Shaoli Song

A Chelator-Free Multifunctional [64Cu]CuS Nanoparticle Platform for Simultaneous Micro-PET/CT Imaging and Photothermal Ablation Therapy

We synthesized and evaluated a novel class of chelator-free [(64)Cu]CuS nanoparticles (NPs) suitable both for PET imaging and as photothermal coupling agents for photothermal ablation. These [(64)Cu]CuS NPs are simple to make, possess excellent stability, and allow robust noninvasive micro-PET imaging. Furthermore, the CuS NPs display strong absorption in the near-infrared (NIR) region (peak at 930 nm); passive targeting prefers the tumor site, and mediated ablation of U87 tumor cells occurs upon exposure to NIR light both in vitro and in vivo after either intratumoral or intravenous injection. The combination of small diameter (∼11 nm), strong NIR absorption, and integration of (64)Cu as a structural component makes these [(64)Cu]CuS NPs ideally suited for multifunctional molecular imaging and therapy.

Copper sulfide nanoparticles as a new class of photoacoustic contrast agent for deep tissue imaging at 1064 nm.

Photoacoustic tomography (PAT) is an emerging molecular imaging modality. Here, we demonstrate use of semiconductor copper sulfide nanoparticles (CuS NPs) for PAT with an Nd:YAG laser at a wavelength of 1064 nm. CuS NPs allowed visualization of mouse brain after intracranial injection, rat lymph nodes 12 mm below the skin after interstitial injection, and CuS NP-containing agarose gel embedded in chicken breast muscle at a depth of ~5 cm. This imaging approach has great potential for molecular imaging of breast cancer.

Effects of photoacoustic imaging and photothermal ablation therapy mediated by targeted hollow gold nanospheres in an orthotopic mouse xenograft model of glioma

Advancements in nanotechnology have made it possible to create multifunctional nanostructures that can be used simultaneously to image and treat cancers. For example, hollow gold nanospheres (HAuNS) have been shown to generate intense photoacoustic signals and induce efficient photothermal ablation (PTA) therapy. In this study, we used photoacoustic tomography, a hybrid imaging modality, to assess the intravenous delivery of HAuNS targeted to integrins that are overexpressed in both glioma and angiogenic blood vessels in a mouse model of glioma. Mice were then treated with near-infrared laser, which elevated tumor temperature by 20.7°C. We found that PTA treatment significantly prolonged the survival of tumor-bearing mice. Taken together, these results show the feasibility of using a single nanostructure for image-guided local tumor PTA therapy with photoacoustic molecular imaging.

Receptor-mediated transcytosis: a mechanism for active extravascular transport of nanoparticles in solid tumors.

Targeted nanoparticle-based delivery systems have been used extensively to develop effective cancer theranostics. However, how targeting ligands affect extravascular transport of nanoparticles in solid tumors remains unclear. Here, we show, using B16/F10 melanoma cells expressing melanocortin type-1 receptor (MC1R), that the nature of targeting ligands, i.e., whether they are agonists or antagonists, directs tumor uptake and intratumoral distribution after extravasation of nanoparticles from tumor vessels into the extravascular fluid space. Pegylated hollow gold nanospheres (HAuNS, diameter=40 nm) coated with MC1R agonist are internalized upon ligand-receptor binding, whereas MC1R antagonist-conjugated HAuNS remain attached on the cell surface. Transcellular transport of agonist-conjugated HAuNS was confirmed by a multilayer tumor cell model and by transmission electron microscopy. MC1R agonist- but not MC1R antagonist-conjugated nanoparticles exhibit significantly higher tumor uptake than nontargeted HAuNS and are quickly dispersed from tumor vessels via receptor-mediated endocytosis and subsequent transcytosis. These results confirm an active transport mechanism that can be used to overcome one of the major biological barriers for efficient nanoparticle delivery to solid tumors.

In vivo small-animal PET/CT of EphB4 receptors using64Cu-labeled peptide

Many solid tumors overexpress EphB4 receptor, a member of the ephrin receptor tyrosine kinase family. Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed against EphB4, and improve patient outcomes. The purpose of this study was to evaluate a novel 64Cu-labeled peptide with high receptor binding affinity for PET of EphB4 receptors. Methods: The EphB4-binding peptide TNYLFSPNGPIARAW (TNYL-RAW) was conjugated with fluorescein isothiocyanate (FITC) and DOTA. DOTA-TNYL-RAW was labeled with 64Cu with high labeling efficiency. The binding affinity of TNYL-RAW and its derivatives to purified recombinant EphB4 was determined using surface plasmon resonance technology. In vitro binding of both FITC-TNYL-RAW and 64Cu-DOTA-TNYL-RAW to cancer cells was assessed by fluorescent microscopy and a radioactivity count method. In vivo biodistribution and small-animal PET/CT were performed in mice bearing EphB4-expressing CT26 and PC-3M tumors as well as EphB4-negative A549 tumors. Results: TNYL-RAW and its derivatives displayed high binding affinity to EphB4, with equilibrium dissociation constant of 1.98–23 nM. In vitro, both FITC-TNYL-RAW and 64Cu-DOTA-TNYL-RAW were selectively taken up by CT26 and PC-3M cells but not by A549 cells. Binding of FITC-TNYL-RAW and 64Cu-DOTA-TNYL-RAW to CT26 and PC-3M cells could be blocked by an excess amount of TNYL-RAW. In vivo, 64Cu-DOTA-TNYL-RAW showed significantly higher uptake in PC-3M tumors than in A549 tumors, with percentages of injected dose per gram of tumor of 0.84 ± 0.09 and 0.44 ± 0.09 at 24 h after radiotracer injection, respectively. Small-animal PET/CT clearly revealed deposition of 64Cu-DOTA-TNYL-RAW in CT26 and PC-3M tumors but not in A549 tumors. Furthermore, uptake of 64Cu-DOTA-TNYL-RAW in both CT26 and PC-3M tumors could be blocked by cold TNYL-RAW. Conclusion: The expression of EphB4 receptors can be noninvasively interrogated by small-animal PET/CT using 64Cu-DOTA-TNYL-RAW.

Peptide-based imaging agents targeting phosphatidylserine for the detection of apoptosis.

A 14-residue phosphatidylserine (PS)-binding peptide FNFRLKAGQKIRFG (PSBP-0) was scanned with Ala. In addition, a radiometal chelator (SAAC) was introduced at selected sites of the lead peptides. Substitution of the Gln(6) residue in PSBP-0 with Ala resulted in a significant increase in binding affinity to PS as determined by surface plasmon resonance sensorgrams. The binding affinity of the resulting peptide FNFRLKAGAKIRFG (PSBP-6, molecular mass = 1623 Da) to PS (K(d) ∼ 100 nM) increased 10-fold as compared to PSBP-0 (K(d) ∼ 1.38 μM). Introduction of SAAC-Re to the N terminus of PSBP-6 further increased the binding affinity of the resulting peptide SAAC(Re)-PSBP-6 (K(d) ∼ 26 nM). SAAC(Re)-PSBP-6 shows specific binding to apoptotic cells in cell-based assays. Biodistribution studies showed significantly higher uptake of SAAC((99 m)Tc)-PSBP-6 in B16/F10 melanoma treated with poly(L-glutamic acid)-paclitaxel than untreated tumors (4.06 ± 0.55% ID/g vs 1.61 ± 0.33% ID/g, P = 0.00011). SAAC((99 m)Tc)-PSBP-6 is a promising probe for noninvasive imaging of apoptotic cells.

Small-Animal PET of Tumor Damage Induced by Photothermal Ablation with 64Cu-Bis-DOTA-Hypericin

The purpose of this study was to investigate the potential application of small-molecular-weight 64Cu-labeled bis-DOTA-hypericin in the noninvasive assessment of response to photothermal ablation therapy. Methods: Bis-DOTA-hypericin was labeled with 64Cu with high efficiency (>95% without purification). Nine mice bearing subcutaneous human mammary BT474 tumors were used. Five mice were injected intratumorally with semiconductor CuS nanoparticles, followed by near-infrared laser irradiation 24 h later (12 W/cm2 for 3 min), and 4 mice were not treated (control group). All mice were intravenously injected with 64Cu-bis-DOTA-hypericin (24 h after laser treatment in treated mice). Small-animal PET images were acquired at 2, 6, and 24 h after radiotracer injection. All mice were killed immediately after the imaging session for biodistribution and histology study. In vitro cell uptake and surface plasmon resonance studies were performed to validate the small-animal PET results. Results: 64Cu-bis-DOTA-hypericin uptake was significantly higher in the treatment group than in the control group. The percentage injected dose per gram of tissue in the treated and control groups was 1.72 ± 0.43 and 0.76 ± 0.19, respectively (P = 0.017), at 24 h after injection. Autoradiography and histology results were consistent with selective uptake of the radiotracer in the necrotic zone of the tumor induced by photothermal ablation therapy. In vitro results showed that treated BT474 cells had a higher uptake of 64Cu-bis-DOTA-hypericin than nontreated cells. Surface plasmon resonance study showed that bis-DOTA-hypericin had higher binding affinity to phosphatidylserine and phosphatidylethanolamine than to phosphatidylcholine. Conclusion: 64Cu-bis-DOTA-hypericin has a potential to image thermal therapy–induced tumor cell damage. The affinity of 64Cu-bis-DOTA-hypericin for injured tissues may be attributed to the breakdown of the cell membrane and exposure of phosphatidylserine or phosphatidylethanolamine to the radiotracer, which binds selectively to these phospholipids.

Theranostic CuS nanoparticles targeting folate receptors for PET image-guided photothermal therapy

Copper sulfide nanoparticles (CuS NPs) have been reported as a single-compartment theranostic nanosystem to visualize and treat tumors simultaneously. However, few studies have investigated the in vivo tumor-targeted delivery of this class of nanoparticles. In this study, we introduced a tumor-specific targeting ligand, folic acid (FA), onto the surface of CuS NPs as a model system to demonstrate the feasibility of actively targeted CuS NPs for positron emission tomography (PET) imaging and PET image-guided photothermal therapy (PTT). A one-pot synthetic method was used for introducing FA to CuS NPs to yield FA-CuS NPs. Biodistribution studies in mice bearing folate receptor-expressing KB tumor showed significantly higher tumor uptake of FA-CuS NPs than non-targeted polyethylene glycol (PEG)-coated PEG-CuS NPs after intravenous injection. Moreover, tumor uptake of FA-CuS NPs could be effectively blocked by free FA. Biodistribution and clearance of 64Cu-labeled FA-CuS NPs (FA-[64Cu]CuS NPs) could be readily visualized by microPET (μPET), which confirmed a significantly higher level of tumor uptake of FA-[64Cu]CuS NPs than non-targeted PEG-[64Cu]CuS NPs. μPET image-guided PTT with FA-CuS NPs mediated substantially greater tumor damage compared with PTT mediated by PEG-CuS NPs. Thus, FA-CuS NPs is a promising candidate for PTT of folate receptor-positive tumors.

Apoptosis imaging probe predicts early chemotherapy response in preclinical models: A comparative study with 18F-FDG PET

Previously, we reported a small-molecular-weight peptide, single amino acid chelae(99mTc)-conjugated phosphatidylserine-binding peptide (SAAC(99mTc)-PSBP-6), with high binding affinity to phosphatidylserine on the surface of apoptotic cells. The purpose of this study was to determine the effectiveness of SAAC(99mTc)-PSBP-6 in detecting apoptosis induced by chemotherapy. Methods: B16/F10 melanoma and 38C13 lymphoma tumor models were used in this study. For each type of tumor model, mice were divided into a group treated for imaging (treated group [TG]) and a control group that was not treated (nontreated group [N-TG]). In the TG, mice bearing murine B16/F10 melanoma received a single dose of intravenous polymeric paclitaxel (equivalent dose, 80 mg/kg), and mice bearing 38C13 xenografts received intraperitoneal cyclophosphamide (100 mg/kg). Mice in the N-TG were given the same volume of saline. γ-imaging 4 h after intravenous injection of SAAC(99mTc)-PSBP-6 and small-animal PET 1 h after intravenous injection of 18F-FDG were performed before chemotherapy and at 1 d after chemotherapy. On day 1, immediately after the apoptosis imaging sessions, 3 mice each in the TGs and N-TGs were killed, and tumor tissues were excised for hematoxylin and eosin histology, autoradiography, and immunohistochemical staining using anti–active caspase 3 and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). The tumor volumes in the remaining mice (n = 5/group) were measured every other day for 7 d. Results: In both tumor models, the uptake of SAAC(99mTc)-PSBP-6 increased significantly on day 1 after treatment, whereas 18F-FDG uptake decreased significantly during the same time. The mean tumor uptake values for SAAC(99mTc)-PSBP-6 increased 142.4% ± 36.9% and 112% ± 42.9% in 38C13 and B16/F10 tumors, respectively (both P < 0.05, pretreatment vs. day 1 after treatment). The mean tumor uptake value for 18F-FDG decreased 67.36% ± 17.52% and 62.82% ± 4.53% in 38C13 and B16/F10 tumors, respectively. The uptake of SAAC(99mTc)-PSBP-6 negatively correlated with 18F-FDG (r = –0.79, P < 0.05). Treated tumors had smaller volumes than untreated controls, treated tumors had significantly higher numbers of apoptotic cells, and tumor uptake of SAAC(99mTc)-PSBP-6 correlated with the number of TUNEL-positive cells. Conclusion: SAAC(99mTc)-PSBP-6 γ-imaging is useful for the early assessment of treatment-induced apoptosis and, thus, may be used as a substitute for 18F-FDG PET for assessing early treatment response.

Radio-photothermal therapy mediated by a single compartment nanoplatform depletes tumor initiating cells and reduces lung metastasis in the orthotopic 4T1 breast tumor model

Tumor Initiating Cells (TICs) are resistant to radiotherapy and chemotherapy, and are believed to be responsible for tumor recurrence and metastasis. Combination therapies can overcome the limitation of conventional cancer treatments, and have demonstrated promising application in the clinic. Here, we show that dual modality radiotherapy (RT) and photothermal therapy (PTT) mediated by a single compartment nanosystem copper-64-labeled copper sulfide nanoparticles ([(64)Cu]CuS NPs) could suppress breast tumor metastasis through eradication of TICs. Positron electron tomography (PET) imaging and biodistribution studies showed that more than 90% of [(64)Cu]CuS NPs was retained in subcutaneously grown BT474 breast tumor 24 h after intratumoral (i.t.) injection, indicating the NPs are suitable for the combination therapy. Combined RT/PTT therapy resulted in significant tumor growth delay in the subcutaneous BT474 breast cancer model. Moreover, RT/PTT treatment significantly prolonged the survival of mice bearing orthotopic 4T1 breast tumors compared to no treatment, RT alone, or PTT alone. The RT/PTT combination therapy significantly reduced the number of tumor nodules in the lung and the formation of tumor mammospheres from treated 4T1 tumors. No obvious side effects of the CuS NPs were noted in the treated mice in a pilot toxicity study. Taken together, our data support the feasibility of a therapeutic approach for the suppression of tumor metastasis through localized RT/PTT therapy.