Induction of complementary immunogenic necroptosis and apoptosis cell death pathways inhibits cancer metastasis and relapse.

Interactions of light-sensitive drugs and materials with Cerenkov radiation-emitting radiopharmaceuticals generate cytotoxic reactive oxygen species (ROS) to inhibit localized and disseminated cancer progression, but the cell death mechanisms underlying this radionuclide stimulated dynamic therapy (RaST) remain elusive. Using ROS-regenerative nanophotosensitizers coated with a tumor-targeting transferrin-titanocene complex (TiO2-TC-Tf) and radiolabeled 2-fluorodeoxyglucose (18FDG), we found that adherent dying cells maintained metabolic activity with increased membrane permeabilization. Mechanistic assessment of these cells revealed that RaST activated the expression of RIPK-1 and RIPK-3, which mediate necroptosis cell death. Subsequent recruitment of the nuclear factors kappa B and the executioner mixed lineage kinase domain-like pseudo kinase (MLKL) triggered plasma membrane permeabilization and pore formation, respectively, followed by the release of cytokines and immunogenic damage-associated molecular patterns (DAMPs). In immune-deficient breast cancer models with adequate stroma and growth factors that recapitulate the human tumor microenvironment, RaST failed to inhibit tumor progression and the ensuing lung metastasis. A similar aggressive tumor model in immunocompetent mice responded to RaST, achieving a remarkable partial response (PR) and complete response (CR) with no evidence of lung metastasis, suggesting active immune system engagement. RaST recruited antitumor CD11b+, CD11c+, and CD8b+ effector immune cells after initiating dual immunogenic apoptosis and necroptosis cell death pathways in responding tumors in vivo. Over time, cancer cells upregulated the expression of negative immune regulating cytokine (TGF-ß) and soluble immune checkpoints (sICP) to challenge RaST effect in the CR mice. Using a signal-amplifying cancer-imaging agent, LS301, we identified latent minimal residual disseminated tumors in the lymph nodes (LNs) of the CR group. Despite increased protumor immunogens in the CR mice, RaST prevented cancer relapse and metastasis through dynamic redistribution of ROS-regenerative TiO2 from bones at the early treatment stage to the spleen and LNs, maintaining active immunity against cancer progression and migration. This study reveals the immune-mechanistic underpinnings of RaST-mediated antitumor immune response and highlights immunogenic reprogramming of tumors in response to RaST. Overcoming apoptosis resistance through complementary necroptosis activation paves the way for strategic drug combinations to improve cancer treatment.

Activation of nano-photosensitizers by Y-90 microspheres to enhance oxidative stress and cell death in hepatocellular carcinoma.

While radioembolization with yttrium-90 (Y-90) microspheres is a promising treatment for hepatocellular carcinoma (HCC), lower responses in advanced and high-grade tumors present an urgent need to augment its tumoricidal efficacy. The purpose of this study was to determine whether clinically used Y-90 microspheres activate light-responsive nano-photosensitizers to enhance hepatocellular carcinoma (HCC) cell oxidative stress and cytotoxicity over Y-90 alone in vitro. Singlet oxygen and hydroxyl radical production was enhanced when Y-90 microspheres were in the presence of several nano-photosensitizers compared to either alone in cell-free conditions. Both the SNU-387 and HepG2 human HCC cells demonstrated significantly lower viability when treated with low activity Y-90 microspheres (0.1-0.2 MBq/0.2 mL) and a nano-photosensitizer consisting of both titanium dioxide (TiO2) and titanocene (TC) labelled with transferrin (TiO2-Tf-TC) compared to Y-90 microspheres alone or untreated cells. Cellular oxidative stress and cell death demonstrated a linear dependence on Y-90 at higher activities (up to 0.75 MBq/0.2 mL), but was significantly more accentuated in the presence of increasing TiO2-Tf-TC concentrations in the poorly differentiated SNU-387 HCC cell line (p < 0.0001 and p = 0.0002 respectively) but not the well-differentiated HepG2 cell line. Addition of TiO2-Tf-TC to normal human hepatocyte THLE-2 cells did not increase cellular oxidative stress or cell death in the presence of Y-90. The enhanced tumoricidal activity of nano-photosensitizers with Y-90 microspheres is a potentially promising adjunctive treatment strategy for certain patient subsets. Applications in clinically relevant in vivo HCC models are underway.

Effects of core titanium crystal dimension and crystal phase on ROS generation and tumour accumulation of transferrin coated titanium dioxide nanoaggregates.

Radionuclide-stimulated therapy (RaST), which is enhanced by Cherenkov radiation, has enabled deep tissue stimulation of UV photosensitizers, providing a new path for cancer treatment. Previous reports have shown UV-active titanium dioxide (TiO2) nanoparticles (NPs) modified with transferrin inhibit tumour growth after orthogonal treatment with Cherenkov radiation-emitting radionuclides such as 18F-fluorodeoxyglucose (FDG). However, poor understanding of TiO2 NP parameters on reactive oxygen species (ROS) generation and particle distribution limits effective therapy. Here we sought to delineate the effects of crystal phase and core TiO2 crystal dimension (cTd) on ROS production and particle morphology. We prepared Transferrin (Tf)-TiO2 nanoaggregates (NAGs) using solvothermally synthesized cTd sizes from 5 to 1000 nm diameter and holo- or apo-transferrin. Holo-transferrin was unable to stabilize TiO2 NPs while apo-transferrin stabilized TiO2 into uniform nanoaggregates (NAGs), which were invariant with differing cTd, averaging 116 ± 1.04 nm for cTds below 100 nm. ROS production increased from 5 to 25 nm cTd, attaining a peak at 25 nm before decreasing with larger sizes. The supra-25 nm ROS production decrease was partially driven by a ~1/r 3 surface area decline. Additionally, amorphous TiO2 of equal core size exhibited a 2.6-fold increase in ROS production compared to anatase NAGs, although limited stability halted further use. Although both 5 and 25 nm anatase cTds formed similarly sized NAGs, 5 nm anatase showed a four-fold higher tumour-to-muscle ratio than the 25 nm NPs in tumour-bearing mice, demonstrating the intricate relationships between physical and biological properties of NAGs. The combined in vivo and ROS results demonstrate that anatase crystals and cTd size of 25 nm or less are ideal particle parameters to balance biodistribution with ROS production efficiency.

Osteotropic Radiolabeled Nanophotosensitizer for Imaging and Treating Multiple Myeloma.

Rapid liver and spleen opsonization of systemically administered nanoparticles (NPs) for in vivo applications remains the Achilles' heel of nanomedicine, allowing only a small fraction of the materials to reach the intended target tissue. Although focusing on diseases that reside in the natural disposal organs for nanoparticles is a viable option, it limits the plurality of lesions that could benefit from nanomedical interventions. Here we designed a theranostic nanoplatform consisting of reactive oxygen (ROS)-generating titanium dioxide (TiO2) NPs, coated with a tumor-targeting agent, transferrin (Tf), and radiolabeled with a radionuclide (89Zr) for targeting bone marrow, imaging the distribution of the NPs, and stimulating ROS generation for cell killing. Radiolabeling of TiO2 NPs with 89Zr afforded thermodynamically and kinetically stable chelate-free 89Zr-TiO2-Tf NPs without altering the NP morphology. Treatment of multiple myeloma (MM) cells, a disease of plasma cells originating in the bone marrow, with 89Zr-TiO2-Tf generated cytotoxic ROS to induce cancer cell killing via the apoptosis pathway. Positron emission tomography/X-ray computed tomography (PET/CT) imaging and tissue biodistribution studies revealed that in vivo administration of 89Zr-TiO2-Tf in mice leveraged the osteotropic effect of 89Zr to selectively localize about 70% of the injected radioactivity in mouse bone tissue. A combination of small-animal PET/CT imaging of NP distribution and bioluminescence imaging of cancer progression showed that a single-dose 89Zr-TiO2-Tf treatment in a disseminated MM mouse model completely inhibited cancer growth at euthanasia of untreated mice and at least doubled the survival of treated mice. Treatment of the mice with cold Zr-TiO2-Tf, 89Zr-oxalate, or 89Zr-Tf had no therapeutic benefit compared to untreated controls. This study reveals an effective radionuclide sensitizing nanophototherapy paradigm for the treatment of MM and possibly other bone-associated malignancies.

Melanocortin 1 Receptor Targeted Imaging of Melanoma With Gold Nanocages and Positron Emission Tomography.

PURPOSE: Melanoma is a lethal skin cancer with unmet clinical needs for targeted imaging and therapy. Nanoscale materials conjugated with targeting components have shown great potential to improve tumor delivery efficiency while minimizing undesirable side effects in vivo. Herein, we proposed to develop targeted nanoparticles for melanoma theranostics. METHOD: In this work, gold nanocages (AuNCs) were conjugated with α-melanocyte-stimulating hormone (α-MSH) peptide and radiolabeled with 64Cu for melanocortin 1 receptor-(MC1R) targeted positron emission tomography (PET) in a mouse B16/F10 melanoma model. RESULTS: Their controlled synthesis and surface chemistry enabled well-defined structure and radiolabeling efficiency. In vivo pharmacokinetic evaluation demonstrated comparable organ distribution between the targeted and nontargeted AuNCs. However, micro-PET/computed tomography (CT) imaging demonstrated specific and improved tumor accumulation via MC1R-mediated delivery. By increasing the coverage density of α-MSH peptide on AuNCs, the tumor delivery efficiency was improved. CONCLUSION: The controlled synthesis, sensitive PET imaging, and optimal tumor targeting suggested the potential of targeted AuNCs for melanoma theranostics.

Hybrid TiO2 -Ruthenium Nano-photosensitizer Synergistically Produces Reactive Oxygen Species in both Hypoxic and Normoxic Conditions.

Photodynamic therapy (PDT) is widely used to treat diverse diseases, but its dependence on oxygen to produce cytotoxic reactive oxygen species (ROS) diminishes the therapeutic effect in a hypoxic environment, such as solid tumors. Herein, we developed a ROS-producing hybrid nanoparticle-based photosensitizer capable of maintaining high levels of ROS under both normoxic and hypoxic conditions. Conjugation of a ruthenium complex (N3) to a TiO2 nanoparticle afforded TiO2 -N3. Upon exposure of TiO2 -N3 to light, the N3 injected electrons into TiO2 to produce three- and four-fold more hydroxyl radicals and hydrogen peroxide, respectively, than TiO2 at 160 mmHg. TiO2 -N3 maintained three-fold higher hydroxyl radicals than TiO2 under hypoxic conditions via N3-facilitated electron-hole reduction of adsorbed water molecules. The incorporation of N3 transformed TiO2 from a dual type I and II PDT agent to a predominantly type I photosensitizer, irrespective of the oxygen content.

In vivo fate tracking of degradable nanoparticles for lung gene transfer using PET and Cerenkov imaging.

Nanoparticles (NPs) play expanding roles in biomedical applications including imaging and therapy, however, their long-term fate and clearance profiles have yet to be fully characterized in vivo. NP delivery via the airway is particularly challenging, as the clearance may be inefficient and lung immune responses complex. Thus, specific material design is required for cargo delivery and quantitative, noninvasive methods are needed to characterize NP pharmacokinetics. Here, biocompatible poly(acrylamidoethylamine)-b-poly(dl-lactide) block copolymer-based degradable, cationic, shell-cross-linked knedel-like NPs (Dg-cSCKs) were employed to transfect plasmid DNA. Radioactive and optical beacons were attached to monitor biodistribution and imaging. The preferential release of cargo in acidic conditions provided enhanced transfection efficiency compared to non-degradable counterparts. In vivo gene transfer to the lung was correlated with NP pharmacokinetics by radiolabeling Dg-cSCKs and performing quantitative biodistribution with parallel positron emission tomography and Cerenkov imaging. Quantitation of imaging over 14 days corresponded with the pharmacokinetics of NP movement from the lung to gastrointestinal and renal routes, consistent with predicted degradation and excretion. This ability to noninvasively and accurately track NP fate highlights the advantage of incorporating multifunctionality into particle design.

Dual-radiolabeled nanoparticle SPECT probes for bioimaging.

A gold nanoparticle was radiolabeled with (125)I and (111)In and functionalized with an MMP9-cleavable peptide to form a multispectral SPECT imaging contrast agent. Peptide cleavage from the nanoprobe by MMP9 was observed in vitro, and distinct pharmacokinetic properties of the contrast agent were observed between tumors with high or low MMP9 expression.

Radioactive 198Au-doped nanostructures with different shapes for in vivo analyses of their biodistribution, tumor uptake, and intratumoral distribution.

With Au nanocages as an example, we recently demonstrated that radioactive (198)Au could be incorporated into the crystal lattice of Au nanostructures for simple and reliable quantification of their in vivo biodistribution by measuring the γ radiation from (198)Au decay and for optical imaging by detecting the Cerenkov radiation. Here we extend the capability of this strategy to synthesize radioactive (198)Au nanostructures with a similar size but different shapes and then compare their biodistribution, tumor uptake, and intratumoral distribution using a murine EMT6 breast cancer model. Specifically, we investigated Au nanospheres, nanodisks, nanorods, and cubic nanocages. After PEGylation, an aqueous suspension of the radioactive Au nanostructures was injected into a tumor-bearing mouse intravenously, and their biodistribution was measured from the γ radiation while their tumor uptake was directly imaged using the Cerenkov radiation. Significantly higher tumor uptake was observed for the Au nanospheres and nanodisks relative to the Au nanorods and nanocages at 24 h postinjection. Furthermore, autoradiographic imaging was performed on thin slices of the tumor after excision to resolve the intratumoral distributions of the nanostructures. While both the Au nanospheres and nanodisks were only observed on the surfaces of the tumors, the Au nanorods and nanocages were distributed throughout the tumors.

Bacterial killing by light-triggered release of silver from biomimetic metal nanorods.

Illumination of noble metal nanoparticles at the plasmon resonance causes substantial heat generation, and the transient and highly localized temperature increases that result from this energy conversion can be exploited for photothermal therapy by plasmonically heating gold nanorods (NRs) bound to cell surfaces. Here, plasmonic heating is used for the first time to locally release silver from gold core/silver shell (Au@Ag) NRs targeted to bacterial cell walls. A novel biomimetic method of preparing Au@Ag core-shell NRs is employed, involving deposition of a thin organic polydopamine (PD) primer onto Au NR surfaces, followed by spontaneous electroless silver metallization, and conjugation of antibacterial antibodies and passivating polymers for targeting to gram-negative and gram-positive bacteria. Dramatic cytotoxicity of S. epidermidis and E. coli cells targeted with Au@Ag NRs is observed upon exposure to light as a result of the combined antibacterial effects of plasmonic heating and silver release. The antibacterial effect is much greater than with either plasmonic heating or silver alone, implying a strong therapeutic synergy between cell-targeted plasmonic heating and the associated silver release upon irradiation. The findings suggest a potential antibacterial use of Au@Ag NRs when coupled with light irradiation, which has not been previously described.

Comparison study of gold nanohexapods, nanorods, and nanocages for photothermal cancer treatment.

Gold nanohexapods represent a novel class of optically tunable nanostructures consisting of an octahedral core and six arms grown on its vertices. By controlling the length of the arms, their localized surface plasmon resonance peaks could be tuned from the visible to the near-infrared region for deep penetration of light into soft tissues. Herein we compare the in vitro and in vivo capabilities of Au nanohexapods as photothermal transducers for theranostic applications by benchmarking against those of Au nanorods and nanocages. While all these Au nanostructures could absorb and convert near-infrared light into heat, Au nanohexapods exhibited the highest cellular uptake and the lowest cytotoxicity in vitro for both the as-prepared and PEGylated nanostructures. In vivo pharmacokinetic studies showed that the PEGylated Au nanohexapods had significant blood circulation and tumor accumulation in a mouse breast cancer model. Following photothermal treatment, substantial heat was produced in situ and the tumor metabolism was greatly reduced for all these Au nanostructures, as determined with (18)F-flourodeoxyglucose positron emission tomography/computed tomography ((18)F-FDG PET/CT). Combined together, we can conclude that Au nanohexapods are promising candidates for cancer theranostics in terms of both photothermal destruction and contrast-enhanced diagnosis.

Chemistry and theranostic applications of radiolabeled nanoparticles for cardiovascular, oncological, and pulmonary research.

Labeling nanoparticles with radionuclides has been widely used to form multifunctional and multivalency agents for various biomedical applications. A variety of nanostructures including inorganic, organic and lipid nanoparticles have been labeled with positron or gamma emitting radioisotopes through versatile radiochemistry in a number of disease models to track their in vivo fate, image biomarkers, and monitor treatment response. This review briefly summarizes the recent applications of nanoparticles labeled with radionuclides for oncological, cardiovascular, and pulmonary theranostics.

Polydopamine-enabled surface functionalization of gold nanorods for cancer cell-targeted imaging and photothermal therapy.

AIM: A novel biomimetic strategy was employed for presenting antibodies on gold nanorods (NRs) to target growth factor receptors on cancer cells for use in photothermal therapy. MATERIALS & METHODS: Polydopamine (PD) was polymerized onto gold NRs, and EGF receptor antibodies (anti-EGFR) were immobilized onto the layer. Cell-binding affinity and light-activated cell death of cancer cells incubated with anti-EGFR-PD-NRs were quantified by optical imaging. RESULTS: PD was deposited onto gold NRs, and antibodies were bound to PD-coated NRs. Anti-EGFR-PD-NRs were stable in media, and were specifically bound to EGFR-overexpressing cells. Illumination of cells targeted with anti-EGFR-PD-NRs enhanced cell death compared with nonirradiated controls and cells treated with antibody-free NRs. CONCLUSION: PD facilitates the surface functionalization of gold NRs with biomolecules, allowing cell targeting and photothermal killing of cancer cells. PD can potentially coat a large variety of nanoparticles with targeting ligands as a strategy for biofunctionalization of diagnostic and therapeutic nanoparticles.

A novel technique for in situ aggregation of Gluconobacter oxydans using bio-adhesive magnetic nanoparticles.

Here, we present a novel technique to immobilize magnetic particles onto whole Gluconobacter oxydans in situ via a synthetic adhesive biomimetic material inspired by the protein glues of marine mussels. Our approach involves simple coating of a cell adherent polydopamine film onto magnetic nanoparticles, followed by conjugation of the polydopamine-coated nanoparticles to G. oxydans which resulted in cell aggregation. After optimization, 21.3 mg (wet cell weight) G. oxydans per milligram of nanoparticle was aggregated and separated with a magnet. Importantly, the G. oxydan aggregates showed high specific activity and good reusability. The facile approach offers the potential advantages of low cost, easy cell separation, low diffusion resistance, and high efficiency. Furthermore, the approach is a convenient platform technique for magnetization of cells in situ by direct mixing of nanoparticles with a cell suspension.

Catechol Redox Induced Formation of Metal Core-Polymer Shell Nanoparticles.

A novel strategy was developed to synthesize polymer-coated metal nanoparticles (NPs) through reduction of metal cations with 3,4-dihydroxyphenylalanine (DOPA)-containing polyethylene glycol (PEG) polymers. Catechol redox chemistry was used to both synthesize metal NPs and simultaneously form a cross-linked shell of PEG polymers on their surfaces. DOPA reduced gold and silver cations into neutral metal atoms, producing reactive quinones that covalently cross-linked the PEG molecules around the surface of the NP. Importantly, these PEG-functionalized metal NPs were stable in physiological ionic strengths and under centrifugation, and hold broad appeal since they absorb and scatter light in aqueous solutions.

Gold nanorods targeted to delta opioid receptor: plasmon-resonant contrast and photothermal agents.

Molecularly targeted gold nanorods were investigated for applications in both diagnostic imaging and disease treatment with cellular resolution. The nanorods were tested in two genetically engineered cell lines derived from the human colon carcinoma HCT-116, a model for studying ligand-receptor interactions. One of these lines was modified to express delta opioid receptor (deltaOR) and green fluorescent protein, whereas the other was receptor free and expressed a red fluorescent protein, to serve as the control. Deltorphin, a high-affinity ligand for deltaOR, was stably attached to the gold nanorods through a thiol-terminated linker. In a mixed population of cells, we demonstrated selective imaging and destruction of receptor-expressing cells while sparing those cells that did not express the receptor. The molecularly targeted nanorods can be used as an in vitro ligand-binding and cytotoxic treatment assay platform and could potentially be applied in vivo for diagnostic and therapeutic purposes with endoscopic technology.

Acid treatment of melanoma cells selects for invasive phenotypes.

Solid tumors become acidic due to hypoxia and upregulated glycolysis. We have hypothesized that this acidosis leads to more aggressive invasive behavior during carcinogenesis (Nature Reviews Cancer 4:891-899, 2004). Previous work on this subject has shown mixed results. While some have observed an induction of metastasis and invasion with acid treatments, others have not. To investigate this, human melanoma cells were acclimated to low pH growth conditions. Significant cell mortality occurred during acclimation, suggesting that acidosis selected for resistant phenotypes. Cells maintained under acidic conditions exhibited a greater range of motility, a reduced capacity to form flank tumors in SCID mice and did not invade more rapidly in vitro, compared to non-selected control cells. However, re-acclimation of these selected cells to physiological pH gave rise to stable populations with significantly higher in vitro invasion. These re-acclimated cells maintained higher invasion and higher motility for multiple generations. Transcriptomic analyses of these three phenotypes revealed significant differences, including upregulation of relevant pathways important for tissue remodeling, cell cycle control and proliferation. These results reinforce the hypothesis that acidosis promotes selection of stable, more invasive phenotypes, rather than inductive changes, which would be reversible.

Metabolite changes in HT-29 xenograft tumors following HIF-1alpha inhibition with PX-478 as studied by MR spectroscopy in vivo and ex vivo.

The hypoxia-inducible transcription factor (HIF-1alpha) plays a central role in tumor development. PX-478 is an experimental anti-cancer drug known to inhibit HIF-1alpha in experimental tumors. The purpose of this study was to identify MRS-visible metabolic biomarkers for PX-478 response prior to phase I/II clinical trials. Single-voxel in vivo localized (1)H spectra were obtained from HT-29 tumor xenografts prior and up to 24 h after treatment with a single dose of PX-478. Profiles of water-soluble and lipophilic metabolites were also examined ex vivo with both (1)H and (31)P spectroscopy for peak identification and to interrogate the underlying biochemistry of the response. The total choline (tCho) resonance was significantly decreased in vivo 12 and 24 h following treatment with PX-478 and this was confirmed with high-resolution (1)H and (31)P MRS. In non-aqueous extracts, significant reductions in cardiolipin, PtdEtn (phosphatidylethanolamine) and PtdI (phosphatidylinositol) were seen in response to PX-478. Although there were trends to a decrease in lactate (and lipid) resonances in vivo and ex vivo, these changes were not significant. This is in contrast to inhibition of in vitro glucose consumption and lactate production by PX-478 in HT-29 cells. The significant and robust change in tCho has identified this as a potential (1)H MRS-visible biomarker for drug response in vivo while high-resolution spectroscopy indicated that GPC, PC, myoI, PE, GPE, CL, PtdEtn and PtdI are potential ex vivo response biomarkers.