Modularly Assembled Upconversion Nanoparticles for Orthogonally Controlled Cell Imaging and Drug Delivery.

Upconversion nanoparticles (UCNPs) have been used effectively as light transducers to convert near-infrared irradiation to short-wavelength emissions for photoactivation in deep tissues. UCNPs with single/multiple emissions under excitation at a single wavelength can be used for simultaneous activation of single or multiple photosensitive molecules only; an ideal multifunctional UCNP nanoplatform should not only have the ability to load multiple molecules but also should activate them at the right time with the right dose when necessary, depending upon the application for which it is used. The control of many biological processes requires complex (simultaneous or subsequent) photoactivation at different time points. Subsequent photoactivation requires UCNPs with orthogonal fluorescence emissions, which can be controlled independently. So far, there are only a few reports about UCNPs with orthogonal emissions. Synthesis of these orthogonal emission nanoparticles is complicated and tedious because nanoparticles with multiple shells need to be synthesized, and different lanthanide ions need to be doped into different shells. Also, there is no flexibility for changing the doped ions and emission profile after the nanoparticles are produced. Here, we have demonstrated a versatile method to modularly assemble individual UCNPs into UCNP clusters (UCNPs-C) with adjustable emissions. The synthesis is much easier, and there is a lot of flexibility in changing the particle size, shape, doped ions, and emission profile. We have demonstrated the use of such UCNPs-C for color encoding at the nanoscale. We further designed orthogonal photoactivatable UCNPs-C (OP-UCNPs-C), which can be independently activated under 980 nm excitation for red emission and 808 nm excitation for UV/blue emission. These OP-UCNPs-C were used for independent activation of processes for cell imaging (980 nm) and drug delivery (808 nm). In comparison to the traditional nonprogrammed activation, a programmed controlled imaging and drug delivery process could guarantee highly targeted and enhanced cell death of cancerous cells.

Upconversion superballs for programmable photoactivation of therapeutics.

Upconversion nanoparticles (UCNPs) are the preferred choice for deep-tissue photoactivation, owing to their unique capability of converting deep tissue-penetrating near-infrared light to UV/visible light for photoactivation. Programmed photoactivation of multiple molecules is critical for controlling many biological processes. However, syntheses of such UCNPs require epitaxial growth of multiple shells on the core nanocrystals and are highly complex/time-consuming. To overcome this bottleneck, we have modularly assembled two distinct UCNPs which can individually be excited by 980/808 nm light, but not both. These orthogonal photoactivable UCNPs superballs are used for programmed photoactivation of multiple therapeutic processes for enhanced efficacy. These include sequential activation of endosomal escape through photochemical-internalization for enhanced cellular uptake, followed by photocontrolled gene knockdown of superoxide dismutase-1 to increase sensitivity to reactive oxygen species and finally, photodynamic therapy under these favorable conditions. Such programmed activation translated to significantly higher therapeutic efficacy in vitro and in vivo in comparison to conventional, non-programmed activation.

Manipulating energy migration within single lanthanide activator for switchable upconversion emissions towards bidirectional photoactivation.

Reliance on low tissue penetrating UV or visible light limits clinical applicability of phototherapy, necessitating use of deep tissue penetrating near-infrared (NIR) to visible light transducers like upconversion nanoparticles (UCNPs). While typical UCNPs produce multiple simultaneous emissions for unidirectional control of biological processes, programmable control requires orthogonal non-overlapping light emissions. These can be obtained through doping nanocrystals with multiple activator ions. However, this requires tedious synthesis and produces complicated multi-shell nanoparticles with a lack of control over emission profiles due to activator crosstalk. Herein, we explore cross-relaxation (CR), a non-radiative recombination pathway typically perceived as deleterious, to manipulate energy migration within the same lanthanide activator ion (Er3+) towards orthogonal red and green emissions, simply by adjusting excitation wavelength from 980 to 808 nm. These UCNPs allow programmable activation of two synergistic light-gated ion channels VChR1 and Jaws in the same cell to manipulate membrane polarization, demonstrated here for cardiac pacing.

Surface protein engineering increases the circulation time of a cell membrane-based nanotherapeutic.

Mammalian cell membranes are often incompatible with chemical modifications typically used to increase circulation half-life. Using cellular nanoghosts as a model, we show that proline-alanine-serine (PAS) peptide sequences expressed on the membrane surface can extend the circulation time of a cell membrane derived nanotherapeutic. Membrane expression of a PAS 40 repeat sequence decreased protein binding and resulted in a 90% decrease in macrophage uptake when compared with non-PASylated controls (P ≤ 0.05). PASylation also extended circulation half-life (t1/2 = 37 h) compared with non-PASylated controls (t1/2 = 10.5 h) (P ≤ 0.005), resulting in ~7-fold higher in vivo serum concentrations at 24 h and 48 h (P ≤ 0.005). Genetically engineered membrane expression of PAS repeats may offer an alternative to PEGylation and provide extended circulation times for cellular membrane-derived nanotherapeutics.

Quasi-Continuous Wave Near-Infrared Excitation of Upconversion Nanoparticles for Optogenetic Manipulation of C. elegans.

Optogenetics is an emerging powerful tool to investigate workings of the nervous system. However, the use of low tissue penetrating visible light limits its therapeutic potential. Employing deep penetrating near-infrared (NIR) light for optogenetics would be beneficial but it cannot be used directly. This issue can be tackled with upconversion nanoparticles (UCNs) acting as nanotransducers emitting at shorter wavelengths extending to the UV range upon NIR light excitation. Although attractive, implementation of such NIR-optogenetics is hindered by the low UCN emission intensity that necessitates high NIR excitation intensities, resulting in overheating issues. A novel quasi-continuous wave (quasi-CW) excitation approach is developed that significantly enhances multiphoton emissions from UCNs, and for the first time NIR light-triggered optogenetic manipulations are implemented in vitro and in C. elegans. The approach developed here enables the activation of channelrhodopsin-2 with a significantly lower excitation power and UCN concentration along with negligible phototoxicity as seen with CW excitation, paving the way for therapeutic optogenetics.

Mesoporous silica-coated upconversion nanocrystals for near infrared light-triggered control of gene expression in zebrafish.

AIM: To develop a platform technology for photoactivation of gene expression in deep tissues. MATERIALS & METHODS: Upconversion nanoparticles (UCNs) were synthesized from rare earth elements like Ytterbium, Yttrium and Thulium. The nanoparticles were then further coated with a layer of mesoporous silica and loaded with photomorpholinos or photocaged plasmids and tested in zebrafish. The UCNs were activated using safe near-infrared (NIR) light which in turn produced UV light locally to enable photoactivation in deep tissues. RESULTS: Light-controlled gene knockdown was demonstrated in an in vivo model, namely zebrafish. UCNs loaded with photomorpholinos were used to knockdown a gene - ntl, which is essential for notochord formation and mesoderm patterning in zebrafish using NIR light. UCN-mediated light-controlled gene expression was also achieved by expressing GFP in tumor cells transplanted into adult zebrafish by irradiating the fish with NIR light. Apart from the delivery and control of genes, the UCNs were also used as imaging agents to image both zebrafish embryos and adult zebrafish. enabled excellent background-free, fluorescent imaging of both embryos and adult zebrafish. CONCLUSION: This technique of controlling gene expression/knockdown through NIR using UCNs is a game changer in the field of genetic manipulation and has the potential of being an excellent, safe and easy to implement tool for developmental biologists to investigate the role of specific genes in development. However, this technique is not restricted to be used only in zebrafish and can be extended for use in other animal models and even for clinical use, in various gene therapy applications.

Luminescent lanthanide nanomaterials: an emerging tool for theranostic applications.

Lanthanide materials have been gaining popularity for use in various theranostic applications, primarily due to their unique optical properties such as narrow emission bands, multiple emission wavelengths, emission tunability, long fluorescence lifetime and large Stokes shift. Apart from these, some lanthanide materials also exhibit magnetic and light-up conversion properties. Such nanomaterials have been used for a wide range of applications ranging from detection of biomarkers, in vitro and in vivo imaging to therapeutic applications. Recently, combined modalities of lanthanide nanomaterials for simultaneous detection/imaging and delivery of therapeutic agents (termed 'theranostics') have been explored. The various advantages and disadvantages of using lanthanide nanomaterials as theranostic agents and potential areas for future development have been discussed in this review.

Upconversion nanoparticles as versatile light nanotransducers for photoactivation applications.

Remote activation of photoactivable therapeutic compounds by light provides a high spatial and temporal control for activating the therapeutic agent. However, photoactivable compounds are mostly responsive towards ultraviolet (UV) or visible light radiation that has poor tissue penetration depth besides being unsafe to the body in the case of UV light. Nanoparticles with energy upconversion hold potential in overcoming this limit by using safe and deeply penetrating near-infrared (NIR) light. These upconversion nanoparticles (UCNs) act as versatile nanotransducers as they convert NIR light to light of shorter wavelengths that can be tuned to the NIR, visible or UV colors to suit different activation wavelengths. Their highly unusual optical properties to fluoresce with near-zero photobleaching, photoblinking and background autofluorescence are unique and an added benefit when used simultaneously as optional imaging agents. This article reviews recent advancements in the use of UCNs for photoactivation of therapeutic agents. Specifically, we discuss the use of these UCNs for activation of light-sensitive/photocaged molecules or photosensitizers for photocontrolled-delivery and photodynamic therapy.

A paradigm shift in the excitation wavelength of upconversion nanoparticles.

The past two decades witnessed the emergence of upconversion nanoparticles as promising luminophores finding multifarious uses from biological studies to solar cells. Progress in their practical use, however, has been hampered by requirements to be excited within a narrow absorption band at around 980 nm. Since the main constituent of biological tissue--water--absorbs strongly in this region, significant reduction in the penetration depth is anticipated as the 980 nm light gets attenuated travelling through tissues, besides also risking tissue damage from the overheating effect. Just recently, remarkable efforts to engineer the excitation of upconversion nanoparticles to a more suitable wavelength for biological applications were reported. This article gives an insightful view on the different ingenious designs that have been reported and their progression towards the development of upconversion nanoparticles with biologically friendlier excitation wavelength.

Near-infrared-light-based nano-platform boosts endosomal escape and controls gene knockdown in vivo.

Current nanoparticle-based gene delivery techniques face two major limitations, namely, endosomal degradation and poor cytosolic release of the nanoparticles and nonspecificity of treatment. These limitations can be overcome with certain light-based techniques, such as photochemical internalization to enable endosomal escape of the delivered nanoparticles and light-controlled gene expression to overcome the nonspecific effects. However, these techniques require UV/visible light, which is either phototoxic and/or has low tissue penetration capabilities, thus preventing their use in deep tissues in a clinical setting. In an effort to overcome these barriers, we have successfully demonstrated a light-based gene delivery system that significantly boosts cytosolic gene delivery, with precise control over gene expression and the potential for use in nonsuperficial tissues. Core-shell fluorescent upconversion nanoparticles excited by highly penetrating near-infrared radiation and emitting simultaneously in the ultraviolet and visible ranges were synthesized and used as remote nanotransducers to simultaneously activate endosomal escape and gene knockdown. Gene knockdown using photomorpholinos was enhanced as much as 30% in vitro compared to the control without endosomal escape facilitation. A similar trend was seen in vivo in a murine melanoma model, demonstrating the enormous clinical potential of this system.

Remote activation of biomolecules in deep tissues using near-infrared-to-UV upconversion nanotransducers.

Controlled activation or release of biomolecules is very crucial in various biological applications. Controlling the activity of biomolecules have been attempted by various means and controlling the activity by light has gained popularity in the past decade. The major hurdle in this process is that photoactivable compounds mostly respond to UV radiation and not to visible or near-infrared (NIR) light. The use of UV irradiation is limited by its toxicity and very low tissue penetration power. In this study, we report the exploitation of the potential of NIR-to-UV upconversion nanoparticles (UCNs), which act as nanotransducers to absorb NIR light having high tissue penetration power and negligible phototoxicity and emit UV light locally, for photoactivation of caged compounds and, in particular, used for photo-controlled gene expression. Both activation and knockdown of GFP was performed in both solution and cells, and patterned activation of GFP was achieved successfully by using upconverted UV light produced by NIR-to-UV UCNs. In-depth photoactivation through tissue phantoms and in vivo activation of caged nucleic acids were also accomplished. The success of this methodology has defined a unique level in the field of photo-controlled activation and delivery of molecules.