Temoporfin-Conjugated PEGylated Poly(N,N-dimethylacrylamide)-Coated Upconversion Colloid for NIR-Induced Photodynamic Therapy of Pancreatic Cancer.

Photodynamic therapy (PDT) has the potential to cure pancreatic cancer with minimal side effects. Visible wavelengths are primarily used to activate hydrophobic photosensitizers, but in clinical practice, these wavelengths do not sufficiently penetrate deeper localized tumor cells. In this work, NaYF4:Yb3+,Er3+,Fe2+ upconversion nanoparticles (UCNPs) were coated with polymer and labeled with meta-tetra(hydroxyphenyl)chlorin (mTHPC; temoporfin) to enable near-infrared light (NIR)-triggered PDT of pancreatic cancer. The coating consisted of alendronate-terminated poly[N,N-dimethylacrylamide-co-2-aminoethylacrylamide]-graft-poly(ethylene glycol) [P(DMA-AEM)-PEG-Ale] to ensure the chemical and colloidal stability of the particles in aqueous physiological fluids, thereby also improving the therapeutic efficacy. The designed particles were well tolerated by the human pancreatic adenocarcinoma cell lines CAPAN-2, PANC-1, and PA-TU-8902. After intratumoral injection of mTHPC-conjugated polymer-coated UCNPs and subsequent exposure to 980 nm NIR light, excellent PDT efficacy was achieved in tumor-bearing mice.

Toxicity of Large and Small Surface-Engineered Upconverting Nanoparticles for In Vitro and In Vivo Bioapplications.

In this study, spherical or hexagonal NaYF4:Yb,Er nanoparticles (UCNPs) with sizes of 25 nm (S-UCNPs) and 120 nm (L-UCNPs) were synthesized by high-temperature coprecipitation and subsequently modified with three kinds of polymers. These included poly(ethylene glycol) (PEG) and poly(N,N-dimethylacrylamide-co-2-aminoethylacrylamide) [P(DMA-AEA)] terminated with an alendronate anchoring group, and poly(methyl vinyl ether-co-maleic acid) (PMVEMA). The internalization of nanoparticles by rat mesenchymal stem cells (rMSCs) and C6 cancer cells (rat glial tumor cell line) was visualized by electron microscopy and the cytotoxicity of the UCNPs and their leaches was measured by the real-time proliferation assay. The comet assay was used to determine the oxidative damage of the UCNPs. An in vivo study on mice determined the elimination route and potential accumulation of UCNPs in the body. The results showed that the L- and S-UCNPs were internalized into cells in the lumen of endosomes. The proliferation assay revealed that the L-UCNPs were less toxic than S-UCNPs. The viability of rMSCs incubated with particles decreased in the order S-UCNP@Ale-(PDMA-AEA) > S-UCNP@Ale-PEG > S-UCNPs > S-UCNP@PMVEMA. Similar results were obtained in C6 cells. The oxidative damage measured by the comet assay showed that neat L-UCNPs caused more oxidative damage to rMSCs than all coated UCNPs while no difference was observed in C6 cells. An in vivo study indicated that L-UCNPs were eliminated from the body via the hepatobiliary route; L-UCNP@Ale-PEG particles were almost eliminated from the liver 96 h after intravenous application. Pilot fluorescence imaging confirmed the limited in vivo detection capabilities of the nanoparticles.

Temoporfin-Conjugated Upconversion Nanoparticles for NIR-Induced Photodynamic Therapy: Studies with Pancreatic Adenocarcinoma Cells In Vitro and In Vivo.

Upconverting nanoparticles are interesting materials that have the potential for use in many applications ranging from solar energy harvesting to biosensing, light-triggered drug delivery, and photodynamic therapy (PDT). One of the main requirements for the particles is their surface modification, in our case using poly(methyl vinyl ether-alt-maleic acid) (PMVEMA) and temoporfin (THPC) photosensitizer to ensure the colloidal and chemical stability of the particles in aqueous media and the formation of singlet oxygen after NIR irradiation, respectively. Codoping of Fe2+, Yb3+, and Er3+ ions in the NaYF4 host induced upconversion emission of particles in the red region, which is dominant for achieving direct excitation of THPC. Novel monodisperse PMVEMA-coated upconversion NaYF4:Yb3+,Er3+,Fe2+ nanoparticles (UCNPs) with chemically bonded THPC were found to efficiently transfer energy and generate singlet oxygen. The cytotoxicity of the UCNPs was determined in the human pancreatic adenocarcinoma cell lines Capan-2, PANC-01, and PA-TU-8902. In vitro data demonstrated enhanced uptake of UCNP@PMVEMA-THPC particles by rat INS-1E insulinoma cells, followed by significant cell destruction after excitation with a 980 nm laser. Intratumoral administration of these nanoconjugates into a mouse model of human pancreatic adenocarcinoma caused extensive necrosis at the tumor site, followed by tumor suppression after NIR-induced PDT. In vitro and in vivo results thus suggest that this nanoconjugate is a promising candidate for NIR-induced PDT of cancer.

Polymer-coated hexagonal upconverting nanoparticles: chemical stability and cytotoxicity.

Large (120 nm) hexagonal NaYF4:Yb, Er nanoparticles (UCNPs) were synthesized by high-temperature coprecipitation method and coated with poly(ethylene glycol)-alendronate (PEG-Ale), poly (N,N-dimethylacrylamide-co-2-aminoethylacrylamide)-alendronate (PDMA-Ale) or poly(methyl vinyl ether-co-maleic acid) (PMVEMA). The colloidal stability of polymer-coated UCNPs in water, PBS and DMEM medium was investigated by dynamic light scattering; UCNP@PMVEMA particles showed the best stability in PBS. Dissolution of the particles in water, PBS, DMEM and artificial lysosomal fluid (ALF) determined by potentiometric measurements showed that all particles were relatively chemically stable in DMEM. The UCNP@Ale-PEG and UCNP@Ale-PDMA particles were the least soluble in water and ALF, while the UCNP@PMVEMA particles were the most chemically stable in PBS. Green fluorescence of FITC-Ale-modified UCNPs was observed inside the cells, demonstrating successful internalization of particles into cells. The highest uptake was observed for neat UCNPs, followed by UCNP@Ale-PDMA and UCNP@PMVEMA. Viability of C6 cells and rat mesenchymal stem cells (rMSCs) growing in the presence of UCNPs was monitored by Alamar Blue assay. Culturing with UCNPs for 24 h did not affect cell viability. Prolonged incubation with particles for 72 h reduced cell viability to 40%-85% depending on the type of coating and nanoparticle concentration. The greatest decrease in cell viability was observed in cells cultured with neat UCNPs and UCNP@PMVEMA particles. Thanks to high upconversion luminescence, high cellular uptake and low toxicity, PDMA-coated hexagonal UCNPs may find future applications in cancer therapy.

Chemical and Colloidal Stability of Polymer-Coated NaYF4:Yb,Er Nanoparticles in Aqueous Media and Viability of Cells: The Effect of a Protective Coating.

Upconverting nanoparticles (UCNPs) are of particular interest in nanomedicine for in vivo deep-tissue optical cancer bioimaging due to their efficient cellular uptake dependent on polymer coating. In this study, particles, ca. 25 nm in diameter, were prepared by a high-temperature coprecipitation of lanthanide chlorides. To ensure optimal dispersion of UCNPs in aqueous milieu, they were coated with three different polymers containing reactive groups, i.e., poly(ethylene glycol)-alendronate (PEG-Ale), poly(N,N-dimethylacrylamide-co-2-aminoethylacrylamide)-alendronate (PDMA-Ale), and poly(methyl vinyl ether-co-maleic acid) (PMVEMA). All the particles were characterized by TEM, DLS, FTIR, and spectrofluorometer to determine the morphology, hydrodynamic size and ξ-potential, composition, and upconversion luminescence. The degradability/dissolution of UCNPs in water, PBS, DMEM, or artificial lysosomal fluid (ALF) was evaluated using an ion-selective electrochemical method and UV-Vis spectroscopy. The dissolution that was more pronounced in PBS at elevated temperatures was decelerated by polymer coatings. The dissolution in DMEM was relatively small, but much more pronounced in ALF. PMVEMA with multiple anchoring groups provided better protection against particle dissolution in PBS than PEG-Ale and PDMA-Ale polymers containing only one reactive group. However, the cytotoxicity of the particles depended not only on their ability to rapidly degrade, but also on the type of coating. According to MTT, neat UCNPs and UCNP@PMVEMA were toxic for both rat cells (C6) and rat mesenchymal stem cells (rMSCs), which was in contrast to the UCNP@Ale-PDMA particles that were biocompatible. On the other hand, both the cytotoxicity and uptake of the UCNP@Ale-PEG particles by C6 and rMSCs were low, according to MTT assay and ICP-MS, respectively. This was confirmed by a confocal microscopy, where the neat UCNPs were preferentially internalized by both cell types, followed by the UCNP@PMVEMA, UCNP@Ale-PDMA, and UCNP@Ale-PEG particles. This study provides guidance for the selection of a suitable nanoparticle coating with respect to future biomedical applications where specific behaviors (extracellular deposition vs. cell internalization) are expected.

Poly(4-Styrenesulfonic Acid-co-maleic Anhydride)-Coated NaGdF4:Yb,Tb,Nd Nanoparticles with Luminescence and Magnetic Properties for Imaging of Pancreatic Islets and ß-Cells.

Novel Yb,Tb,Nd-doped GdF3 and NaGdF4 nanoparticles were synthesized by a coprecipitation method in ethylene glycol (EG) in the presence of the poly(4-styrenesulfonic acid-co-maleic anhydride) stabilizer. The particle size and morphology, crystal structure, and phase change were controlled by adjusting the PSSMA concentration and source of fluoride anions in the reaction. Doping of Yb3+, Tb3+, and Nd3+ ions in the NaGdF4 host nanoparticles induced luminescence under ultraviolet and near-infrared excitation and high relaxivity in magnetic resonance (MR) imaging (MRI). In vitro toxicity of the nanoparticles and their cellular uptake efficiency were determined in model rat pancreatic ß-cells (INS-1E). As the NaGdF4:Yb,Tb,Nd@PSSMA-EG nanoparticles were non-toxic and possessed good luminescence and magnetic properties, they were applicable for in vitro optical and MRI of isolated pancreatic islets in phantoms. The superior contrast was achieved for in vivo T2*-weighted MR images of the islets transplanted under the kidney capsule to mice in preclinical trials.

PMVEMA-coated upconverting nanoparticles for upconversion-linked immunoassay of cardiac troponin.

Surface engineering of upconverting nanoparticles (UCNPs) is crucial for their bioanalytical applications. Here, an antibody specific to cardiac troponin I (cTnI), an important biomarker for acute myocardial infection, was covalently immobilized on the surface of UCNPs to prepare a label for the detection of cTnI biomarker in an upconversion-linked immunoassay (ULISA). Core-shell UCNPs (NaYF4:Yb,Tm@NaYF4) were first coated with poly(methyl vinyl ether-alt-maleic acid) (PMVEMA) and then conjugated to antibodies. The morphology (size and uniformity), hydrodynamic diameter, chemical composition, and amount of coating on the of UCNPs, as well as their upconversion luminescence, colloidal stability, and leaching of Y3+ ions into the surrounding media, were determined. The developed ULISA allowed reaching a limit of detection (LOD) of 0.13 ng/ml and 0.25 ng/ml of cTnI in plasma and serum, respectively, which represents 12- and 2-fold improvement to conventional enzyme-linked immunosorbent based on the same immunoreagents.

Multimodal fluorescently labeled polymer-coated GdF3 nanoparticles inhibit degranulation in mast cells.

Multimodal gadolinium fluoride nanoparticles belong to potential contrast agents useful for bimodal optical fluorescence and magnetic resonance imaging. However, the metallic nature of the nanoparticles, similarly to some paramagnetic iron oxides, might induce allergic and anaphylactic reactions in patients after administration. A reduction of these adverse side effects is a priority for the safe application of the nanoparticles. Herein, we prepared paramagnetic poly(4-styrenesulfonic acid-co-maleic acid) (PSSMA)-stabilized GdF3 nanoparticles with surface modified by Atto 488-labeled poly(styrene-grad-2-dimethylaminoethyl acrylate)-block-poly(2-dimethylaminoethyl acrylate) (PSDA-A488) with reactive amino groups for introduction of an additional imaging (luminescence) modality and possible targeting of anticancer drugs. The saturation magnetization of GdF3@PSSMA particles according to SQUID magnetometry reached 157 Am2 kg-1 at 2 K and magnetic field of 7 T. GdF3@PSSMA-PSDA-A488 nanoparticles were well tolerated by human cervical adenocarcinoma (HeLa), mouse bone marrow-derived mast cells (BMMC), and rat basophilic mast cells (RBL-2H3); the particles also affected cell morphology and protein tyrosine phosphorylation in mast cells. Moreover, the nanoparticles interfered with the activation of mast cells by multivalent antigens and inhibited calcium mobilization and cell degranulation. These findings show that the new multimodal GdF3-based nanoparticles possess properties useful for various imaging methods and might minimize mast cell degranulation incurred after future nanoparticle diagnostic administration.

Poly(N,N-dimethylacrylamide)-coated upconverting NaYF4:Yb,Er@NaYF4:Nd core-shell nanoparticles for fluorescent labeling of carcinoma cells.

Upconverting luminescent lanthanide-doped nanoparticles (UCNP) belong to promising new materials that absorb infrared light able to penetrate in the deep tissue level, while emitting photons in the visible or ultraviolet region, which makes them favorable for bioimaging and cell labeling. Here, we have prepared upconverting NaYF4:Yb,Er@NaYF4:Nd core-shell nanoparticles, which were coated with copolymers of N,N-dimethylacrylamide (DMA) and 2-(acryloylamino)-2-methylpropane-1-sulfonic acid (AMPS) or tert-butyl [2-(acryloylamino)ethyl]carbamate (AEC-Boc) with negative or positive charges, respectively. The copolymers were synthesized by a reversible addition-fragmentation chain transfer (RAFT) polymerization, reaching Mn ~ 11 kDa and containing ~ 5 mol% of reactive groups. All copolymers contained bisphosphonate end-groups to be firmly anchored on the surface of NaYF4:Yb,Er@NaYF4:Nd core-shell nanoparticles. To compare properties of polymer coatings, poly(ethylene glycol)-coated and neat UCNP were used as a control. UCNP with various charges were then studied as labels of carcinoma cells, including human hepatocellular carcinoma HepG2, human cervical cancer HeLa, and rat insulinoma INS-1E cells. All the particles proved to be biocompatible (nontoxic); depending on their ξ-potential, the ability to penetrate the cells differed. This ability together with the upconversion luminescence are basic prerequisites for application of particles in photodynamic therapy (PDT) of various tumors, where emission of nanoparticles in visible light range at ~ 650 nm excites photosensitizer.

Colloidally Stable P(DMA-AGME)-Ale-Coated Gd(Tb)F3:Tb3+(Gd3+),Yb3+,Nd3+ Nanoparticles as a Multimodal Contrast Agent for Down- and Upconversion Luminescence, Magnetic Resonance Imaging, and Computed Tomography.

Multimodal imaging, integrating several modalities including down- and up-conversion luminescence, T 1- and T 2(T 2*)-weighted MRI, and CT contrasting in one system, is very promising for improved diagnosis of severe medical disorders. To reach the goal, it is necessary to develop suitable nanoparticles that are highly colloidally stable in biologically relevant media. Here, hydrophilic poly(N,N-dimethylacrylamide-N-acryloylglycine methyl ester)-alendronate-[P(DMA-AGME)-Ale]-coated Gd(Tb)F3:Tb3+(Gd3+),Yb3+,Nd3+ nanoparticles were synthesized by a coprecipitation method in ethylene glycol (EG) followed by coating with the polymer. The particles were tho-roughly characterized by a dynamic light scattering (DLS), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), X-ray energy dispersive spectroscopy (EDAX), selected area electron diffraction (SAED), elemental ana-lysis and fluorescence spectroscopy. Aqueous particle dispersions exhibited excellent colloidal stability in water and physiological buffers. In vitro toxicity assessments suggested no or only mild toxicity of the surface-engineered Gd(Tb)F3:Tb3+(Gd3+),Yb3+,Nd3+ particles in a wide range of concentrations. Internalization of the particles by several types of cells, including HeLa, HF, HepG2, and INS, was confirmed by a down- and up-conversion confocal microscopy. Newly developed particles thus proved to be an efficient contrast agent for fluorescence imaging, T 1- and T 2(T 2*)-weighted magnetic resonance imaging (MRI), and computed tomography (CT).

Multimodal PSSMA-Functionalized GdF3 : Eu3+ (Tb3+ ) Nanoparticles for Luminescence Imaging, MRI, and X-Ray Computed Tomography.

Biocompatible poly(4-styrenesulfonic acid-co-maleic acid)-stabilized GdF3 : Eu3+ (Tb3+ ) nanoparticles were obtained by a one-step coprecipitation method in ethylene glycol or water. The particles are very small (3 nm), have a narrow size distribution, and were detectable by fluorescence, magnetic resonance, and X-ray contrast imaging. These properties allow multimodal imaging, which has prospective applications in the simultaneous and detailed detection of diseased tissues.