Tumor-targeting Ligand Research Articles

Abstract 3236: Rejuvenation of exhausted CAR-T cells with a targeted TLR7 agonist

Abstract CAR T cell therapies have achieved remarkable responses in leukemias, but their potencies in solid tumors have been disappointing. Two proposed causes of CAR T cell failures in solid tumors are: 1) CAR-T cells may become dysfunctional or “exhausted” upon chronic exposure to tumor antigen and/or immunosuppressive factors such as MDSCs, TAMs, Tregs and inhibitory cytokines, and 2) cancer cells mutate to no longer express the tumor antigen recognized by the CAR. We have described a CAR T cell in which the CAR contains the usual activation motifs, but expresses an scFv against FITC instead of the usual scFv to a tumor antigen. The resulting CAR T cell kills and proliferates only when it encounters a cancer cell labeled with FITC. To achieve this FITC labeling, we construct multiple FITC-ligand conjugates (adaptor) by conjugating FITC to one of 10 different low molecular weight (<1kDa) tumor targeting ligands that we have developed over the past 30 years. By bridging between the anti-FITC CAR T cell and the receptor-expressing cancer cell, the adapter mediates rapid CAR T cell engagement and killing of the cancer cell. Moreover, by administering a cocktail of different tumor-specific adapters, we have shown that we can eradicate heterogeneous solid tumors that have lost their primary tumor antigen; i.e. addressing the second cause of CAR T cell failure in solid tumors. To address the first probable cause of CAR T cell failure, we exploit the natural endocytosis of the anti-FITC scFV CAR to deliver a FITC-drug conjugate into the CAR T cell, wherein the attached drug is a potent TLR7 agonist. By concentrating the TLR7 agonist within the CAR T cell, we demonstrate that an exhausted/dysfunctional CAR T cell can be rejuvenated to a functional and tumor-killing state, leading to renewed eradication of the solid tumor. For initial refinement of this strategy in vitro, we generated exhausted CAR T cells by their continuous transfer (every 12 hours) to fresh MDA-MB-231 cells in presence of FITC-folate adaptor while monitoring for appearance of exhaustion markers (PD-1+Tim3+LAG3+) and loss of cell cytotoxicity. We then documented the abilities of both nontargeted and FITC-targeted TLR7 agonists to reverse the above CAR T cell exhaustion. Then, to evaluate whether the same FITC-TLR7 agonists might reverse exhaustion/dysfunction in vivo, we treated NSG mice bearing KB tumors with the same anti-FITC CAR T cells and waited until the above hallmarks of CAR T cell dysfunction/exhaustion became prominent. Upon tail vein injection of the same FITC-TLR7 agonist, we found that the T cell exhaustion/dysfunction markers were reversed and tumor killing was restored. Taken together, we conclude that CAR T cell eradication of solid tumors can be achieved by use of a universal CAR T cell with multiple adapters that allow recognition of multiple tumor antigens plus administration of a CAR T cell targeted TLR7 agonist that can reverse exhaustion/dysfunction induced by the tumor microenvironment. Citation Format: Philip S. Low, Qian Luo, John Victor Napoleon, Boning Zhang. Rejuvenation of exhausted CAR-T cells with a targeted TLR7 agonist [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3236.

Mitochondrion-Directed Nanoparticles Loaded with a Natural Compound and a microRNA for Promoting Cancer Cell Death via the Modulation of Tumor Metabolism and Mitochondrial Dynamics.

Mitochondrial dysfunction may cause cancer and metabolic syndrome. Ellagic acid (abbreviated as E), a phytochemical, possesses anticancer activity. MicroRNA 125 (miR-125) may regulate metabolism. However, E has low aqueous solubility, and miR-125 is unstable in a biological fluid. Hence, this study aimed to develop nanoparticle formulations for the co-treatment of miR-125 and E. These nanoparticles were modified with one mitochondrion-directed peptide and a tumor-targeted ligand, and their modulating effects on mitochondrial dysfunction, antitumor efficacy, and safety in head and neck cancer (HNC) were evaluated. Results revealed that miR-125- and E-loaded nanoparticles effectively targeted cancer cells and intracellular mitochondria. The co-treatment significantly altered cellular bioenergetics, lipid, and glucose metabolism in human tongue squamous carcinoma SAS cells. This combination therapy also regulated protein expression associated with bioenergenesis and mitochondrial dynamics. These formulations also modulated multiple pathways of tumor metabolism, apoptosis, resistance, and metastasis in SAS cells. In vivo mouse experiments showed that the combined treatment of miR-125 and E nanoparticles exhibited significant hypoglycemic and hypolipidemic effects. The combinatorial therapy of E and miR-125 nanoparticles effectively reduced SAS tumor growth. To our best knowledge, this prospective study provided a basis for combining miRNA with a natural compound in nanoformulations to regulate mitochondrial dysfunction and energy metabolism associated with cancer.

Water-soluble superparamagnetic dysprosium-doped iron oxide flowerlike nanoclusters for high-resolution MR imaging

Magnetic nanomaterials have great application prospects in water treatment, disease diagnosis and therapy, surface adsorption and other fields. Herein, the water-soluble, biocompatible and superparamagnetic dysprosium-doped iron oxide flowerlike nanoclusters (DyIO NCs) were synthesized by thermal decomposition of iron oleate and dysprosium oleate in high-boiling solvent of phenyl ether with α,ω-dicarboxyl poly(ethylene glycol) as a capping agent. The size of the nanoclusters and the amount of Dy ions in the clusters can be controlled by simply adjusting the reaction parameters. The DyIO NCs exhibited excellent colloid stability under physiological conditions. The saturation magnetization and transverse relaxivity (r2) of DyIO NCs were significantly improved by the doping of Dy ions. The r2 value of 21 nm Dy0.209Fe2.791O4 NCs was 485.6 mM−1s−1, which is 9 times that of Dy-doped iron oxide nanoparticles (55.6 mM−1s−1), 4 times that of commercial Feridex (123.6 mM−1s−1) and 2 times that of iron oxide nanoclusters (253.9 mM−1s−1). In vivo imaging showed that DyIO NCs exhibited long blood circulation time and excellent enhancement for T2-weighted magnetic resonance imaging. In addition, the DyIO NCs can be functionalized with tumor targeting ligand cyclic Arg-Gly-Asp peptide and fluorescein isothiocyanate isomer I. This study provides an effective strategy for the development of new sensitive nanoprobes and drug delivery systems, and other applications requiring strong magnetic responses.

Multifunctional Molecular Therapeutic Agent for Targeted and Controlled Dual Chemo- and Photodynamic Therapy.

A novel molecular therapeutic agent was designed and synthesized, which contains three functional components, namely, a zinc(II) phthalocyanine substituted with a 2,4-dinitrobenzenesulfonate (DNBS) group as a glutathione (GSH)-activated photosensitizer, a chemo-prodrug based on combretastatin A-4 (CA4) with a singlet oxygen-cleavable aminoacrylate linker, and a biotin moiety as a tumor-targeting ligand. The conjugate showed preferential uptake toward the biotin-receptor-positive HepG2 cells compared with the low biotin-receptor-expressed HCT-116 cells used as the negative control, resulting in the restoration of the fluorescence emission and singlet oxygen generation upon removal of the DNBS group by intracellular GSH. The singlet oxygen produced not only induced a significant photodynamic effect against HepG2 cells but also triggered the cascaded release of the chemocytotoxic CA4, leading to synergistic cytotoxicity as shown by the less-than-unity combination index.

A novel bi-modal probe based on BaHoF5 and Cu-doped QDs with enhanced CT contrast efficiency and fluorescent brightness for tumor-targeting imaging.

In this work, a novel bi-modal imaging probe with enhanced CT contrast efficiency and FL brightness was constructed, in which the combination of a binary CT contrast agent BaHoF5 and Cu-doped QDs served as a vehicle; hyaluronic acid (HA) was employed as a tumor-targeting ligand. With its CT contrast efficiency about 2.1- and 3.9-fold higher than PEG-BaHoF5 and Iohexol, the CT contrast efficiency and the fluorescent brightness of the bi-modal probe were both enhanced. Likewise, its fluorescent brightness is almost 6-fold brighter after Cu-doped QDs loading. The most important contribution of thiswork lies on the proposed strategy. The inherent contradiction of the imaging sensitivity of CT and FL imaging iswell balanced and a great CT/FL bi-modal imaging performance issimultaneously obtained even atlow concentration (400μg/mL) of the probe, which was superior to the previous CT/FL bi-modal probes. Moreover, since BaHoF5 as a binary CT contrast agent was introduced instead of conventional Au and Bi2S3, the CT/FL bi-modal probe would be more suitable for different patients under different operation voltages. In addition, the in vitro tumor cell imaging also demonstrated a good photo-stability, FL brightness, and tumor-targeting capability of the probe, indicating its great potential in practical bi-modal imaging for further tumor diagnosis and therapy. Graphical abstract A novel bi-modal imaging probe with enhanced CT contrast efficiency and FL brightness was fabricated, in which its CT contrast efficiency was about 2.1- and 3.9-fold higher than PEG-BaHoF5 and Iohexol, respectively, and its fluorescent brightness almost 6-fold brighter after Cu-doped QDs loading.

Multifunctional oxygen-enriching nano-theranostics for cancer-specific magnetic resonance imaging and enhanced photodynamic/photothermal therapy

The combination of photodynamic therapy (PDT) and photothermal therapy (PTT) has attracted much interest in recent years, but non-specific distribution of photosensitizers and intrinsic tumor hypoxic microenvironment have continued to limit its therapeutic efficiency. We herein report a nano-theranostic system, denoted as Ce6-CuS/MSN@PDA@MnO2-FA NPs, which combines PDT, PTT, magnetic resonance (MR) imaging with hypoxia-relieving and tumor-targeting functionalities. Central to this design is the use of mussel-inspired polydopamine (PDA) coating to encapsulate the chlorin e6 (Ce6) and copper sulfide nanoparticles (CuS NPs) loaded mesoporous silica nanoparticle (MSN) core. The PDA coating not only acts as pH sensitive gatekeeper to prevent the premature release of Ce6 under non-acidic tumor microenvironment (TME), but also facilitates post-functionalization so that hypoxia-relieving MnO2 nano-sheets and tumor-targeting ligand folic acid-PEG-thiol (FA-PEG-SH) can be decorated on the outer part of the drug system. In vitro and in vivo measurements clearly demonstrated that all these functionalities worked synergistically as expected. The system, having a low dark cytotoxicity, can be effectively internalized by 4T1 cells and decrease the cell viability to 2% upon 660 nm/808 nm laser irradiation. Tumors in 4T1 tumor-bearing mice can almost be completely destroyed in 2 weeks via combined PDT/PTT. Together with the TME-sensitive MR imaging performance demonstrated, Ce6-CuS/MSN@PDA@MnO2-FA NPs represent a multifunctional prototype which holds great potential to be developed into clinical theranostics.

Size-adaptable and ligand (biotin)-sheddable nanocarriers equipped with avidin scavenging technology for deep tumor penetration and reduced toxicity

The conventional active-targeting nano-chemotherapy suffers from poor tumor tissue penetration and non-negligible toxicity due to the size/ligand dilemmas and insufficient target selectivity. In this report, a stimuli-responsive size-adaptable and ligand (biotin)-sheddable drug delivery system (DDS) combined with two-step strategy of biotin-avidin system was designed to seek a balance between tumor targeting and penetration as well as to self-scavenge the nonresponsive nanocarriers in normal tissues. This DDS was composed of ‘multi-seed’ polymeric liposomes (ASL-BIO-MPL) with asulacrine-loaded micelles as seeds in their aqueous cavities. The shell of such liposomes was modified with MMP-9 cleavable polymer-polypeptide functionalized with the tumor targeting ligand biotin. ASL-BIO-MPL could disintegrate into mixture of irregularly-shaped liposomes (~200 nm) and scattered tiny micelles (~40 nm) after incubation with MMP-9. The fluorescence-labeled BIO-MPL could travel to the center of the 4T1 breast tumor spheroids under the action of MMP-9, possibly benefited from the relay of released tiny micelles. Conversely, neither the biotin-modified micelles nor non-MMP-9-responsive multi-seed liposomes could penetrate into the spheroids possibly due to the potent binding-site barrier of biotin and large size, respectively. In tumor-bearing mice, ASL-BIO-MPL exhibited the strongest drug penetrability and thus the optimal inhibition of tumor growth compared to other formulations. Following administration of avidin with a rational dosage regimen, the number of apoptotic cells in normal tissues induced by ASL-BIO-MPL reduced without affecting their targeting effect, suggesting the followed administration of adivin could scavenge the DDS in non-target site. Overall, the size/ligand adapting MPL system combined with two-step strategy of biotin-avidin may provide potential avenues for nanocarriers to enhance deep tumor tissue targeting and protect normal tissues.

The impact of size and surface ligand of gold nanorods on liver cancer accumulation and photothermal therapy in the second near-infrared window

Gold nanorods (GNRs) with longitudinal surface plasmon resonance (LSPR) peaks in second near-infrared (NIR-II) window have attracted a great amount of attention as photothermal transducer because of their inherently excellent photothermal transition efficiency, high biocompatibility and versatile surface functionalization. One key question for the application of these GNRs against tumors in vivo is which size/shape and surface ligand conjugation are promising for circulation and tumor targeting. In this study, we prepared a series of gold nanorods (GNRs) of similar aspect ratio and LSPR peaks, and thus similar photothermal transfer efficiency under irradiation of 980 nm laser, but with tunable size in width and length. The obtained GNRs were subjected to surface modification with PEG and tumor targeting ligand lactoferrin. With these tailor-designed GNRs in hand, we have the chance to study the impact of dimension and surface property of the GNRs on their internalization via tumor cells, photothermal cytotoxicity in vitro, blood circulation and tissue distribution pattern in vivo. As a result, the GNRs with medium size (70 nm in length and 11.5 nm in width) and surface PEG/LF modification (GNR70@PEG-LF) exhibit the fastest cell internalization via HepG2 cells and best photothermal outcome in vitro. The GNR70@PEG-LF also display long circulation time and the highest tumor accumulation in vivo, due to the synergetic effect of surface coating and dimension. Finally, tumor ablation ability of the GNRs under irradiation of 980 nm light were validated on mice xenograft model, suggesting their potential photothermal therapy against cancer in NIR-II window.

Combined Modality Therapy Based on Hybrid Gold Nanostars Coated with Temperature Sensitive Liposomes to Overcome Paclitaxel-Resistance in Hepatic Carcinoma.

In this study, we prepared gold nanostar (GNS) composite nanoparticles containing siRNA of cyclooxygenase-2(siCOX-2) that were modified by tumor targeting ligand 2-deoxyglucose (DG) and transmembrane peptide 9-poly-D-arginine (9R) to form siCOX-2(9R/DG-GNS). Paclitaxel loaded temperature sensitive liposomes (PTX-TSL) were surface-modified to produce PTX-TSL-siCOX-2(9R/DG-GNS) displaying homogeneous star-shaped structures of suitable size (293.93 nm ± 3.21) and zeta potentials (2.47 mV ± 0.22). PTX-TSL-siCOX-2(9R/DG-GNS) had a high thermal conversion efficiency under 808 nm laser radiation and a superior transfection efficiency, which may be related to the targeting effects of DG and increased heat induced membrane permeability. COX-2 expression in HepG2/PTX cells was significantly suppressed by PTX-TSL-siCOX-2(9R/DG-GNS) in high temperatures. The co-delivery system inhibited drug-resistant cell growth rates by ≥77% and increased the cell apoptosis rate about 47% at elevated temperatures. PTX-TSL and siCOX-2 loaded gold nanostar particles, therefore, show promise for overcoming tumor resistance.

Targeting the EGF-Receptor and CD38 in Solid and Haematological Malignancies with Nanobody-Based Heavy Chain Antibodies and AAV Vectors

Abstract Background Although monoclonal antibodies binding the EGF-receptor (cetuximab, panitumumab) or CD38 (daratumumab) show therapeutic efficacy, many cancer patients still develop resistance. Therefore, new therapies are necessary. Nanobodies are highly soluble immunoglobulin domains derived from heavy chain antibodies (hcAbs) that naturally occur in camelids. Replication defective AAV (adeno associated virus) vectors, when equipped with a tumor targeting ligand, can be used for the delivery of (suicide) genes to tumor cells. Methods We generated recombinant chimeric nanobody-based human IgG1 hcAbs, by fusing EGFR- or CD38-specific nanobodies derived from immunized llamas to the hinge, CH2 and CH3 domains of human IgG1 (1, 2). To promote cytotoxic effector functions we introduced CDC (complement dependent cytotoxicity)-enhancing and ADCC (antibody-dependent cell-mediated cytotoxicity)-enhancing mutations into the CH2 and CH3 domains of human IgG1. As a second strategy to target CD38-overexpressing cells, we inserted CD38-specific nanobodies into the VP1 capsid protein of AAV2 (3). We analyzed the capacity of these constructs to target EGFR and CD38 in tumor cell lines and multiple myeloma (MM) patient bone marrow samples using proliferation, CDC, ADCC and AAV-transduction assays. Results EGFR-specific hcAbs effectively blocked the proliferation of tumor cell lines (HNSCC, head and neck squamous cell carcinoma and mCRC, metastatic colorectal cancer) expressing WT EGFR and cetuximab/panitumumab-escape variants of EGFR. EGFR- and CD38-specific hcAbs mediated effective ADCC towards tumor cell lines (HNSCC, mCRC and MM respectively). HcAbs carrying the hexabody mutation were more potent at inducing CDC than parental hcAbs. Display of CD38-specific nanobodies on the AAV capsid resulted in a 20- to 100-fold enhanced transduction of myeloma cells in patient bone marrow samples. Some of these results have recently been published (1, 2) or accepted for publication (3). We will present updated data on tumor cell lines and patient samples. Conclusion Nanobody-based hcAbs and nanobody-displaying AAV hold promise as novel tools to target solid and hematological tumors. Legal entity responsible for the study Friedrich Koch-Nolte. Funding DFG (German Research Foundation) and SFB (Collaborative Research Centres). Disclosure The author has declared no conflicts of interest.

Dual-Functionalized Janus Mesoporous Silica Nanoparticles with Active Targeting and Charge Reversal for Synergistic Tumor-Targeting Therapy.

Janus nanoparticles with an anisotropic feature concentrated multiple properties on a single carrier, providing synergistic effects. In this study, dual-functionalized Janus nanoparticles (HA-JMSN/DOX-DMMA) were constructed with a tumor-targeting ligand (hyaluronic acid, HA) modified on the one side and a charge reversal group (2,3-dimethylmaleic anhydride, DMMA) on the other side. The drug release of HA-JMSN/DOX-DMMA was positively correlated with the acidity of the environment. The cytotoxicity and cell uptake of HA-JMSN/DOX-DMMA were superior to the isotropous nanoparticles. The endocytosis pathway of HA-JMSN/DOX-DMMA involved the clathrin-mediated endocytosis (HA) and the micropinocytosis (DMMA) at the same time, which indicated that they both participated in the interaction between nanoparticles and tumor cells. After being injected intravenously in mice, the distribution of HA-JMSN/DOX-DMMA in tumor was enhanced significantly. The antitumor therapy study in vivo showed that HA-JMSN/DOX-DMMA inhibited tumor growth and improved the survival rate of tumor-bearing mice effectively. In general, HA-JMSN/DOX-DMMA could take the synergistic effect of active targeting and charge reversal to deliver drug in tumor cells and kill them efficiently, which was a promising antitumor nanodrug.

Transferrin-Functionalized Microemulsions Coloaded with Coix Seed Oil and Tripterine Deeply Penetrate To Improve Cervical Cancer Therapy.

Tumor-targeted ligand modification and nanosized coloaded drug delivery systems are promising for cancer therapy. In this study, we showed that coix seed oil and tripterine coloaded microemulsions with a transferrin modification (Tf-CT-MEs) could improve the treatment of cervical cancer. Tf-CT-MEs exhibited good stability in serum and a notably synergistic antiproliferation effect. In the HeLa xenograft tumor-bearing mouse model, Tf-CT-MEs accumulated at tumor sites and penetrated deeply in tumor tissues. Tf-CT-MEs had superior anticancer efficacy in vivo, which greatly slowed the growth of tumors (***p < 0.001 vs saline). We also found that Tf-CT-MEs inhibited tumor cell proliferation, enhanced antiangiogenesis, and induced apoptosis by regulating bax/bcl-2 and the activating caspase-3 pathway. Tf-CT-MEs decreased by 27.7, 26.9, 61.2, and 42.5% of concentrations of TGF-β1, CCL2, TNF-α, and IL-6 in serum, respectively. In addition, Tf-CT-MEs showed little toxicity in vital organs. These results were due to the improved drug delivery efficiency. Collectively, Tf-CT-MEs enhance tumor-targeting, facilitate deep penetration of drugs, and have promising potential as an efficient treatment for cervical cancer.

Construction of a Targeting Nanoparticle of 3',3″-Bis-Peptide-siRNA Conjugate/Mixed Lipid with Postinserted DSPE-PEG2000-cRGD.

The cyclic Arg-Gly-Asp (cRGD) peptides are widely used as tumor-targeting ligands due to their specific binding ability to integrin αvβ3, which is overexpressed on the surface of various cancer cells and the endothelial cells of new blood vessels within tumor tissues. In this paper, the postinsertion strategy of DSPE-PEG2000-cRGD has been applied to the nanoparticles of 3',3″-bis-peptide-siRNA (pp-siRNA) encapsulated by gemini-like cationic lipid (CLD) and neutral cytosin-1-yl lipid (DNCA) from our lab. It was confirmed that the nanoparticles of pp-siRNA/CLD/DNCA/DSPE-PEG2000-cRGD (PCNR) were able to specifically target tumor cells with highly expressed integrin αvβ3; moreover, it efficiently downregulated the levels of BRAF mRNA and the BRAF protein and inhibited cell proliferation in A375 cells, in comparison with the nontargeted nanocomplex of pp-siRNA/CLD/DNCA/cRAD (PCNA). The uptake pathways of PCNR are mostly dependent on CvME-mediated endocytosis and macropinocytosis in A375 cells, which could bypass lysosome or quickly lead to the lysosomal escape to reduce siRNA degradation. Finally, the biodistribution study showed that PCNR exhibited a high ability to accumulate in tumor tissues. These results suggest that the nanocomplex of PCNR is promising to be highly effective in the treatment of melanomas including their mutation.

Peptide-Decorated Ultrasmall Superparamagnetic Nanoparticles as Active Targeting MRI Contrast Agents for Ovarian Tumors

Magnetic resonance imaging (MRI) is widely applied in medical research and diagnosis, and a MRI contrast medium plays a crucial role in improving the sensitivity of detection. Ultrasmall superparamagnetic iron oxides (USPIOs) exhibit the potential as a T2 enhancement contrast medium for MRI due to their excellent magnetic response performance; however, to endow them with specific tumor targetability, long-term circulation performance has always been a hot topic in this field. In this study, a well-designed procedure of chemical coprecipitation, surface modification, and peptide grafting was applied to prepare the active tumor-targeting USPIOs@F127-WSG, in which Pluronic F127 (F127) and the peptide WSGPGVWGASVK (peptide-WSG) were selected as the template agent and the ovarian tumor-targeting ligand, respectively. The results showed that single USPIOs@F127-WSG particles were Fe3O4 nanoparticles regulated by the confinement effect of F127 micelles with a uniform globular morphology and size (∼9 nm), and peptide-WSG was grafted for their tumor targetability. USPIOs@F127-WSG particles presented superparamagnetic behavior with high T2 relaxivity (r2 = 278.15 mM-1 s-1) and in vitro targetability for SKOV-3 cells due to the special binding between peptide-WSG and specific receptors of SKOV-3. The test results in vivo verified the targetability of USPIOs@F127-WSG by their specific aggregation in the tumor regions, leading to the T2-weighted MRI contrast enhancement. These outstanding properties indicate that USPIOs@F127-WSG have great potential to be applied as the active tumor-targeting contrast agent for MRI.

Application of silicon dioxide nanoparticles modified with tumor-targeting ligands for cellular delivery of nucleoside triphosphate analogues

A key advantage of amino-modified SiO2 nanoparticles for delivery of phosphorylated nucleosides is a broad possibility for functionalization. It can be modified with ligands currently investigated in targeted drug delivery. To improve the efficacy for intracellular delivery, SiO2 nanoparticles were functionalized with tumor-targeting ligands folic acid, biotin or 5-fluorouracil. Studies of accumulation of these conjugates in HCT116 colon carcinoma cells revealed that the uptake of modified conjugates was significantly bigger compared to unmodified nanoparticles, with the biotinylated conjugate as the preferred compound. The nanocomposites of biotin modified SiO2 and 2′,3′-dideoxyuridine triphosphate showed a pronounced antiproliferative potency relative to the unmodified nanocomposites. Thus, multi-functionalization of SiO2 nanoparticle based conjugates has a major potential for delivery of nucleoside triphosphate analogues, therefore tentatively enhancing their bioactivity.

Mixed Lanthanide Oxide Nanoparticles Coated with Alginate-Polydopamine as Multifunctional Nanovehicles for Dual Modality: Targeted Imaging and Chemotherapy.

Integrating anticancer drugs and diagnostic agents in a polymer nanosystem is an emerging and promising strategy for improving cancer treatment. However, the development of multifunctional nanoparticles (NPs) for an "all-in-one" platform characterized by specific targeting, therapeutic efficiency, and imaging feedback remains an unmet clinical need. In this study, pH-responsive mixed-lanthanide-based multifunctional NPs were fabricated based on simple metal-ligand interactions for simultaneous cancer cell imaging and drug delivery. We investigated two new systems of alginate-polydopamine complexed with either terbium/europium or dysprosium/erbium oxide NPs (Tb/Eu@AlgPDA or Dy/Er@AlgPDA NPs). Tb/Eu@AlgPDA NPs were then functionalized with the tumor-targeting ligand folic acid (FA) and loaded with the anticancer drug doxorubicin (DOX) to form FA-Tb/Eu@AlgPDA-DOX NPs. Using such systems, the mussel-inspired property of PDA was introduced to improve tumor targetability and penetration, in addition to active targeting (via FA-folate receptor interactions). Determining the photoluminescence efficiency showed that the Tb/Eu@AlgPDA system was superior to the Dy/Er@AlgPDA system, presenting intense and sharp emission peaks on the fluorescence spectra. In addition, compared to Dy/Er@AlgPDA NPs (82.4%), Tb/Eu@AlgPDA NPs exhibited negligible cytotoxicity with >93.3% HeLa cell viability found in MTT assays at NP concentrations of up to 0.50 mg/mL and high biocompatibility when incubated with zebrafish (Danio rerio) embryos and larvae. The FA-Tb/Eu@AlgPDA-DOX system exhibited a pH-responsive and sustained drug-release pattern. In a spheroid model of HeLa cells, the FA-Tb/Eu@AlgPDA-DOX system showed a better penetration efficiency and spheroid growth-inhibitory effect than free DOX. After incubation with zebrafish embryos, the FA-Tb/Eu@AlgPDA-DOX system also showed improved antitumor efficacies versus the other experimental groups in HeLa tumor cell xenografted zebrafish. Therefore, our results suggested that FA-Tb/Eu@AlgPDA-DOX NPs are promising multifunctional nanocarriers with therapeutic capacity for tumor targeting and penetration.

Fabrication of folate‐phytosterol‐carboxymethyl cellulose nanoparticles derived from plant material as carrier of anticancer drug

In this work, folate-phytosterol-carboxymethyl cellulose nanoparticles (FPCMC NPs) derived from plant material were fabricated and investigated as carrier of hydrophobic anticancer drugs. Firstly, hydrophobic phytosterol was grafted onto the framework of water-soluble carboxymethyl cellulose. Then folate, as tumor-targeting ligand, was coupled to the phytosterol-carboxymethyl cellulose to become self-assembled FPCMC NPs. The physicochemical properties of the fabricated FPCMC NPs were characterized. Doxorubicin (DOX) was selected as model anticancer drug that was entrapped in prepared FPCMC NPs with satisfactory loading content (7%) and loading efficiency (71.2%). The in vitro drug release test showed that the release amount of DOX from drug-loaded FPCMC NPs at pH 5.3 was much higher than that at pH 6.5 or pH 7.4. The research results indicated that the fabricated FPCMC NPs had the potential as nanocarrier of hydrophobic anticancer drugs for further experimental study.

Synthesis, characterisation and bioconjugation of [109Cd]CdSe/ZnS core/shell quantum dots as “proof of principle” for the potential development of an anti-cancer theranostic

This work describes the synthesis and analysis of radioactive multifunctional nanoparticle probes based on CdSe/ZnS semiconductor quantum dots (QDs) for potential in vitro and in vivo anti-cancer theranostics. The synthetic design involved the radiosynthesis of luminescent CdSe/ZnS QDs using the SPECT imaging agent Cd-109 as the cadmium elemental source. These radioactive nanoparticle materials were then rendered water dispersed and bioconjugated to the tumor-targeting ligand Rituximab.

Abstract 4775: Optimizing the potency and dosing design for ARO-HIF2: An RNAi therapeutic for clear cell renal cell carcinoma

Abstract Background: Clear cell renal cell carcinoma (ccRCC) frequently involves the inactivation of the von Hippel-Lindau (VHL) tumor suppressor. Loss of VHL functions lead to the accumulation of hypoxia-inducible factors (HIFs). HIF2α has been regarded as a key tumorigenic driver of ccRCC and an attractive therapeutic target. Arrowhead has developed a RNA interference therapeutic (HIF2 RNAi) to selectively target and silence HIF2α expression, using a proprietary targeted-RNAi molecule (TRiM™) delivery platform for the treatment of ccRCC. The TRiM™ based Hif2 construct comprises a highly potent RNAi trigger using stabilization chemistries, targeting ligands to facilitate delivery, and structures to enhance pharmacokinetics (PK). The optimization of HIF2 RNAi to enhance the potency and safety profile to maximize the potential clinical success is described. Methods: Functional optimization of HIF2 RNAi was evaluated in an orthotopic ccRCC tumor xenograft model established with A498 ccRCC cells that stably expresses the reporter gene SEAP (secreted embryonic alkaline phosphatase) as a serum biomarker for monitoring tumor growth. HIF2 RNAi was delivered by intravenous injections. HIF2α gene silencing was evaluated by isolating tumor RNA and measuring relative gene expression by qRT-PCR. Results: We demonstrate that to achieve deep HIF2α mRNA knockdown (KD), functionalizing HIF2 RNAi with PK enhancement and tumor targeting ligand (TTL) is required. Optimization of the HIF2 RNAi construct enabled a 10-fold improvement in potency. Evaluation of a loading dose regimen improved overall HIF2α mRNA KD compared to a single administration of equal total dosage. Utilizing this strategy, we demonstrated that silencing of HIF2α mRNA (85% KD) resulted in tumor growth inhibition in the A498 xenograft model. Significant improvement in overall survival was also seen in a patient derived xenograft model. Histology evaluation of tumor samples revealed extensive tumor destruction with clusters of apoptotic cells and necrosis. Follow-up studies suggest that loading doses can be administered four hours apart without loss in potency. This allows dosing to be completed in one day and may be more acceptable in clinical settings. The maximum HIF2α mRNA KD after a single dose of HIF2 RNAi was achieved about 7 days after dosing and sustained for about one week in the xenograft model. This suggests that dosing can likely be less frequent in clinical settings. An exploratory toxicity study in rats predicts a wide safety margin. Conclusions: We demonstrate that the TRiM™ delivery platform can be utilized to deliver a RNAi therapeutic selectively targeting HIF2α for the treatment of ccRCC. This represents a novel therapeutic approach either as a monotherapy or in combination with other therapies in seeking better tolerated and/or more effective treatment for ccRCC. Citation Format: So C. Wong, Anthony Nicholas, Jeff Carlson, Dongxu Shu, Che Liu, Rui Chu, Amanda Frankiewicz, Holly Hamilton, Casi Schienebeck, Aaron Andersen, Matthew Fowler-Watters, Stephanie Bertin, Xiaokai Li, Bo Chen, Josh Schumacher, Julia Hegge, Bruce Given, Zhen Li. Optimizing the potency and dosing design for ARO-HIF2: An RNAi therapeutic for clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4775.

Theranostic Nanoparticles for RNA-Based Cancer Treatment.

Certain genetic mutations lead to the development of cancer through unchecked cell growth and division. Cancer is typically treated through surgical resection, radiotherapy, and small-molecule chemotherapy. A relatively recent approach to cancer therapy involves the use of a natural process wherein small RNA molecules regulate gene expression in a pathway known as RNA interference (RNAi). RNA oligomers pair with a network of proteins to form an RNA-induced silencing complex, which inhibits the translation of mRNA into proteins, thereby controlling the expression of gene products. Synthetically produced RNA oligomers may be designed to target and silence specific oncogenes to provide cancer therapy. The primary challenges facing the use of the RNAi pathway for cancer therapy are the safe and efficacious delivery of RNA payloads and their release at pertinent sites within disease-causing cells. Nucleases are abundant in the bloodstream and intracellular environment, and therapeutic RNA sequences often require a suitable carrier to provide protection from degradation prior to reaching their site of action in the body. The use of metal core nanoparticles (NPs) serving as targeted delivery vehicles able to shield and direct RNA payloads to their intended destinations have recently gained favor. Biological barriers present in the body establish a size prerequisite for drug delivery vehicles; to overcome recognition by the body's immune system and to gain access to intracellular environments, drug carriers must be small (< 100 nm). Iron oxide and gold core NPs can be synthesized with a high degree of control to create uniform ultrasmall drug delivery vehicles capable of bypassing key biological barriers. While progress is being made in size control of liposomal and polymer NPs, such advances still lag in comparison to the exquisite tunability and time stability of size engineering achievable with metal core NPs at bulk scales. Further, unlike lipid- and viral-based transfection agents, the biodistribution of metal core NPs can be traced using noninvasive imaging techniques that capitalize on the interaction of electromagnetic radiation and the inorganic atoms at the core of the NPs. Finally, metal core NPs have been shown to match the transfection efficiency of conventional RNA-delivery vehicles while also providing less immunogenicity and minimal side effects through the addition of tumor-targeting ligands on their surface. This Account reviews recent advances in the use of iron oxide and gold NPs for RNAi therapy. An overview of the different types of RNA-based therapies is provided along with a discussion of the advantages and current limitations of the technique. We highlight design considerations for the use of iron oxide and gold NP carriers in RNAi, including a discussion of the importance of size and its role in traversing biological barriers, NP surface modifications required for targeted delivery and RNA payload release, and auxiliary properties supporting imaging functionality for treatment monitoring. Applications of NPs for combination therapies including the pairing of RNAi with chemotherapy, photothermal therapy, immunotherapy, and radiotherapy are explored through examples. Finally, future perspectives are provided with a focus on the current limitations and the potential for clinical translation of iron oxide and gold NPs in RNAi therapy.