Marites P. Melancon

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.

Cancer theranostics with near-infrared light-activatable multimodal nanoparticles.

Nanomaterials that interact with light provide a unique opportunity for applications in biophotonic nanomedicine. Image-guided therapies could be designed based on multifunctional nanoparticles (NPs). Such NPs have a strong and tunable surface plasmon resonance absorption in the near-infrared region and can be detected using multiple imaging modalities (magnetic resonance imaging, nuclear imaging, and photoacoustic imaging). These novel nanostructures, once introduced, are expected to home in on solid tumors either via a passive targeting mechanism (i.e., the enhanced permeability and retention effect) or via an active targeting mechanism facilitated by ligands bound to their surfaces. Once the NPs reach their target tissue, their activity can then be turned on using an external stimulus. For example, photothermal conducting NPs primarily act by converting light energy into heat. As a result, the temperature in the treatment volume is elevated above the thermal damage threshold, which kills the cells. This process, termed photothermal ablation therapy (PTA), is effective, but it is also unlikely to kill all tumor cells when used alone. In addition to PTA, photothermal conducting NPs can also efficiently trigger the release of drugs and activate RNA interference. A multimodal approach, which permits simultaneous PTA therapy, chemotherapy, and therapeutic RNA interference, has the potential to completely eradicate residual diseased cells. In this Account, we provide an up-to-date review of the synthesis and characterization, functionalization, and in vitro and in vivo evaluation of NIR lightactivatable multifunctional nanostructures used for imaging and therapy. We emphasize research on hollow gold nanospheres, magnetic core-shell gold nanoshells, and semiconductor copper monosulfide NPs. We discuss three types of novel drug delivery systems in which hollow gold nanospheres are used to mediate controlled drug release.

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.

Targeted multifunctional gold-based nanoshells for magnetic resonance-guided laser ablation of head and neck cancer

Image-guided thermal ablation of tumors is becoming a more widely accepted minimally invasive alternative to surgery for patients who are not good surgical candidates, such as patients with advanced head and neck cancer. In this study, multifunctional superparamagnetic iron oxide coated with gold nanoshell (SPIO@Au NS) that have both optical and magnetic properties was conjugated with the targeting agent, C225 monoclonal antibody, against epidermal growth factor receptor (EGFR). C225-SPIO@Au NS have an average a diameter of 82 ± 4.4 nm, contain 142 ± 15 antibodies per nanoshell, have an absorption peak in the near infrared (~800 nm), and have transverse relaxivity (r(2)) of 193 and 353 mM(-1) s(-1) versus Feridex™ of 171 and 300 mM(-1) s(-1), using 1.5 T and 7 T MR scanners, respectively. Specific targeting of the synthesized C225-SPIO@Au NS was tested in vitro using A431 cells and oral cancer cells, FaDu, OSC19, and HN5, all of which overexpress EGFR. Selective binding was achieved using C225-SPIO@Au NS but not with the non-targeting PEG-SPIO@Au NS and blocking group (excess of C225 + C225-SPIO@Au NS). In vivo biodistribution on mice bearing A431 tumors also showed selective targeting of C225-SPIO@Au NS compared with the non-targeting and blocking groups. The selective photothermal ablation of the nanoshells shows that without laser treatment there were no cell death and among the groups that were treated with laser at a power of 36 W/cm(2) for 3 min, only the cells treated with C225-SPIO@Au NS had cell killing (p < 0.001). In summary, successful synthesis and characterization of targeted C225-SPIO@Au NS demonstrating both superparamagnetic and optical properties has been achieved. We have shown both in vitro and in vivo that these nanoshells are MR-active and can be selectively heated up for simultaneous imaging and photothermal ablation therapy.

Selective uptake and imaging of aptamer- and antibody-conjugated hollow nanospheres targeted to epidermal growth factor receptors overexpressed in head and neck cancer.

The purpose of this study was to compare the binding affinity and selective targeting of aptamer- and antibody-coated hollow gold nanospheres (HAuNS) targeted to epidermal growth factor receptors (EGFR). EGFR-targeting aptamers were conjugated to HAuNS (apt-HAuNS) by attaching a thiol-terminated single-stranded DNA to the HAuNS and then adding the complementary RNA targeted to EGFR. Apt-HAuNS was characterized in terms of size, surface charge, absorption, and number of aptamers per particle. The in vivo pharmacokinetics, in vivo biodistribution, and micro-SPECT/CT imaging of 111In-labeled apt-HAuNS and anti-EGFR antibody (C225)-conjugated HAuNS were evaluated in nude mice bearing highly malignant human OSC-19 oral tumors. 111In-labeled PEG-HAuNS was used as a control (n = 5/group). Apt-HAuNS did not have an altered absorbance profile or size (λmax = 800 nm; diameter = 55 nm) compared to C225-HAuNS or PEG-HAuNS. The surface charge became more negative upon conjugation of the aptamer (−51.4 vs −19.0 for PEG-HAuNS and −25.0 for C225-HAuNS). The number of aptamers/particle was ∼250. In vitro cell binding and in vivo biodistribution showed selective binding of the apt-HAuNS to EGFR. μSPECT/CT imaging confirmed that there was more tumor uptake of apt-HAuNS than C225-HAuNS. Aptamer is a promising ligand for image-guided delivery of nanoparticles for treatment of tumor cells overexpressing EGFR.

Theranostics with multifunctional magnetic gold nanoshells: Photothermal therapy and T2* magnetic resonance imaging

Objectives:To investigate the multifunctional imaging and therapeutic capabilities of core-shell nanoparticles composed of a superparamagnetic iron oxide (SPIO) core and a gold shell (SPIO@AuNS). Materials and Methods:The magnetic/optical properties of SPIO@AuNS were examined both in an agar gel phantom and in vivo by evaluating contrast-enhanced magnetic resonance imaging (MRI) and by measuring near-infrared (NIR) light-induced temperature changes mediated by SPIO@AuNS. In addition, the biodistribution and pharmacokinetics of 111In-labeled SPIO@AuNS after intravenous injection in mice bearing A431 tumors were evaluated in the presence and absence of an external magnet. Results:In agar phantoms containing SPIO@AuNS, a significant contrast enhancement in T2-weighted MRI was observed and a linear increase in temperature was observed with increasing concentration and laser output power when irradiated with NIR light centered at an 808 nm. In vivo, T2*-MRI delineated SPIO@AuNS and magnetic resonance temperature imaging of the same tumors revealed significant temperature elevations when intratumorally injected with SPIO@AuNS (1 × 1011 particles/mouse) and irradiated with NIR light (65.70°C ± 0.69°C vs. 44.23°C ± 0.24°C for saline + laser). Biodistribution studies in mice intravenously injected with 111In-labeled-SPIO@AuNS(1 × 1013 particles/mouse) had an approximately 2-fold increase in SPIO@AuNS delivered into tumors in the presence of an external magnet compared with tumors without the magnet. Conclusions:Owing to its ability to mediate efficient photothermal ablation of cancer cells under MRI guidance, as well as the ability to be directed to solid tumors with an external magnetic field gradient, multifunctional SPIO@AuNS is a promising theranostic nanoplatform.

Challenges to effective cancer nanotheranostics

Advances in nanotechnology for oncology will arise from an increased understanding of the interaction between nanomaterials and biological systems; refinement of multifunctional nanocomposites for applications such as simultaneous imaging and therapy (theranostics); and harnessing of the unique physicochemical properties arising from nanoscale effects which distinguish them from small-molecular-weight molecules in the detection and destruction of cancer cells with high selectivity and efficiency. The major challenges in successful clinical translation of tumor specific nanoparticle delivery include overcoming various biological barriers and demonstrating enhanced therapeutic efficacy over the current standard of care in the clinic. For many nanoparticle mediated theranostic applications, image guidance can play a crucial role not only in exploiting the cancer specific imaging capabilities of these novel particles, but in planning, targeting, monitoring and verifying treatment delivery, thus enhancing the safety and efficacy of these emerging procedures.

In vitro and in vivo mapping of drug release after laser ablation thermal therapy with doxorubicin-loaded hollow gold nanoshells using fluorescence and photoacoustic imaging

Doxorubicin-loaded hollow gold nanoshells (Dox@PEG-HAuNS) increase the efficacy of photothermal ablation (PTA) not only by mediating efficient PTA but also through chemotherapy, and therefore have potential utility for local anticancer therapy. However, in vivo real-time monitoring of Dox release and temperature achieved during the laser ablation technique has not been previously demonstrated before. In this study, we used fluorescence optical imaging to map the release of Dox from Dox@PEG-HAuNS and photoacoustic imaging to monitor the tumor temperature achieved during near-infrared laser-induced photothermal heating in vitro and in vivo. In vitro, treatment with a 3-W laser was sufficient to initiate the release of Dox from Dox@PEG-HAuNS (1:3:1 wt/wt, 1.32 × 10(12)particles/mL). Laser powers of 3 and 6W achieved ablative temperatures of more than 50°C. In 4T1 tumor-bearing nude mice that received intratumoral or intravenous injections of Dox@PEG-HAuNS, fluorescence optical imaging (emission wavelength = 600 nm, excitation wavelength = 500 nm) revealed that the fluorescence intensity in surface laser-treated tumors 24h after treatment was significantly higher than that in untreated tumors (p = 0.015 for intratumoral, p = 0.008 for intravenous). Similar results were obtained using an interstitial laser to irradiate tumors following the intravenous injection of Dox@PEG-HAuNS (p = 0.002 at t = 24h). Photoacoustic imaging (acquisition wavelength = 800 nm) revealed that laser treatment caused a substantial increase in tumor temperature, from 37 °C to ablative temperatures of more than 50 °C. Ex vivo analysis revealed that the fluorescence intensity of laser-treated tumors was twice as high as that of untreated tumors (p = 0.009). Histological analysis confirmed that intratumoral injection of Dox@PEG-HAuNS and laser treatment caused significantly more tumor necrosis compared to tumors that were not treated with laser (p<0.001). On the basis of these findings, we conclude that fluorescence optical imaging and photoacoustic imaging are promising approaches to assessing Dox release and monitoring temperature, respectively, after Dox@PEG-HAuNS-mediated thermal ablation therapy.

Near-infrared light modulated photothermal effect increases vascular perfusion and enhances polymeric drug delivery

Hyperthermia, which is heating of tumors above 43°C for about 30min, has been known to modulate vascular permeability for enhanced chemotherapy. However, it is not clear whether a similar effect exists when temperature at tumor sites is elevated above 43°C, such as temperature achieved in laser-induced photothermal ablation (PTA) therapy. Also, the effect of timing of chemotherapeutic drug administration following heating in the efficiency of drug delivery is not established. In this study, we investigated the impact of near infrared (NIR) laser irradiated anti-EGFR monoclonal antibody C225-conjugated hollow gold nanospheres (C225-HAuNS) on vascular permeability and subsequent tumor uptake of a water-soluble polymer using combined MRI, ultrasound and optical imaging approaches. Magnetic temperature imaging showed a maximum temperature of 65.2±0.10 °C in A431 tumor xenograft of mice treated with C225-HAuNS plus laser and 47.0±0.33 °C in tumors of mice treated with saline plus laser at 4 W/cm² for 3 min (control) at 2 mm from the light incident surface. Dynamic contrast enhanced (DCE) MRI demonstrated greater than 2-fold increase of DTPA-Gd in the initial area under the curve (IAUC₉₀) in mice injected with C225-HAuNS and exposed to NIR laser compared with control mice at 3 min after laser treatment. Similarly, Power Doppler (PD) ultrasound revealed a 4- to 6-fold increase in percentage vascularization in mice treated with C225-HAuNS plus NIR laser compared to control mice and confirmed increased vascular perfusion immediately after laser treatment. Twenty-four hours later, the blood perfusion was shut down. On optical imaging, tumor uptake of PG-Gd-NIR813, which is the model polymeric drug used, was significantly higher (p-value<0.05) in mice injected with PG-Gd-NIR813 at 5 min after laser treatment than in mice injected with PG-Gd-NIR813 at 24h after laser treatment and the saline-treated mice. In conclusion, laser irradiation of tumors after intravenous injection of C255-HAuNS induces a thermally mediated modulation of the vascular perfusion, which enhances the delivery of polymeric drugs to the tumors at the time phototherapy is initiated.

Development of a macromolecular dual-modality MR-optical imaging for sentinel lymph node mapping.

Objective:To evaluate the effectiveness of a dual magnetic resonance-near infrared fluorescence optical imaging agent, poly(l-glutamic acid)-DTPA-Gd-NIR813, for both preoperative and intraoperative visualization and characterization of sentinel lymph nodes (SLN) in mice. Materials and Methods:Poly(l-glutamic acid) was conjugated with DTPA-Gd and NIR813 dye to obtain PG-DTPA-Gd-NIR813. To confirm drainage into the SLNs, this agent was injected subcutaneously into the front paw of nude mice followed by isosulfan blue (n = 6). Furthermore, PG-DTPA-Gd-NIR813 was injected subcutaneously at doses of 0.002 mmol Gd/kg (4.8 nmol eq. NIR813) and 0.02 mmol Gd/kg (48 nmol eq. NIR813) (n = 3/dose). To differentiate metastatic from nonmetastatic lymph nodes, nude mice bearing human oral squamous cell carcinoma (DM14) were injected intralingually with 0.02 mmol Gd/kg PG-DTPA-Gd-NIR813 (n = 3). Pre- and postcontrast images were taken using 4.7 T Bruker MRI scanner and Xenogen optical imaging system. The status of lymph nodes resected under the guidance of optical imaging was confirmed by histologic examinations. Results:PG-DTPA-Gd-NIR813 colocalized with isosulfan blue, indicating drainage to the SLN. After subcutaneous injection, axiliary and branchial lymph nodes were clearly visualized with both T1-weighted MR and optical imaging within 3 minutes of contrast injection, even at the lowest dose tested (0.002 mmol Gd/kg). After intralingual injection in tumor-bearing mice, MR imaging identified 4 of the 6 superficial cervical lymph nodes, whereas near-infrared fluorescence (NIRF) optical imaging identified all 6 cervical nodes. The pattern of contrast enhancement of SLN visualized in MR images showed a characteristic ring-shaped appearance with a central filling defect, possibly resulting from nodal infiltration of metastatic lesions. Histopathologic examination of the SLNs resected under NIRF imaging guidance revealed micrometastases in all 6 SLNs identified by NIRF imaging. Conclusions:The dual modality imaging method demonstrated in this study represents an effective technique for localization and characterization of SLN.