Tb-Doped core-shell-shell nanophosphors for enhanced X-ray induced luminescence and sensitization of radiodynamic therapy.

The development of radiation responsive materials, such as nanoscintillators, enables a variety of exciting new theranostic applications. In particular, the ability of nanophosphors to serve as molecular imaging agents in novel modalities, such as X-ray luminescence computed tomography (XLCT), has gained significant interest recently. Here, we present a radioluminescent nanoplatform consisting of Tb-doped nanophosphors with an unique core/shell/shell (CSS) architecture for improved optical emission under X-ray excitation. Owing to the spatial confinement and separation of luminescent activators, these CSS nanophosphors exhibited bright optical luminescence upon irradiation. In addition to standard physiochemical characterization, these CSS nanophosphors were evaluated for their ability to serve as energy mediators in X-ray stimulated photodynamic therapy, also known as radiodynamic therapy (RDT), through attachment of a photosensitizer, rose bengal (RB). Furthermore, cRGD peptide was used as a model targeting agent against U87 MG glioblastoma cells. In vitro RDT efficacy studies suggested the RGD-CSS-RB in combination with X-ray irradiation could induce enhanced DNA damage and increased cell killing, while the nanoparticles alone are well tolerated. These studies support the utility of CSS nanophosphors and warrants their further development for theranostic applications.

Development of a Pemetrexed/Folic Acid Nanoformulation: Synthesis, Characterization, and Efficacy in a Murine Colorectal Cancer Model.

The folate analogue pemetrexed (PEM) is an approved therapeutic for non-small cell lung cancer and malignant pleural mesothelioma with the potential for broader application in combination therapies. Here, we report the development of a nanoformulation of PEM and its efficacy against the CT26 murine colorectal cancer cell line in vitro and in vivo. Utilizing layer-by-layer deposition, we integrate PEM, along with folic acid (FA), onto a fluorescent polystyrene nanoparticle (NP) substrate. The final nanoformulation (PEM/FA-NP) has a size of ∼40 nm and a zeta potential of approximately -20 mV. Cell uptake studies indicated increased uptake in vitro for the PEM/FA-NP compared to the uncoated NP, likely due to the presence of PEM and FA. Viability studies were performed to determine the potency of the PEM/FA-NP formulation against CT26 cells. Syngeneic CT26 tumors in BALB/c mice showed reduced growth when treated once daily (2.1 mg/kg PEM) for 3 days with PEM/FA-NP versus the vehicle (uncoated) control, with no observable signs of systemic toxicity associated with the nanoformulation. Although the current study size is limited (n = 4 animals for each group), the overall performance and biocompatibility of the PEM/FA-NP observed suggest that further optimization and larger-scale studies may be warranted for this novel formulation.

Formulation of Folate-Modified Raltitrexed-Loaded Nanoparticles for Colorectal Cancer Theranostics.

Multifunctional nanoparticles (NPs) that enable the imaging of drug delivery and facilitate cancer cell uptake are potentially powerful tools in tailoring oncologic treatments. Here we report the development of a layer-by-layer (LbL) formulation of folic acid (FA) and folate antimetabolites that have been well-established for enhanced tumor uptake and as potent chemotherapeutics, respectively. To investigate the uptake of LbL coated NPs, we deposited raltitrexed (RTX) or combined RTX-FA on fluorescent polystyrene NPs. The performance of these NP formulations was evaluated with CT26 murine colorectal cancer (CRC) cells in vitro and in vivo to examine both uptake and cytotoxicity against CRC. Fluorescence microscopy and flow cytometry indicated an increased accumulation of the coated NP formulations versus bare NPs. Ex vivo near-infrared (NIR) fluorescence imaging of major organs suggested the majority of NPs accumulated in the liver, which is typical of a majority of NP formulations. Imaging of the CRC tumors alone showed a higher average fluorescence from NPs accumulated in animals treated with the coated NPs, with the majority of RTX NP-treated animals showing the consistently-highest mean tumoral accumulation. Overall, these results contribute to the development of LbL formulations in CRC theranostic applications.

Inverse-micelle synthesis of doxorubicin-loaded alginate/chitosan nanoparticles and in vitro assessment of breast cancer cytotoxicity.

Naturally-derived polysaccharides, such as alginate and chitosan, can be assembled to form nanocarriers for the delivery of therapeutic agents. Here we exploit the electrostatic complexation of alginate/chitosan in a water-in-oil (w/o) emulsion process to produce doxorubicin (DOX)-loaded nanoparticles (~80 nm) with exceptional spherical morphology and uniformity. This robust synthetic route utilizes an aqueous phase dispersed in a cyclohexane/dodecylamine organic phase and is capable of encapsulating DOX in the nanoparticle solution. The uptake and efficacy of this novel formulation was evaluated in a murine breast cancer cell line, 4T1, with comparable 72 h IC50 values of the nanoparticle solution (0.15 µg/mL) and free DOX (0.13 µg/mL). Overall, the favorable performance, physiochemical properties, and their facile production support these nanocarriers as promising platform for the delivery of aqueous soluble drugs.

Enhancing chemoradiation of colorectal cancer through targeted delivery of raltitrexed by hyaluronic acid coated nanoparticles.

Combined modality therapy incorporating raltitrexed (RTX), a thymidylate synthase inhibitor, and radiation can lead to improved outcome for rectal cancer patients. To increase delivery and treatment efficacy, we formulated a hyaluronic acid (HA) coated nanoparticle encapsulating RTX (HARPs) through layer-by-layer assembly. These particles were determined to have a diameter of ∼115 nm, with a polydispersity index of 0.112 and a zeta potential of -22 mV. Cell uptake in CT26 cells determined through flow cytometry showed a ∼5-fold increase between untargeted and HA-coated particles. Through viability and DNA damage assays, we assessed the potency of the free RTX and HARPs, and found increased DNA damage in cells treated with the RTX-loaded nanoparticles administered concurrently with radiation. In vivo efficacy through tumor growth inhibition was investigated in a syngeneic murine colorectal cancer model. Nanoparticle treatment showed no acute toxicity in vivo, and all treatments showed survival benefits for their respective groups compared to controls. HARPs alone slowed tumor growth, although not significantly. Radiation alone and in combination with the HARPs showed significant growth delay. Notably, the combination treatment significantly hindered tumor progression relative to the HARPs highlighting the benefit of this multipronged treatment. These results provide a foundation for loading RTX in a nanoparticle formulation, and establish a combined radiation and drug dosing schedule to determine optimal tumor growth delay and subsequent treatment efficacy.

Micellar Formulation of Talazoparib and Buparlisib for Enhanced DNA Damage in Breast Cancer Chemoradiotherapy.

Chemoradiation is an effective combined modality therapeutic approach that utilizes principles of spatial cooperation to combat the adaptability associated with cancer and to potentially expand the therapeutic window. Optimal therapeutic efficacy requires intelligent selection and refinement of radiosynergistic pharmaceutical agents, enhanced delivery methods, and temporal consideration. Here, a monodisperse sub-20 nm mixed poloxamer micelle (MPM) system was developed to deliver hydrophobic drugs intravenously, in tandem with ionizing radiation. This report demonstrates in vitro synergy and enhanced radiosensitivity when two molecularly targeted DNA repair inhibitors, talazoparib and buparlisib, are encapsulated and combined with radiation in a 4T1 murine breast cancer model. Evaluation of in vivo biodistribution and toxicity exhibited no reduction in particle accumulation upon radiation and a lack of both acute and chronic toxicities. In vivo efficacy studies suggested the promise of combining talazoparib, buparlisib, and radiation to enhance survival and control tumor growth. Tissue analysis suggests enhanced DNA damage leading to apoptosis, thus increasing efficacy. These findings highlight the challenges associated with utilizing clinically relevant inclusion criteria and treatment protocols because complete tumor regression and extended survival were masked by an aggressively metastasizing model. As with clinical treatment regimens, the findings here establish a need for further optimization of this multimodal platform.

Nanoalginates via Inverse-Micelle Synthesis: Doxorubicin-Encapsulation and Breast Cancer Cytotoxicity.

Crosslinked-biopolymer nanoparticles provide a convenient platform for therapeutic encapsulation and delivery. Here, we present a robust inverse-micelle process to load water-soluble drugs into a calcium-crosslinked alginate matrix. The utility of the resulting nanoalginate (NALG) carriers was assessed by a doxorubicin (DOX) formulation (NALG-DOX) and evaluating its potency on breast cancer cells (4T1). This facile synthesis process produced doxorubicin-containing particles of ~ 83 nm by hydrodynamic size and zeta potential ~ 7.2 mV. The cyclohexane/dodecylamine microemulsion yielded uniform and spherical nanoparticles as observed by electron microscopy. The uptake of the drug from the NALG-DOX formulation in 4T1 cells was observed by fluorescence microscopy employing doxorubicin's inherent fluorescence. Therapeutic efficacy of the NALG-DOX against 4T1 cells was demonstrated qualitatively through a LIVE/DEAD fluorescence assay and quantitatively via cell viability assay (Alamar Blue). In addition, IC50 values were determined, with encapsulated doxorubicin having a slightly higher value. No toxicity of the empty NALG carrier was observed. Overall, these results demonstrate the utility of this synthesis process for encapsulation of hydrophilic therapeutics and NALG to function as a drug carrier.