bis([1,2,5]thiadiazole)) (PDTPTBT), was synthesized and used as a photothermal agent for PTT of bacterial infections. PDTPTBT was encapsulated into liposomes (L-PDTPTBT) for improved water dispersibility. Upon 808 nm NIR irradiation, L-PDTPTBT can eliminate multiple bacteria including the Gram-positive methicillin-resistant Staphylococcus aureus and Enterococcus faecalis, the Gram-negative Escherichia coli and Pseudomonas aeruginosa. Serious damage of bacterial membrane and leakage of cytoplasm is observed after photothermal treatment using L-PDTPTBT. The potential of the formulation has been demonstrated in two infected animal models: (i) a subcutaneous abscess model and (ii) a diabetic skin infection model. In the diabetic skin infection model, the death of mice is largely suppressed and the wounds can heal more quickly with treatment of L-PDTPTBT under NIR irradiation. The excellent photothermal bactericidal ability and low cytotoxicity make L-PDTPTBT potential candidate for treating MDR bacterial infections in the future.

A novel system for analyzing indocyanine green (ICG) fluorescence spectra enables deeper lung tumor localization during thoracoscopic surgery.

Background: Palpation of tumors during thoracoscopic surgery remains difficult, and identification of deep-seated tumors may be impossible. This preclinical study investigated the usefulness of a novel indocyanine green (ICG) fluorescence spectroscopy system for tumor localization. Methods: ICG was diluted to 5.0×10-2 mg/mL in fetal bovine serum (FBS) and mixed with silicone resin to prepare pseudo-tumors. Sponges of different densities and a porcine lung were placed on top of the pseudo-tumors, which were examined using a novel fluorescence spectroscopy system and a near-infrared (NIR) camera. Spectra were measured for different sponge and lung thicknesses, and the lung spectra were measured during both inflation and deflation. Results: The fluorescence spectroscopy system was able to identify tumors at depths ≥15 mm, while the NIR system was not. The spectroscopy system also detected tumors at greater depths when the density of the intervening material was lower. Depending on the density and thickness of the intervening material, the system could detect spectra as deep as 40 mm for sponges and 30 mm for lungs. Conclusions: This new fluorescence spectroscopy system can be used to identify lung tumors up to a depth of 30 mm in experiments using pseudo-tumors and a porcine lung, which may aid in tumor identification during thoracoscopic surgery.

Thermo- and pH-sensitive nanoparticles of poly (N-isopropylacrylamide-decenoic acid-1-vinylimidazole) for ultrasound switchable fluorescence imaging.

We herein report the synthesis of poly (9-decenoic acid-1-vinylimidazole-N-isopropylacrylamide) nanoparticles containing indocyanine green (ICG) in one pot and in water phase throughout the reaction. We have shown that copolymers of 9-decenoic acid and 1-vinylimidazole, or 9-decenoic acid alone, have an enhanced sensitivity to pH values between 7.4 and 6.8 and are superior to the widely used acrylic acid. We have also shown that incorporation of acidic comonomers leads to the favorable outcome of a higher fluorescence signal intensity in lower pH values, whereas the opposite is true of basic comonomers, where the fluorescence signal intensity is lower at low pH values. It was shown that to keep the pH response favorable the molar ratio of basic comonomers to acidic comonomers should roughly equal 1:4. We controlled the lower critical solution temperature (LCST) of the nanoparticles from around 30 to 38°C for different applications by adding acrylamide comonomers. Finally, the nanoparticles at varying pH values, when imaged by an ultrasound switchable fluorescence (USF) imaging system, showed pH sensitivity and thermosensitivity at physiological and tumor pH.