The assembly of a photoluminescent nanocomplex based on upconversion nanoparticles

Upconversion nanoparticles possess the ability to convert low energy photons to high energy photons, with the advantages of no autofluorescence, reduced photodamage, deeper tissue penetration, and extended photostability. Therefore, upconversion nanoparticles are considered ideal probes for framing potential theranostic agents. Generally, oleic acid has been used as a capping agent to fabricate high-quality upconversion nanoparticles. However, the hydrophobic oleic acid ligands on the surface of the nanoparticles continue to be a barrier when used in biological applications. Herein, we modified the surface of oleic acid coated upconversion (OA-NaGdF4:Yb/Er) nanoparticles to be hydrophilic using a novel one-step solvent assisted mechanochemical (SAM) milling ligand exchange strategy. Normal ligand exchange processes are tedious and take one to two days to complete, but the SAM ligand exchange strategy presented here is facile, rapid, and takes less than 1 h. This surface modified citric acid coated upconversion (CA-NaGdF4:Yb/Er) nanoparticles further ensures dispersibility in water and good biocompatibility, as revealed by electron microscopy, ATR-FTIR spectroscopy, zeta potential measurement, upconversion luminescence studies, and cytotoxicity assessment. Besides, the size and shape of the nanoparticles were retained after surface modification. Moreover, the monodispersed CA-NaGdF4:Yb/Er nanoparticles exhibit intrinsic paramagnetic behavior which makes them suitable to be used as a contrast enhancer for T 1-weighted MRI. In addition, the measured CT numbers (in HU) increased linearly with increasing the concentration of the surface modified CA-NaGdF4:Yb/Er nanoparticles, indicating their plausibility as a CT contrast agent. The current findings suggest that the SAM ligand exchange strategy could be used to fabricate hydrophilic and biocompatible upconversion nanoparticles for bioimaging applications.

Multi-function up-conversion luminescent Bi4Ti3O12 nanoparticles sensitized by Nd3+ and Yb3+

We assessed the influence of Yb3+ and Er3+ dopant concentration on the relative spectral distribution, quantum yield (ΦUC), and decay kinetics of the upconversion luminescence (UCL) and particle brightness (BUC) for similarly sized (33 nm) oleate-capped β-NaYF4:Yb3+,Er3+ upconversion (UC) nanoparticles (UCNPs) in toluene at broadly varied excitation power densities (P). This included an Yb3+ series where the Yb3+ concentration was varied between 11%-21% for a constant Er3+ concentration of 3%, and an Er3+ series, where the Er3+ concentration was varied between 1%-4% for a constant Yb3+ concentration of 14%. The results were fitted with a coupled rate equation model utilizing the UCL data and decay kinetics of the green and red Er3+ emission and the Yb3+ luminescence at 980 nm. An increasing Yb3+ concentration favors a pronounced triphotonic population of 4F9/2 at high P by an enhanced back energy transfer (BET) from the 4G11/2 level. Simultaneously, the Yb3+-controlled UCNPs absorption cross section overcompensates for the reduction in ΦUC with increasing Yb3+ concentration at high P, resulting in an increase in BUC. Additionally, our results show that an increase in Yb3+ and a decrease in Er3+ concentration enhance the color tuning range by P. These findings will pave the road to a deeper understanding of the energy transfer processes and their contribution to efficient UCL, as well as still debated trends in green-to-red intensity ratios of UCNPs at different P.

Controlled synthesis and luminescence properties of β-NaGdF4: Yb3+, Er3+ upconversion nanoparticles

Purpose:Near‐IR absorptive up‐converting nanoparticles (UCNPs) is a novel contrast for optical‐ECT that allows auto‐fluorescence‐free 3D imaging of labeled cells in a matrix of large (∼1cm3) unsectioned normal tissue. This has the potential to image small metastases or dormant cells that is difficult with down‐converting fluorescing dyes due to auto‐fluorescence. The feasibility of imaging UCNP in agarose phantoms and a mouse lung is demonstrated, aided by a 3D‐printed optical‐ECT stage designed to excite UCNP in a mouse lung.Methods:The UCNP, NaYF4:Yb/Er (20/2%), studied in this work up‐converts 980nm light to visible light peaking sharply at ∼540nm. To characterize the UCNP emission as a function of UCNP concentration, cylindrical 2.5%wt agarose phantoms infused with UCNP at concentrations of 25µg/mL and 50µg/mL were exposed to 1.5W 980nm laser coupled to an optical fiber. The fiber was held stably at 1cm above the stage via a custom 3D‐printed stage. An optically cleared lung harvested from a BALBc mice was then injected with 100µL of 1mg/mL UCNP solution ex vivo. Tomographic imaging of the UCNP emission in lung was performed.Results:The laser beam tract is visualized within the agarose phantom. A line profile of UCNP emission at 25µg/mL versus 50µg/mL shows that increasing the UCNP concentration increases emission count. UCNPs injected into a cleared mouse lung disperse throughout the respiratory tract, allowing for visualization and 3D reconstruction. Excitation before and after UCNP injection shows the tissue exhibits no auto‐fluorescence at 980nm, allowing clear view of the UCNP without any obscuring features such as conventional down‐converting fluorescent tags.Conclusion:We confirm that up‐conversion in tissue circumvents completely tissue auto‐fluorescence, which allowed background‐free 3D reconstruction of the UCNP distribution. We also confirm that raising the UCNP concentration increases emission and that UCNPs are retained in agarose samples during the optical clearing process.

The luminescence spectra of upconversion nanoparticles (UCNPs) and ZnCdS nanoparticles (ZnCdSNPs) were measured and analyzed in a wide temperature range: from room to human body and further to a hyperthermic temperature resulting in tissue morphology change. The results show that the luminescence signal of UCNPs and ZnCdSNPs placed within the tissue is reasonably good sensitive to temperature change and accompanied by phase transitions of lipid structures of adipose tissue. The most likely that the multiple phase transitions are associated with the different components of fat cells, such as phospholipids of cell membrane and lipids of fat droplets. In the course of fat cell heating, lipids of fat droplet first transit from a crystalline form to a liquid crystal form and then to a liquid form, which is characterized by much less scattering. The results of phase transitions of lipids were observed as the changes in the slope of the temperature dependence of the intensity of luminescence of the film with nanoparticles embedded into tissue. The obtained results confirm a high sensitivity of the luminescent UCNPs and ZnCdSNPs to the temperature variations within thin tissue samples and show a strong potential for the controllable tissue thermolysis.

Background The optogenetics manipulation of the heart based on the visible light is limited in the therapeutic potential because of the low tissue penetration. Near-infrared (NIR) light has deeper tissue penetration capabilities but radiates at unsuitable wavelengths, while upconversion nanoparticles (UCNPs) absorb NIR light to convert visible light. Purpose We aimed to investigate the efficient NIR control of the rat heart in vivo via UCNPs mediated cardiac optogenetics. Methods Systemic delivery via jugular vein injection of (AAV9-CAG-hChR2 (H134R)-mCherry) were performed in SD rats to achieve sufficient Channelrhodopsin-2 (ChR2) transfer throughout the whole heart. UCNPs of NaYF4:Yb/Tm with optimal excitation wavelength at 975nm were chosen to emit upconverted blue light. Different concentrations of UCNPs cyclohexane solution were embed in composite polydimethylsiloxane films to make flexible substrates for cardiac optogenetics study in open-chest rats (n=3). The UCNPs film was attached to the right ventricle and the 980nm NIR illumination was applied. Results The upconversion luminescence spectra of four concentrations (2.5, 5, 10 and 20mg/ml) of NaYF4:Yb/Tm scanned under 980nm excitation at 0.5w showed similar peaks around 475, 645 and 695nm. Emission intensity increased with the UCNPs concentration (Figure 1). The NIR-upconverted blue light from the freestanding films embedded with 2.5 and 5mg/ml UCNPs failed to capture the heart till the peak output power of the NIR laser, and the hearts were successfully captured and paced by the upconverted blue light from 10 and 20mg/mL UCNPs films (20 pulses in 8Hz with 20ms duration were repeated 3 times with the interval of 1s). However the NIR power was lower on 10mg/mL UCNPs film than the 20mg/mL one (0.93±0.11w vs 1.71±0.75w). Therefore, UCNPs film with concentration of 10mg/mL NaYF4:Yb/Tm were used for efficient cardiac optogenetic pacing by NIR light from a 400um optical fiber. Optogenetics capture of the ventricle was achieved at different NIR power, pulse duration and flash frequency. The strength-duration curve summarized the minimal NIR irradiance power of 8Hz flash required for 100% capture at different pulse duration (2, 5, 10, 20 and 50ms). Notably the longer the pulse duration was, the lower the light intensity required. Furthermore, the increasing flash frequency (6, 7, 8 and 10Hz) of the NIR light setting at 1.66w (2-fold threshold power) and 20ms duration induced sufficient cardiac pacing (Figure 2). The efficient NIR control of the heart Conclusion We demonstrated the successful NIR photo-activation of ChR2 expressed in the heart by the upconverted blue light via UCNPs, which resulted in a flexible UCNPs-assisted cardiac optogenetic approach for optical control of heart activity. We believe that these advances in cardiac optogenetic toolbox not only represent a novel practical application of UCNPs, but also open up new possibilities for remote or tissue penetrating heart control. Acknowledgement/Funding The national natural science foundation of China (81772044)

Bioimaging and distribution of unmodified and polyethylene glycol-modified up-converting SrF2:Yb3+,Er3+ nanoparticles in Tenebrio molitor larvae and their impact on lifespan, moulting, and metabolism.

Upconversion nanoparticles (UCNPs) are utilized extensively for biomedical imaging, sensing, and therapeutic applications, yet the molecular weight of UCNPs has not previously been reported. Herein, we present a theory based upon the crystal structure of UCNPs to estimate the molecular weight of UCNPs: enabling insight into UCNP molecular weight for the first time. We estimate the theoretical molecular weight of various UCNPs reported in the literature, predicting that spherical NaYF4 UCNPs ~ 10 nm in diameter will be ~1 MDa (i.e. 106 g/mol), whereas UCNPs ~ 45 nm in diameter will be ~100 MDa (i.e. 108 g/mol). We also predict that hexagonal crystal phase UCNPs will be of greater molecular weight than cubic crystal phase UCNPs. Additionally we find that a Gaussian UCNP diameter distribution will correspond to a lognormal UCNP molecular weight distribution. Our approach could potentially be generalised to predict the molecular weight of other arbitrary crystalline nanoparticles: as such, we provide stand-alone graphic user interfaces to calculate the molecular weight both UCNPs and arbitrary crystalline nanoparticles. We expect knowledge of UCNP molecular weight to be of wide utility in biomedical applications where reporting UCNP quantity in absolute numbers or molarity will be beneficial for inter-study comparison and repeatability.

The increasing interest in upconverting nanoparticles (UCNPs) in biodiagnostics and therapy fuels the development of biocompatible UCNPs platforms. UCNPs are typically nanocrystallites of rare-earth fluorides codoped with Yb3+ and Er3+ or Tm3+. The most studied UCNPs are based on NaYF4 but are not chemically stable in water. They dissolve significantly in the presence of phosphates. To prevent any adverse effects on the UCNPs induced by cellular phosphates, the surfaces of UCNPs must be made chemically inert and stable by suitable coatings. We studied the effect of various phosphonate coatings on chemical stability and in vitro cytotoxicity of the Yb3+,Er3+-codoped NaYF4 UCNPs in human endothelial cells obtained from cellular line Ea.hy926. Cell viability of endothelial cells was determined using the resazurin-based assay after the short-term (15 min), and long-term (24 h and 48 h) incubations with UCNPs dispersed in cell-culture medium. The coatings were obtained from tertaphosphonic acid (EDTMP), sodium alendronate and poly(ethylene glycol)-neridronate. Regardless of the coating conditions, 1 − 2 nm-thick amorphous surface layers were observed on the UCNPs with transmission electron microscopy. The upconversion fluorescence was measured in the dispersions of all UCNPs. Surafce quenching in aqueous suspensions of the UCNPs was reduced by the coatings. The dissolution degree of the UCNPs was determined from the concentration of dissolved fluoride measured with ion-selective electrode after the ageing of UCNPs in water, physiological buffer (i.e., phosphate-buffered saline—PBS) and cell-culture medium. The phosphonate coatings prepared at 80 °C significantly suppressed the dissolution of UCNPs in PBS while only minor dissolution of bare and coated UCNPs was measured in water and cell-culture medium. The viability of human endothelial cells was significantly reduced when incubated with UCNPs, but it increased with the improved chemical stability of UCNPs by the phosphonate coatings with negligible cytotoxicity when coated with EDTMP at 80 °C.

In clinic, the application of photodynamic therapy (PDT) in deep tissue is severely constrained by the limited penetration depth of visible light needed for activating the photosensitizer (PS). In this Article, a merocyanine 540 (MC540) and upconverting nanoparticle (UCN) coloaded functional polymeric liposome nanocarrier, (MC540 + UCN)/FPL, was designed and constructed successfully for solving this problem in PDT. Compared with the conventional approaches using UCNs absorbing PSs directly, the combination of UCN and polymeric liposome has unique advantages. The UCN core as a transducer can convert deep-penetrating near-infrared light to visible light for activating MC540. The functional polymeric liposome shell decorated with folate as a nanoshield can keep the UCN and MC540 stable, protect them from being attacked, and help them get into cells. The results show that (MC540 + UCN)/FPL is an individual nanosphere with an average size of 26 nm. MC540 can be activated to produce singlet oxygen successfully by upconverting fluorescence emitted from UCNs. After (MC540 + UCN)/FPL was modified with folate, the cell uptake efficiency increased obviously. More interestingly, in the PDT effect test, the (MC540 + UCN)/FPL nanocarrier further improved the inhibition effect on tumor cells by anchoring targeting folate and transactivating transduction peptide. Our data suggest that the (MC540 + UCN)/FPL nanocarrier may be a useful nanoplatform for future PDT treatment in deep-cancer therapy based on upconversion mechanism.

Lanthanide‐doped upconversion nanoparticles (UCNPs) exhibit unique luminescence properties, making them promising for applications in displays, sensors, security labels, and solar cells. Embedding UCNPs in polymer films can enhance their functionality; however, the properties of the polymer matrix significantly influence the dispersion and loading capacity of UCNPs, ultimately affecting optical performance. In this study, we investigate the incorporation of UCNPs into two distinct polymer matrices, poly(3‐hexylthiophene) (P3HT) and poly(methyl methacrylate) (PMMA), via spin coating at different speeds. Our findings demonstrate that UCNP dispersion and monodispersity are governed by polymer polarity, viscosity, and UCNP concentration in the suspension. To enhance UCNP loading, multiple spin coatings were explored. In UCNP−P3HT films, the volume fraction of UCNPs increased from 26.1% to 51.4% after three consecutive spin coatings, while maintaining a uniform distribution. In contrast, the lower miscibility and higher viscosity of PMMA restricted UCNP loading to 12.0% before significant clustering occurred. Although multiple spin coatings increased the total UCNP content in PMMA films, the volume fraction decreased to 8.0% due to film thickening. This comparative analysis highlights the critical role of polymer matrix properties in UCNP embedding and provides valuable insights for optimizing UCNP−polymer composites for advanced optical applications.

Gd(3+)-ion-doped upconversion nanoparticles (UCNPs), integrating the advantages of upconversion luminescence and magnetic resonance imaging (MRI) modalities, are capturing increasing attention because they are promising to improve the accuracy of diagnosis. The embedded Gd(3+) ions in UCNPs, however, have an indistinct MRI enhancement owing to the inefficient exchange of magnetic fields with the surrounding water protons. In this study, a novel approach is developed to improve the MR imaging sensitivity of Gd(3+)-ion-doped UCNPs. Bovine serum albumin (BSA) bundled with DTPA-Gd(3+) (DTPA(Gd)) is synthesized both as the MR imaging sensitivity synergist and phase-transfer ligand for the surface engineering of UCNPs. The external Gd(3+) ion attachment strategy is found to significant improve the MR imaging sensitivity of Gd(3+)-ion-doped UCNPs. The relaxivity analysis shows that UCNPs@BSA·DTPA(Gd) exhibit higher relaxivity values than do UCNPs@BSA without DTPA(Gd) moieties. Another relaxivity study discloses a striking message that the relaxivity value does not always reflect the realistic MRI enhancement capability. The high concentration of Gd(3+)-ion-containing UCNPs with further surface-engineered BSA·DTPA(Gd) (denoted as UCNPs-H@BSA·DTPA(Gd)) exhibits a more pronounced MRI enhancement capability compared to the other two counterparts [UCNPs-N@BSA·DTPA(Gd) and UCNPs-L@BSA·DTPA(Gd) (-N and -L are denoted as zero and low concentrations of Gd(3+) ion doping, respectively)], even though it holds the lowest r1 of 1.56 s(-1) per mmol L(-1) of Gd(3+). The physicochemical properties of UCNPs are essentially maintained after BSA·DTPA(Gd) surface decoration with good colloidal stability, in addition to improving the MR imaging sensitivity. In vivo T1-weighted MRI shows potent tumor-enhanced MRI with UCNPs-H@BSA·DTPA(Gd). An in vivo biodistribution study indicates that it is gradually excreted from the body via hepatobiliary and renal processing with no obvious toxicity. It could therefore be concluded, with improved MR imaging sensitivity by an internal and external incorporation of Gd(3+) strategy, that UCNPs-H@BSA·DTPA(Gd) presents great potential as an alternative in tumor-targeted MR imaging.

Purpose: Nanoparticle based drug delivery systems have shown promising applications in cancer treatments. Among various nanoparticles, upconversion nanoparticles (UCNPs) allow for the conversion of near infrared (NIR) excitation to localized UV/visible emission providing unprecedented control over UCNP based delivery platforms for deep tissue therapeutic payload release, both spatially and temporally. This study reports the layer-by-layer engineering of UCNP based siRNA and miRNA delivery systems for application in cancer therapy. Experimental Design: To obtain the nanoparticle core, NaYF4:Yb,Er UCNPs were synthesized via thermal decomposition method. The morphology and size of the UCNPs were assessed by transmission electron microscopic (TEM). The upconversion fluorescence property was studied by fluorescence spectroscopy using a 980 nm laser as the excitation source. To enable gene delivery, the naked UCNPs were then surface functionalized with biocompatible polymers using polyacrylic acid (PAA) and poly allylamine hydrochloride (PAH). Surface functionalization was accomplished through layer-by-layer assembly of polyelectrolyte multilayers. Successful surface functionalization was demonstrated by measuring zeta potential after assembly of each layer. Biocompatibility of the developed delivery system was tested by MTT assay. Gel electrophoresis was employed to determine the loading capacity of the delivery system. We then tested the siRNA, miRNA, and EGFP expression vector transfection efficiency in different cancer cell lines. Results: The diameter of the synthesized UCNPs was ∼40 nm. Upon 980 nm excitation, green emission and red emission were identified from the fluorescence spectrum. Zeta potential of the delivery system was ∼+38 mV, indicating its capability to absorb negatively charged siRNA, miRNA, or EGFP vector by electrostatic interaction. The MTT assay demonstrated good biocompatibility. Significant retardation of siRNA, miRNA, or EGFP plasmid in gel electrophoresis assay was observed when mixed with PAA and PAH coated UCNPs. In contrast, UCNP-PAA without PAH coating did not retard siRNA, miRNA, or EGFP plasmid. Confocal laser scanning microscopy results provided direct evidence of siRNA, miRNA, or EGFP plasmid transfection. Conclusions: In this proof-of-concept study, a layer-by-layer engineered UCNP based siRNA and miRNA delivery system was successfully developed. Our novel delivery system opens up preparation of advanced UCNP based photoresponsive delivery systems that allow remote, precise, and trackable control over therapeutic payload (e.g., drug, gene) release for better cancer theranostics. Citation Format: Lin Min, YAN GAO, Francis J. Hornicek, Mansoor M. Amiji, Zhenfeng Duan. Layer-by-layer engineering of upconversion nanoparticle based siRNA and miRNA delivery system for cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-102. doi:10.1158/1538-7445.AM2015-LB-102

ABSTRACTY2O3 and Gd2O3 upconversion nanoparticles (UCN) co-doped with Yb3+ and Er3+ can absorb and upconvert near infrared (NIR) radiation into visible light. These UCN find application in bioimaging, as an important tool to diagnose and visualize cancer cells. The UCN can be used as biolabels to identify the cells; the nanoparticles can be coated and functionalized with ligands that bind to receptors on the surface of the cell. In this project, the UCN were synthesized by sol-gel method and subsequently coated with a thin silica shell by using the Stöber method. The core-shell UCN were functionalized with amine group to enable folic acid conjugation. The functionalized core-shell nanoparticles were analyzed by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and luminescence measurements. Concentrations of bare and coated/functionalized UCN between 0.001 µg/mL and 1 µg/mL were tested on two different cell lines from human cervix carcinoma (HeLa) and human colorectal adenocarcinoma (DLD-)1 with colorimetric assay based on the MTT reagent (methy-134 thiazolyltetrazolium). The results show good luminescence spectra on all core-shell UCN. The MTT assays show that some concentrations of bare UCN of Y2O3: Er, Yb (1%, 1% mol) and Gd2O3 were cytotoxic for cervical adenocarcinoma cells (HeLa). For human colorectal adenocarcinoma all UCN are non-cytotoxic. The UCN with silica-aminosilane functionalization (APTS/TEOS) were non-cytotoxic on both cell lines.