MAGE-Targeted Gold Nanoparticles for Ultrasound Imaging-Guided Phototherapy in Melanoma.

Gold nanorods exhibit a wide variety of applications such as tumor molecular imaging and photothermal therapy (PTT) due to their tunable optical properties. Several studies have demonstrated that the combination of other therapeutic strategies may improve PTT efficiency. A method called optical droplet vaporization (ODV) was considered as another noninvasive imaging and therapy strategy. Via the ODV method, superheated perfluorocarbon droplets can be vaporized to a gas phase for enhancing ultrasound imaging; meanwhile, this violent process can cause damage to cells and tissue. In addition, active targeting through the functionalization with targeting ligands can effectively increase nanoprobe accumulation in the tumor area, improving the sensitivity and specificity of imaging and therapy. Our study prepared a nanoparticle loaded with gold nanorods and perfluorinated hexane and conjugated to a monoclonal antibody (MAGE-1 antibody) to melanoma-associated antigens (MAGE) targeting melanoma, investigated the synergistic effect of PTT/ODV therapy, and monitored the therapeutic effect using ultrasound. The prepared MAGE-Au-PFH-NPs achieved complete eradication of tumors. Meanwhile, the MAGE-Au-PFH-NPs also possess significant ultrasound imaging signal enhancement, which shows the potential for imaging-guided tumor therapy in the future.

Rational Design of a Robust Antibody-like Small-Molecule Inhibitor Nanoplatform for Enhanced Photoimmunotherapy.

Immune checkpoint blockade of the programmed cell death-ligand 1/programmed cell death-1 (PD-L1/PD-1) pathway via an antibody is a potent strategy for T cell remodeling. Nevertheless, the potency of the antibody is partly compromised by its high price, instability, risk of autoimmune disease, and so forth. Small-molecule inhibitors are interesting alternatives to antibodies. However, tumor-specific delivery of small-molecule inhibitors to the target site for boosting the interruption of the PD-L1/PD-1 pathway is rarely reported. Herein, we designed a tumor-specific delivery nanoplatform that could efficiently deliver the small-molecule inhibitor to the precise target site, greatly enhancing the blocking effect of the PD-L1/PD-1 pathway. Hyaluronic acid (HA) was conjugated with chlorin e6 (Ce6), resulting in a HA-Ce6 conjugate (HC). The nanoplatform was constructed by the HC micelles with the encapsulation of small-molecule inhibitor, BMS 202 (BMS), to form BMS/HC micelles. The target property of HA, combined with the hyaluronidase-induced degradation of HA in the tumor site, enables the as-prepared micelles with tumor-specific delivery of BMS for blocking the PD-L1/PD-1 pathway. With cooperative treatment with the photosensitizer Ce6, the present therapeutic nanoplatform demonstrated excellent photoimmunotherapy for tumor regression in distant tumors and lung metastasis. This strategy of tumor-specific delivery of small-molecule inhibitors provides an effective pathway to strengthen the blocking efficacy of PD-L1/PD-1 on effective photoimmunotherapy.

The Application of the in-Situ Hyperthermia Emission from Acoustic Nanodroplets for Theranostic Dual-Imaging and Antitumor Modalities.

In this study, we have developed a theranostic nanocarrier that can emit heat upon the exposure to ultrasound (US) irradiation as well as the generation of a contrast signal that can be detected with ultrasonography. The prepared acoustic nanodroplets (NDs) made with liquid perfluporopentane (PFPn) had an average size of 197.7 ± 3.6 nm in diameter and were stable in vitro for 60 min. US irradiation at 2 W.cm-2 induced phase change of NDs into bubbles in vitro. On the other hand, the intra-tumor injection of NDs in combination with US irradiation induced thermal emission in situ in B16BL6 melanoma tumor implanted into mice and the emission areas have mostly covered the tumor site. Also, the combination between NDs and US irradiation has inhibited the tumor growth. Under this condition, the heat shock protein (HSP70) in tumor was significantly upregulated after 6 h of the treatment of NDs with US. Thus, we have developed a therapeutic system with multiple theranostic modalities composed of acoustic NDs and US irradiation applicable to the tumor treatment on the external surface of the body.

Size-Transformable Hyaluronan Stacked Self-Assembling Peptide Nanoparticles for Improved Transcellular Tumor Penetration and Photo-Chemo Combination Therapy.

Size-transformable nanomedicine has the potential to overcome systemic and local barriers, leading to efficient accumulation and penetration throughout the tumor tissue. However, the design of this type of nanomedicine was seldom based on active targeting and intracellular size transformation. Here, we report an intracellular size-transformable nanosystem, in which small and positively charged nanoparticles (<30 nm) prepared from the self-assembly of an amphiphilic hexadecapeptide derivative was coated by folic acid- and dopamine-decorated hyaluronan (HA) to form large and negatively charged nanoparticles (∼130 nm). This nanosystem has been proven to improve the blood circulation half-life of the drug and prevent premature intravascular drug leakage from the nanocarrier. Once accumulated in the tumor, the nanoparticles were prone to HA- and folic acid-mediated cellular uptake, followed by intracellular size transformation and discharge of transformed small nanoparticles. The size-transformable nanosystem facilitated the transcytosis-mediated tumor penetration and improved the internalization of nanoparticles by cells and the intracellular release of 7-ethyl-10 hydroxycamptothecin. With an indocyanine green derivative as the intrinsic component of the amphiphilic polymer, the nanosystem has exhibited additional theranostic functions: photoacoustic imaging, NIR-laser-induced drug release, and synergistic chemotherapy and phototherapy, leading to a 50% complete cure rate in a subcutaneous B16 melanoma model. This nanosystem with multimodalities and efficient tumor penetration has shown potentials in improving anticancer efficacy.

IR780-based light-responsive nanocomplexes combining phase transition for enhancing multimodal imaging-guided photothermal therapy.

Near-infrared (NIR) light-triggered photothermal therapy (PTT) has been widely applied for treating cancer. The combination of nanotechnology and NIR has shown great promise for promoting the efficacy of PTT. However, PTT alone could not completely ablate the tumors and easily causes tumor recurrence. To overcome this challenge, many studies have been performed to enhance PTT, including combining chemical therapy and radiotherapy, both of which have side effects on the body. To reduce the side effects and enhance PTT, a new infrared IR780-based nanocomplex combining liquid fluorocarbon perfluoropentane (PFP) has been synthesized for enhancing multimodal imaging-guided PTT. Under NIR irradiation, the size changes of PFP-loaded nanobubbles transforming into microbubbles allow ultrasound (US) imaging, showing boundaries and internal information of tumors. The breakup process and cascade reaction of phase transition can improve intratumoral permeation and retention of nanoparticles in nonmicrovascular tissue and damage the cell membranes of tumors, further enhancing PTT to kill tumor cells. The strong absorption in the NIR field of IR780-loaded NPs allows not only photoacoustic (PA) imaging but also NIR fluorescence (NIRF) imaging, which provides more anatomical information about tumors. This nanocomplex exhibits good biocompatibility and nontoxicity, strong PA/US/NIRF imaging contrast, excellent liquid-gas transition and a photothermal effect. This finding provides a new method to enhance multimodal imaging-guided cancer nanotheranostics.

Nanoclusters of crystallographically aligned nanoparticles for magnetic thermotherapy: aqueous ferrofluid, agarose phantoms and ex vivo melanoma tumour assessment.

Magnetic hyperthermia is an oncological therapy where magnetic nanostructures, under a radiofrequency field, act as heat transducers increasing tumour temperature and killing cancerous cells. Nanostructure heating efficiency depends both on the field conditions and on the nanostructure properties and mobility inside the tumour. Such nanostructures are often incorrectly bench-marketed in the colloidal state and using field settings far off from the recommended therapeutic values. Here, we prepared nanoclusters composed of iron oxide magnetite nanoparticles crystallographically aligned and their specific absorption rate (SAR) values were calorimetrically determined in physiological fluids, agarose-gel-phantoms and ex vivo tumours extracted from mice challenged with B16-F0 melanoma cells. A portable, multipurpose applicator using medical field settings; 100 kHz and 9.3 kA m-1, was developed and the results were fully analysed in terms of nanoclusters' structural and magnetic properties. A careful evaluation of the nanoclusters' heating capacity in the three milieus clearly indicates that the SAR values of fluid suspensions or agarose-gel-phantoms are not adequate to predict the real tissue temperature increase or the dosage needed to heat a tumour. Our results show that besides nanostructure mobility, perfusion and local thermoregulation, the nanostructure distribution inside the tumour plays a key role in effective heating. A suppression of the magnetic material effective heating efficiency appears in tumour tissue. In fact, dosage had to be increased considerably, from the SAR values predicted from fluid or agarose, to achieve the desired temperature increase. These results represent an important contribution towards the design of more efficient nanostructures and towards the clinical translation of hyperthermia.

Comparative preclinical evaluation of 68Ga-NODAGA and 68Ga-HBED-CC conjugated procainamide in melanoma imaging.

Malignant melanoma is the most aggressive form of skin cancer. The early detection of primary melanoma tumors and metastases using non-invasive PET imaging determines the outcome of this disease. Previous studies have shown that benzamide derivatives (e.g. procainamide) conjugated with PET radionuclides specifically bind to melanin pigment of melanoma tumors. 68Ga chelating agents can have high influence on physiological properties of 68Ga labeled bioactive molecules, as was experienced during the application of HBED-CC on PSMA ligand. The aim of this study was to assess this concept in the case of the melanin specific procaindamide (PCA) and to compare the melanin specificity of 68Ga-labeled PCA using HBED-CC and NODAGA chelators under in vitro and in vivo conditions. Procainamide (PCA) was conjugated with HBED-CC and NODAGA chelators and was labeled with Ga-68. The melanin specificity of 68Ga-HBED-CC-PCA and 68Ga-NODAGA-PCA was investigated in vitro and in vivo using amelanotic (MELUR and A375) and melanin containing (B16-F10) melanoma cell lines. Tumor-bearing mice were prepared by subcutaneous injection of B16-F10, MELUR and A375 melanoma cells into C57BL/6 and SCID mice. 21±2days after tumor cell inoculation and 90min after intravenous injection of the 68Ga-labelledlabeled radiopharmacons whole body PET/MRI scans were performed. 68Ga-NODAGA-PCA and 68Ga-HBED-CC-PCA were produced with excellent radiochemical purity (98%). In vitro experiments demonstrated that after 30 and 90min incubation time 68Ga-NODAGA-PCA uptake of B16-F10 cells was significantly (p≤0.01) higher than the 68Ga-HBED-CC-conjugated PCA accumulation in the same cell line. Furthermore, significant difference (p≤0.01 and 0.05) was found between the uptake of melanin negative and positive cell lines using 68Ga-NODAGA-PCA and 68Ga-HBED-CC-PCA. In vivo PET/MRI studies using tumor models revealed significantly (p≤0.01) higher 68Ga-NODAGA-PCA uptake (SUVmean: 0.46±0.05, SUVmax: 1.96±0.25,T/M ratio: 40.7±4.23) in B16-F10 tumors in contrast to 68Ga-HBED-CC-PCA where the SUVmean, SUVmax and T/M ratio were 0.13±0.01, 0.56±0.11 and 11.43±1.24, respectively. Melanin specific PCA conjugated with NODAGA chelator showed higher specific binding properties than conjugated with HBED-CC. The chemical properties of the bifunctional chelators used for 68Ga-labeling of PCA determine the biological behaviour of the probes. Due to the high specificity and sensitivity 68Ga-labeled PCA molecules are promising radiotracers in melanoma imaging.

Graphene oxide-incorporated pH-responsive folate-albumin-photosensitizer nanocomplex as image-guided dual therapeutics.

The objective of this study was to develop an active-targeted, pH-responsive albumin-photosensitizer-incorporated graphene oxide nanocomplex as an image-guided theranostic agent for dual therapies. Herein, bovine serum albumin (BSA)-cis-aconityl pheophorbide-a (c-PheoA) conjugate was complexed with graphene oxide (GO) at ratios of 1:1, 1:0.5, and 1:0.1 with the mean hydrodynamic diameter of the resulting complex being 100-200nm. Further, with the 1:0.5 ratio, we developed a folate-BSA-c-PheoA conjugate:GO complex incorporated free PheoA (PheoA+GO:FA-BSA-c-PheoA NC) with a mean hydrodynamic diameter of 182.0±33.2nm. The release study showed that the photosensitizer from the nanocomplex was released rapidly at pH5.5 compared to that at pH7.4 when incubated for 24h. Cellular uptake results showed that the PheoA+GO:FA-BSA-c-PheoA NCs was readily taken up by B16F10 and MCF7 cancer cells. In vitro phototoxicity results showed that PheoA+GO:FA-BSA-c-PheoA NC has a higher efficacy against cancer cells than free PheoA, thereby demonstrating the synergistic effect of PS and GO in response to a single laser of 670nm. In vivo and ex vivo bioimaging results showed that fluorescence signals of higher intensity were observed in the tumor area of mice treated with PheoA+GO:FA-BSA-c-PheoA NC than those in the tumor of mice treated with free PheoA, thereby suggesting that the targeted nanocomplex selectively accumulated in the tumor area compared to free PheoA. Through antitumor study, PheoA+GO:FA-BSA-c-PheoA NC showed a synergistic effect in tumor-bearing mice by a single 671nm laser treatment. These results demonstrate that our prepared PheoA+GO:FA-BSA-c-PheoA NC can be used as a theranostic agent in phototherapies and for the photodiagnosis of cancer.

Nano-graphene oxide-mediated In vivo fluorescence imaging and bimodal photodynamic and photothermal destruction of tumors.

Cancer is one of the major life-threatening diseases among human beings. Developing a simple, cost-effective and biocompatible approach to treat cancers using ultra-low doses of light is a grand challenge in clinical cancer treatments. In this study, we report for the first time that nano-sized graphene oxide (GO) exhibits single-photon excitation wavelength dependent photoluminescence in the visible and short near-infrared (NIR) region, suitable for in vivo multi-color fluorescence imaging. We also demonstrate in both in vitro and in vivo experiments to show that nano GO can sensitize the formation of singlet oxygen to exert combined nanomaterial-mediated photodynamic therapeutic (NmPDT) and photothermal therapy (NmPTT) effects on the destruction of B16F0 melanoma tumors in mice using ultra-low doses (∼0.36 W/cm(2)) of NIR (980 nm) light. The average half-life span of the mice treated by the GO-PEG-folate-mediated NmPDT effects is beyond 30 days, which is ∼1.8 times longer than the mice treated with doxorubicin (17 days). Overall, the current study points out a successful example of using GO-PEG-folate nanocomposite as a theranostic nanomedicine to exert simultaneously in vivo fluorescent imaging as well as combined NmPDT and NmPTT effects for clinical cancer treatments.

Microdosed Lipid-Coated (67)Ga-Magnetite Enhances Antigen-Specific Immunity by Image Tracked Delivery of Antigen and CpG to Lymph Nodes.

Development of vaccines to prevent and treat emerging new pathogens and re-emerging infections and cancer remains a major challenge. An attractive approach is to build the vaccine upon a biocompatible NP that simultaneously acts as accurate delivery vehicle and radiotracer for PET/SPECT imaging for ultrasensitive and quantitative in vivo imaging of NP delivery to target tissues/organs. Success in developing these nanovaccines will depend in part on having a "correct" NP size and accommodating and suitably displaying antigen and/or adjuvants (e.g., TLR agonists). Here we develop and evaluate a NP vaccine based on iron oxide-selective radio-gallium labeling suitable for SPECT((67)Ga)/PET((68)Ga) imaging and efficient delivery of antigen (OVA) and TLR 9 agonists (CpGs) using lipid-coated magnetite micelles. OVA, CpGs and rhodamine are easily accommodated in the hybrid micelles, and the average size of the construct can be controlled to be ca. 40 nm in diameter to target direct lymphatic delivery of the vaccine cargo to antigen presenting cells (APCs) in the lymph nodes (LNs). While the OVA/CpG-loaded construct showed effective delivery to endosomal TLR 9 in APCs, SPECT imaging demonstrated migration from the injection site to regional and nonregional LNs. In correlation with the imaging results, a range of in vitro and in vivo studies demonstrate that by using this microdosed nanosystem the cellular and humoral immune responses are greatly enhanced and provide protection against tumor challenge. These results suggest that these nanosystems have considerable potential for image-guided development of targeted vaccines that are more effective and limit toxicity.

Evaluation of the radiolabeled boronic acid-based FAP inhibitor MIP-1232 for atherosclerotic plaque imaging.

Research towards the non-invasive imaging of atherosclerotic plaques is of high clinical priority as early recognition of vulnerable plaques may reduce the incidence of cardiovascular events. The fibroblast activation protein alpha (FAP) was recently proposed as inflammation-induced protease involved in the process of plaque vulnerability. In this study, FAP mRNA and protein levels were investigated by quantitative polymerase chain reaction and immunohistochemistry, respectively, in human endarterectomized carotid plaques. A published boronic-acid based FAP inhibitor, MIP-1232, was synthetized and radiolabeled with iodine-125. The potential of this radiotracer to image plaques was evaluated by in vitro autoradiography with human carotid plaques. Specificity was assessed with a xenograft with high and one with low FAP level, grown in mice. Target expression analyses revealed a moderately higher protein level in atherosclerotic plaques than normal arteries correlating with plaque vulnerability. No difference in expression was determined on mRNA level. The radiotracer was successfully produced and accumulated strongly in the FAP-positive SK-Mel-187 melanoma xenograft in vitro while accumulation was negligible in an NCI-H69 xenograft with low FAP levels. Binding of the tracer to endarterectomized tissue was similar in plaques and normal arteries, hampering its use for atherosclerosis imaging.

Gadolinium-loaded chitosan nanoparticles as magnetic resonance imaging contrast agents for the diagnosis of tumor.

The aim of our study was to prepare gadolinium loaded chitosan nanoparticles (Gd-CSNPs) for magnetic resonance imaging (MRI). The chitosan nanoparticles (CSNPs) were prepared by ionic gelation method with sodium tripolyphosphate. The Gd ions were conjugated to the surface of CSNPs through diethylenetriamine pentaacetic acid (DTPA) using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to obtain Gd-CSNPs. The physicochemical properties of CSNPs and Gd-CSNPs were measured by transmission electron microscope, dynamic light scattering and inductively coupled plasma optical emission spectroscopy, respectively. The cell toxicity evaluation was performed in mouse B16 cells by MTT assay. The T1-weighed magnetic resonance images were measured by a 3.0 T Sigma scanner. The morphologies of the CSNPs and Gd-CSNPs were spherical or ellipsoidal in shape. The mean sizes of the CSNPs and Gd-CSNPs were 110.9 +/- 6.8 nm and 153.0 +/- 7.5 nm, respectively. The zeta potentials of the CSNPs and Gd-CSNPs were 22.30 +/- 0.77 mV and 13.91 +/- 4.26 mV, respectively. The relaxation rates of Gd-CSNPs and Magnevist were 7.509 mM(-1) x s(-1) and 3.052 mM(-1) x s(-1), respectively. The Gd-CSNPs exhibited high T1 relaxivity and no obvious cytotoxicity was observed under the experimental concentrations in mouse B16 melanoma cells. These results indicated that the Gd-CSNPs had great potential as MRI contrast agents for the early diagnosis of tumor.

Preclinical evaluation of an 131I-labeled benzamide for targeted radiotherapy of metastatic melanoma.

Radiolabeled benzamides are attractive candidates for targeted radiotherapy of metastatic melanoma as they bind melanin and exhibit high tumor uptake and retention. One such benzamide, N-(2-diethylamino-ethyl)-4-(4-fluoro-benzamido)-5-iodo-2-methoxy-benzamide (MIP-1145), was evaluated for its ability to distinguish melanin-expressing from amelanotic human melanoma cells, and to specifically localize to melanin-containing tumor xenografts. The binding of [(131)I]MIP-1145 to melanoma cells in vitro was melanin dependent, increased over time, and insensitive to mild acid treatment, indicating that it was retained within cells. Cold carrier MIP-1145 did not reduce the binding, consistent with the high capacity of melanin binding of benzamides. In human melanoma xenografts, [(131)I]MIP-1145 exhibited diffuse tissue distribution and washout from all tissues except melanin-expressing tumors. Tumor uptake of 8.82% injected dose per gram (ID/g) was seen at 4 hours postinjection and remained at 5.91% ID/g at 24 hours, with tumor/blood ratios of 25.2 and 197, respectively. Single photon emission computed tomography imaging was consistent with tissue distribution results. The administration of [(131)I]MIP-1145 at 25 MBq or 2.5 GBq/m(2) in single or multiple doses significantly reduced SK-MEL-3 tumor growth, with multiple doses resulting in tumor regression and a durable response for over 125 days. To estimate human dosimetry, gamma camera imaging and pharmacokinetic analysis was performed in cynomolgus monkeys. The melanin-specific binding of [(131)I]MIP-1145 combined with prolonged tumor retention, the ability to significantly inhibit tumor growth, and acceptable projected human dosimetry suggest that it may be effective as a radiotherapeutic pharmaceutical for treating patients with metastatic malignant melanoma.

Lymph flow from murine footpad tumors before and after sublethal hyperthermia.

The effect of local hyperthermia (43.5 degrees C for 1 h) on lymph flow from B16-F10 tumor-bearing foot pads of C57BL/6 mice was measured by monitoring the clearance of 99mTc-labeled human serum albumin. The foot was represented by a single-compartment model enabling a quantitative computation of lymphatic flow from the tumor to regional lymph nodes. Lymphatic flow from untreated tumors was 0.0059 +/- 0.0011 ml/min cm3 compared to 0.0118 +/- 0.0027 ml/min cm3 lymphatic flow from tumors immediately following heating. Morphological alterations in tumor blood vessels result in their high vascular permeability. The increase in lymphatic clearance from tumors after sublethal hyperthermia is compatible with the increase in interstitial fluid formation in tumors based on Starling's Law.

125I-iodinated benzazepines bind to melanin: implications for the noninvasive localization of pigmented melanomas.

Both the 5-R and the 5-S enantiomers of [125I]2,3,4,5-tetrahydro-8-iodo-3-methyl-5-phenyl-1H-3-benzazepin- 7-ol bind to melanin. The interaction between the 5-R enantiomer and melanin permits visualization of melanomas in mice with a noninvasive imaging procedure. Two lines of evidence suggest that the interaction between iodinated ligands and melanin is not related to the D-1 dopamine receptor, a known target for the 5-R enantiomer: first, melanin binds both enantiomers of the 125I-iodinated benzazepine while the D-1 receptor binds only the 5-R enantiomer; second, the melanin binding site displays only a 5-fold difference in affinity towards the R- and S-enantiomers of SCH 23390 while the D-1 receptor displays a 100-fold difference in affinity towards these two molecules. Because both enantiomers of the iodinated benzazepine bind to a human pigmented melanoma, we propose that such compounds may be of use in the diagnosis of pigmented melanoma: in addition, we discuss the possible application of these molecules as a supplement to existing technology for the localization of pigmented melanomas.