Enhanced Delivery of Radiolabeled Polyoxazoline into Tumors via Self-Aggregation under Hyperthermic Conditions.

In order to develop a radiopharmaceutical for internal radiotherapy that had a high anticancer effect while exposing normal tissues to low radiation levels, we synthesized a radiolabeled polyoxazoline (POZ), a thermoresponsive polymer, and established a novel drug delivery system for targeting tumors by accelerating the accumulation of the radiolabeled POZ via self-aggregation under hyperthermic (42-43 °C) conditions. By living-cationic polymerization using 2-ethyl-2-oxazoline and 2-isopropyl-2-oxazoline, POZ derivatives (Et-IspPOZ) (10, 20, and 30 kDa) with lower critical solution temperatures (LCSTs) of 37-38 °C were synthesized; the POZ derivatives were soluble at the body temperature but self-aggregated upon heat treatment (42-43 °C). Next, the indium-111 (111In)-labeled Et-IspPOZ was prepared, and the effect of molecular weight and injected POZ dose on the accumulation of radioactivity in the tumors was investigated upon intravenous injection of probes under hyperthermic conditions in colon 26-bearing mice. The uptake of radioactivity in tumors was increased when the molecular weight of POZ was greater than 20 kDa, while it was independent of the injected POZ dose (4-40 nmol). The amount of radioactivity retained in the tumor did not change for up to 3 h after exposure to heat treatment was stopped. Furthermore, the tumor uptake of the Et-IspPOZ derivative with an LCST greater than 42 °C was significantly lower than that of Et-IspPOZ, which had an LCST of 37-38 °C, suggesting the involvement of the self-aggregation of POZ on tumor uptake. Finally, the intratumoral localization of fluorescence-labeled Et-IspPOZ was evaluated using in vivo confocal laser microscopy. Many bright fluorescence spots were observed in the heat-treated tumors nearby and within blood vessels. In conclusion, the high tumor uptake of radiolabeled Et-IspPOZ was elucidated under hyperthermic conditions; thereby, the possibility of developing a novel internal radiotherapy using radiolabeled POZ derivatives was demonstrated.

Indocyanine Green-Labeled Polysarcosine for in Vivo Photoacoustic Tumor Imaging.

Photoacoustic (PA) imaging has been considered an attractive imaging modality for sensitive and in-depth imaging of biomolecules with a high resolution in vivo. PA imaging probes utilizing fluorescence dyes, including indocyanine green (ICG), have been proposed to enhance PA signal intensity. On the other hand, nanomicelles modified with polysarcosine (PSar), a biocompatible hydrophilic polymer, on their surface have previously achieved rapid tumor uptake, suggesting active transport of PSar into tumor tissues. Thus, we hypothesized that PSar-based materials might be utilized as diagnostic probes for targeting tumors and therefore evaluated the potential of PSar labeled with an ICG derivative, ICG-PSar, as a PA imaging probe for targeting cancer. In this study, ICG-PSars with differing molecular weights (10, 20, and 30 kDa) were synthesized. In vitro cellular uptake studies using ICG-PSar demonstrated rapid uptake in colon26 tumor cells partially via macropinocytosis-mediated endocytosis. In vivo fluorescence imaging and biodistribution study indicated that ICG-PSar30k exhibited high accumulation in the tumor (8.4% dose/g), with high tumor-to-blood ratios reaching 4.6 at 24 h post injection of the probe. Finally, in vivo PA imaging studies showed that PA signal increased in tumors (251%) but not in blood vessels, achieving high contrast tumor imaging at 24 h after ICG-PSar30k probe injection. These results suggest that ICG-PSar has potential as a tumor-targeting PA imaging probe.

In vivo photoacoustic imaging of cancer using indocyanine green-labeled monoclonal antibody targeting the epidermal growth factor receptor.

Photoacoustic (PA) imaging is an attractive imaging modality for sensitive and depth imaging of biomolecules with high resolution in vivo. The aim of this study was to evaluate the effectiveness of an anti-epidermal growth factor receptor (EGFR) monoclonal antibody (panitumumab; Pan) labeled with indocyanine green derivative (ICG-EG4-Sulfo-OSu), Pan-EG4-ICG, as a PA imaging probe to target cancer-associated EGFR. In vitro PA imaging studies demonstrated that Pan-EG4-ICG yielded high EGFR-specific PA signals in EGFR-positive cells. To determine the optimal injection dose and scan timing, we investigated the biodistribution of radiolabeled Pan-EG4-ICG (200-400 µg) in A431 tumor (EGFR++)-bearing mice. The highest tumor accumulation (29.4% injected dose/g) and high tumor-to-blood ratio (2.1) was observed 7 days after injection of Pan-EG4-ICG (400 µg). In in vivo PA imaging studies using Pan-EG4-ICG (400 µg), the increase in PA signal (114%) was observed in A431 tumors inoculated in the mammary glands 7 days post-injection. Co-injection of excess Pan resulted in a 35% inhibition of this PA signal, indicating the EGFR-specific accumulation. In conclusion, the ICG-labeled monoclonal antibody (i.e., panitumumab) has the potential to enhance target-specific PA signal, leading to the discrimination of aggressiveness and metastatic potential of tumors and the selection of effective therapeutic strategies.

Development of anti-HER2 fragment antibody conjugated to iron oxide nanoparticles for in vivo HER2-targeted photoacoustic tumor imaging.

Photoacoustic (PA) imaging is a promising imaging modality that provides biomedical information with high sensitivity and resolution. Iron oxide nanoparticles (IONPs) have been regarded as remarkable PA contrast agents because of their low toxicity and biodegradable properties. However, IONP delivery is restricted by its modest leakage and retention in tumors. In this study, we designed IONPs (20nm, 50nm, and 100nm) conjugated with anti-HER2 moieties [whole IgG, single-chain fragment variable (scFv), and peptide] for HER2-targeted PA tumor imaging. The binding affinity, cellular uptake, and in vivo biodistribution were examined. We propose 20-nm anti-HER2 scFv-conjugated IONPs (SNP20) as a novel PA contrast agent. SNP20 demonstrated high affinity and specific binding to HER2-expressing cells; it selectively visualized HER2-positive tumors in PA imaging studies. These data indicate that SNP20 is a potential PA contrast agent for imaging of HER2-expressing tumors. FROM THE CLINICAL EDITOR: Iron oxide nanoparticles have been demonstrated to be good contrast agents for tumor imaging. They may also be useful in photoacoustic (PA) imaging, which can provide high sensitivity data and image resolution. The authors here coupled iron oxide nanoparticles with anti-HER2 antibody fragment and showed significant retention of these nanoparticles in tumors. This combination may provide another option for enhanced imaging of tumors.

Biomimetic engineering of modular bispecific antibodies for biomolecule immobilization.

Modular bispecific antibodies (BsAb's) that interact directly with a gold surface were engineered for immobilization on biosensing devices. The BsAb's consist of the variable fragments of antigold and antilysozyme antibodies connected via one of three linkers derived from naturally occurring proteins. The BsAb's were bound tightly to both the gold surface and to lysozyme, thus functioning as interface molecules between lysozyme and the gold surface without a substantial loss of antigen-binding activity. The antigen-binding capacity (the ratio of the amount of immobilized lysozyme to the amount of immobilized BsAb) on the gold surface reached 82%. An analysis of the correlation between binding capacity and linker characteristics indicated that the presence of a long, rigid linker sequence derived from a cellulase resulted in a higher antigen-binding capacity than did the presence of a long but relatively flexible glycine-rich linker. This result suggests a strategy for designing linkers suitable for BsAb-based biomolecular immobilization.

Sensitive Photoacoustic/Magnetic Resonance Dual Imaging Probe for Detection of Malignant Tumors.

In order to completely remove tumors in surgeries, probes are needed both preoperatively and intraoperatively. For tumor diagnosis, magnetic resonance imaging (MRI) has been widely used as a precise preoperative method, and photoacoustic imaging (PAI) is a recently emerged intraoperative (or preoperative) method, which detects ultrasonic waves thermoelastically induced by optical absorbers irradiated by laser. Iron oxide nanoparticles (IONPs) can be used as both MR and PA imaging probes. In order to improve the sensitivity of IONPs as MR/PA imaging probes, we newly prepared liposomes encapsulated with a number of IONPs (Lipo-IONPs). Interestingly, Lipo-IONPs showed 2.6 and 3.8-times higher PA and MR signals, respectively, compared to dispersed IONPs at the same concentration. Furthermore, trastuzumab (Tra) (anti-human epidermal growth factor receptor 2 (EGFR2; HER2) monoclonal antibody) was introduced onto the surface of liposomes for detection of HER2 related to tumor malignancy. In an cellular uptake study, Tra-Lipo-IONPs were taken up by HER2-positive tumor cells and HER2-specific MR/PA dual imaging was achieved. Finally, a biodistribution study using radiolabeled Tra-Lipo-IONPs showed HER2-specific tumor accumulation. In conclusion, we demonstrated the usefulness of Lipo-IONPs as platforms for sensitive MR/PA dual imaging and the possibility of HER2-specific tumor MR/PA imaging using Tra-Lipo-IONPs.

Brachytherapy with Intratumoral Injections of Radiometal-Labeled Polymers That Thermoresponsively Self-Aggregate in Tumor Tissues.

Brachytherapy is a type of radiotherapy wherein titanium capsules containing therapeutic radioisotopes are implanted within tumor tissues, enabling high-dose radioirradiation to tumor tissues around the seeds. Although marked therapeutic effects have been demonstrated, brachytherapy needs a complicated implantation technique under general anesthesia and the seeds could migrate to other organs. The aim of this study was to establish a novel brachytherapy using biocompatible, injectable thermoresponsive polymers (polyoxazoline [POZ]) labeled with 90Y, which can self-aggregate above a specific transition temperature (Tt), resulting in long-term intratumoral retention of radioactivity and therapeutic effect. Therefore, we evaluated the tumor retention of radiolabeled POZ derivatives and their therapeutic effects. Methods: Using oxazoline derivatives with ethyl (Et), isopropyl (Isp), and propyl (Pr) side chains, we synthesized EtPOZ, IspPOZ, Isp-PrPOZ (heteropolymer), and PrPOZ and measured their characteristic Tts. The intratumoral retention of 111In-labeled POZ was evaluated until 7 d after injection in nude mice bearing PC-3 human prostate cancer. The intratumoral localization of 111In-labeled POZ derivatives was investigated by an autoradiographic study. Furthermore, a therapeutic study using 90Y-labeled Isp-PrPOZ was performed, and tumor growth and survival rate were evaluated. Results: The Tts of EtPOZ, IspPOZ, Isp-PrPOZ, and PrPOZ (∼20 kDa) were greater than 70°C, 34°C, 25°C, and 19°C, respectively. In the intratumoral injection study, Isp-PrPOZ and PrPOZ (2,000 µM) with Tts lower than tumor temperature (33.5°C under anesthesia) showed a significantly higher retention of radioactivity at 1 d after injection (73.6% and 73.9%, respectively) than EtPOZ (5.6%) and IspPOZ (15.8%). Even at low injected dose (100 µM), Isp-PrPOZ exhibited high retention (68.3% at 1 d). The high level of radioactivity of Isp-PrPOZ was retained in the tumor 7 d after injection (69.5%). The autoradiographic study demonstrated that the radioactivity of 111In-labeled Isp-PrPOZ and PrPOZ was localized in a small area. In the therapeutic study using 90Y-labeled Isp-PrPOZ, significant suppression of tumor growth and prolonged survival rate were achieved in an injection dose-dependent manner compared with that observed for the vehicle-injected group and nonradioactive Isp-PrPOZ-injected group. Conclusion: The injectable 90Y-labeled Isp-PrPOZ was retained for a prolonged period within tumor tissues via self-aggregation and exhibited marked therapeutic effect, suggesting its usefulness for brachytherapy.

Polyoxazoline multivalently conjugated with indocyanine green for sensitive in vivo photoacoustic imaging of tumors.

Photoacoustic imaging, which enables high-resolution imaging in deep tissues, has lately attracted considerable attention. For tumor imaging, photoacoustic probes have been proposed to enhance the photoacoustic effect to improve detection sensitivity. Here, we evaluated the feasibility of using a biocompatible hydrophilic polymer, polyoxazoline, conjugated with indocyanine green (ICG) as a tumor-targeted photoacoustic probe via enhanced permeability and retention effect. ICG molecules were multivalently conjugated to partially hydrolyzed polyoxazoline, thereby serving as highly sensitive photoacoustic probes. Interestingly, loading multiple ICG molecules to polyoxazoline significantly enhanced photoacoustic signal intensity under the same ICG concentration. In vivo biodistribution studies using tumor bearing mice demonstrated that 5% hydrolyzed polyoxazoline (50 kDa) conjugated with ICG (ICG/polyoxazoline = 7.8), P14-ICG7.8, showed relatively high tumor accumulation (9.4%ID/g), resulting in delivery of the highest dose of ICG among the probes tested. P14-ICG7.8 enabled clear visualization of the tumor regions by photoacoustic imaging 24 h after administration; the photoacoustic signal increased in proportion with the injected dose. In addition, the signal intensity in blood vessels in the photoacoustic images did not show much change, which was attributed to the high tumor-to-blood ratios of P14-ICG7.8. These results suggest that polyoxazoline-ICG would serve as a robust probe for sensitive photoacoustic tumor imaging.

In Vivo HER2-Targeted Magnetic Resonance Tumor Imaging Using Iron Oxide Nanoparticles Conjugated with Anti-HER2 Fragment Antibody.

PURPOSE: The feasibility of iron oxide nanoparticles (IONPs) conjugated with anti-epidermal growth factor receptor 2 (HER2) single-chain antibody (scFv-IONPs) as novel HER2-targeted magnetic resonance (MR) contrast agents was investigated. PROCEDURES: The scFv-IONPs were prepared and identified. For in vitro MRI, NCI-N87 (HER2 high expression) and SUIT2 (low expression) cells were incubated with scFv-IONPs. For in vivo MRI, NCI-N87 and SUIT2 tumor-bearing mice were intravenously injected with scFv-IONPs and imaged before and 24 h post-injection. RESULTS: The scFv-IONPs demonstrated high transverse relaxivity (296.3 s-1 mM-1) and affinity toward HER2 (KD = 11.7 nM). In the in vitro MRI, NCI-N87 cells treated with scFv-IONPs exhibited significant MR signal reduction (44.6 %) than SUIT2 cells (6.8 %). In the in vivo MRI, decrease of MR signals in NCI-N87 tumors (19.3 %) was more notable than that in SUIT2 tumors (6.2 %). CONCLUSIONS: The scFv-IONPs enabled HER2-specific tumor MR imaging, suggesting the potential of scFv-IONPs as a robust HER2-targeted MR contrast agent.

Feasibility of poly(ethylene glycol) derivatives as diagnostic drug carriers for tumor imaging.

Poly(ethylene glycol) (PEG) is an artificial but biocompatible hydrophilic polymer that has been widely used in clinical products. To evaluate the feasibility of using PEG derivative itself as a tumor imaging carrier via an enhanced permeability and retention (EPR) effect, we prepared indium-111-labeled PEG ((111)In-DTPA-PEG) and indocyanine green (ICG)-labeled PEG (ICG-PEG) with PEG molecular weights of 5-40kDa and investigated their in vivo biodistribution in colon26 tumor-bearing mice. Thereafter, single-photon emission computed tomography (SPECT) and photoacoustic (PA) imaging studies were performed. The in vivo biodistribution studies demonstrated increased tumor uptake and a prolongation of circulation half-life as the molecular weight of PEG increased. Although the observed differences in in vivo biodistribution were dependent on the labeling method ((111)In or ICG), the tumor-to-normal tissue ratios were comparable. Because PEG-based probes with a molecular weight of 20kDa (PEG20) showed a preferable biodistribution (highest accumulation among tissues excised and relatively high tumor-to-blood ratios), an imaging study using (111)In-DTPA-PEG20 and ICG-PEG20 was performed. Colon26 tumors inoculated in the right shoulder were clearly visualized by SPECT 24h after administration. Furthermore, PA imaging using ICG-PEG20 also detected tumor regions, and the detected PA signals increased in proportion with the injected dose. These results suggest that PEG derivatives (20kDa) serve as robust diagnostic drug carriers for tumor imaging.

Development of photostabilized asymmetrical cyanine dyes for in vivo photoacoustic imaging of tumors.

Photoacoustic imaging (PAI) contributes to tumor diagnosis through the use of PAI probes that effectively accumulate in tumors. Previously, we developed a symmetrical cyanine dye, IC7-1-Bu, which showed high potential as a PAI probe because of its high tumor targeting ability and sufficient in vivo PA signal. However, IC7-1-Bu lacks photostability for multiple laser irradiations, so we developed stabilized PAI probes using IC7-1-Bu as a lead compound. We focused on the effect of singlet oxygen (1O2) generated by excited PAI probes on probe degeneration. We introduced a triplet-state quencher (TSQ) moiety into IC7-1-Bu to quench 1O2 generation and designed three IC-n-T derivatives with different linker lengths (n indicates linker length). The IC-n-T derivatives emitted in vitro PA signals that were comparable to IC7-1-Bu and significantly reduced 1O2 generation while showing improved photostability against multiple irradiations. Of the three derivatives evaluated, IC-5-T accumulated in tumors effectively to allow clear PAI of tumors in vivo. Furthermore, the photostability of IC-5-T was 1.5-fold higher than that of IC7-1-Bu in in vivo sequential PAI. These results suggest that IC-5-T is a potential PAI probe for in vivo sequential tumor imaging.

Preclinical evaluation of a novel cyanine dye for tumor imaging with in vivo photoacoustic imaging.

Photoacoustic imaging (PA imaging or PAI) has shown great promise in the detection and monitoring of cancer. Although nanocarrier-based contrast agents have been studied for use in PAI, small molecule contrast agents are required due to their ease of preparation, costeffectiveness, and low toxicity. Here, we evaluated the usefulness of a novel cyanine dye IC7-1-Bu as a PAI contrast agent without conjugated targeting moieties for in vivo tumor imaging in a mice model. Basic PA characteristics of IC7-1-Bu were compared with indocyanine green (ICG), a Food and Drug Administration approved dye, in an aqueous solution. We evaluated the tumor accumulation profile of IC7-1-Bu and ICG by in vivo fluorescence imaging. In vivo PAI was then performed with a photoacoustic tomography system 24 and 48 h after intravenous injection of IC7-1-Bu into tumor bearing mice. IC7-1-Bu showed about a 2.3-fold higher PA signal in aqueous solution compared with that of ICG. Unlike ICG, IC7-1-Bu showed high tumor fluorescence after intravenous injection. In vivo PAI provided a tumor to background PA signal ratio of approximately 2.5 after intravenous injection of IC7-1-Bu. These results indicate that IC7-1-Bu is a promising PAI contrast agent for cancer imaging without conjugation of targeting moieties.

Development of human serum albumin conjugated with near-infrared dye for photoacoustic tumor imaging.

Photoacoustic (PA) imaging has emerged as a noninvasive diagnostic method which detects ultrasonic waves thermoelastically induced by optical absorbers irradiated with laser. For tumor diagnosis, PA contrast agent has been proposed to enhance the PA effect for detecting tumors sensitively. Here, we prepared a human serum albumin (HSA) conjugated with indocyanine green (ICG) as a PA contrast agent allowing enhanced permeability and retention effect for sensitive tumor imaging. The feasibility of PA imaging with HSA-ICG to detect allografted tumors was evaluated in tumor-bearing mice. In vivo fluorescence imaging and radiolabeled biodistribution study showed that the biodistribution dramatically changed as the number of ICG bound to HSA increased, and the maximum accumulation of ICG was achieved when around three ICG molecules were loaded on an HSA. In vivo PA imaging demonstrated a tumor-selective and dose-dependent increase of PA signal intensity in mice injected with HSA-ICG (R2 = 0.88, 387% increase for HSA-ICG, 104 nmol ICG). In conclusion, HSA-ICG clearly visualized the allografted tumors with high tumor-to-background ratios having high quantitative and spatial resolution for the sensitive PA imaging of tumors. HSA-ICG could be useful as a favorable contrast agent for PA tumor imaging for the management of cancer.