Tether-free optogenetic control of insulin secretion using an upconversion nanoparticle-doped hydrogel platform.

Glucagon-like peptide-1 (GLP-1), as a molecular therapeutic, induces glucose-dependent stimulation of insulin secretion, which has drawn significant attention in treating type II diabetes. However, it always suffers from hurdles such as short half-lives or instability. Thus, producing such therapeutics endogenously, as and when needed, is beneficial. Optogenetics-based production of GLP-1 offers an attractive alternative, wherein, the cell lines such as HEK293T can be genetically modified to bring the expression of the gene of interest under visible light control. However, the need for blue light for activation necessitates the implantation of invasive optical fibers owing to high tissue scattering and low depth of penetration through biological tissue at this wavelength. Here, we overcome this problem by proposing an upconversion nanoparticle (UCNP)-based system. HEK293T cells, rewired to produce GLP-1 under blue light illumination, were co-encapsulated with UCNPs in a hydrogel. The UCNPs act as near-infrared (NIR) to blue light nano-transducers, allowing deep penetration toward implementing a tether-free optogenetic gene expression platform. This platform is particularly powerful for thick gel implants (>3 mm) that cannot be illuminated throughout using a blue light source. Moreover, the GLP-1 produced in this platform was sufficient to increase insulin secretion in rat insulinoma cells, providing a powerful and controllable therapeutic tool for diabetes.

Towards translational optogenetics.

Optogenetics is widely used to interrogate the neural circuits underlying disease and has most recently been harnessed for therapeutic applications. The optogenetic toolkit consists of light-responsive proteins that modulate specific cellular functions, vectors for the delivery of the transgenes that encode the light-responsive proteins to targeted cellular populations, and devices for the delivery of light of suitable wavelengths at effective fluence rates. A refined toolkit with a focus towards translational uses would include efficient and safer viral and non-viral gene-delivery vectors, increasingly red-shifted photoresponsive proteins, nanomaterials that efficiently transduce near-infrared light deep into tissue, and wireless implantable light-delivery devices that allow for spatiotemporally precise interventions at clinically relevant tissue depths. In this Review, we examine the current optogenetics toolkit and the most notable preclinical and translational uses of optogenetics, and discuss future methodological and translational developments and bottlenecks.

A Flexi-PEGDA Upconversion Implant for Wireless Brain Photodynamic Therapy.

Near-infrared (NIR) activatable upconversion nanoparticles (UCNPs) enable wireless-based phototherapies by converting deep-tissue-penetrating NIR to visible light. UCNPs are therefore ideal as wireless transducers for photodynamic therapy (PDT) of deep-sited tumors. However, the retention of unsequestered UCNPs in tissue with minimal options for removal limits their clinical translation. To address this shortcoming, biocompatible UCNPs implants are developed to deliver upconversion photonic properties in a flexible, optical guide design. To enhance its translatability, the UCNPs implant is constructed with an FDA-approved poly(ethylene glycol) diacrylate (PEGDA) core clad with fluorinated ethylene propylene (FEP). The emission spectrum of the UCNPs implant can be tuned to overlap with the absorption spectra of the clinically relevant photosensitizer, 5-aminolevulinic acid (5-ALA). The UCNPs implant can wirelessly transmit upconverted visible light till 8 cm in length and in a bendable manner even when implanted underneath the skin or scalp. With this system, it is demonstrated that NIR-based chronic PDT is achievable in an untethered and noninvasive manner in a mouse xenograft glioblastoma multiforme (GBM) model. It is postulated that such encapsulated UCNPs implants represent a translational shift for wireless deep-tissue phototherapy by enabling sequestration of UCNPs without compromising wireless deep-tissue light delivery.

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.

In vivo wireless photonic photodynamic therapy.

An emerging class of targeted therapy relies on light as a spatially and temporally precise stimulus. Photodynamic therapy (PDT) is a clinical example in which optical illumination selectively activates light-sensitive drugs, termed photosensitizers, destroying malignant cells without the side effects associated with systemic treatments such as chemotherapy. Effective clinical application of PDT and other light-based therapies, however, is hindered by challenges in light delivery across biological tissue, which is optically opaque. To target deep regions, current clinical PDT uses optical fibers, but their incompatibility with chronic implantation allows only a single dose of light to be delivered per surgery. Here we report a wireless photonic approach to PDT using a miniaturized (30 mg, 15 mm3) implantable device and wireless powering system for light delivery. We demonstrate the therapeutic efficacy of this approach by activating photosensitizers (chlorin e6) through thick (>3 cm) tissues inaccessible by direct illumination, and by delivering multiple controlled doses of light to suppress tumor growth in vivo in animal cancer models. This versatility in light delivery overcomes key clinical limitations in PDT, and may afford further opportunities for light-based therapies.

Near-IR photoactivation using mesoporous silica-coated NaYF4:Yb,Er/Tm upconversion nanoparticles.

Photoactivation is a process in which light is used to 'activate' photolabile therapeutics. As a therapeutic strategy, its advantages are that it is noninvasive and that a high degree of spatial and temporal control is possible. However, conventional photoactivation techniques are hampered by the limited penetration depth of the UV and visible lights to which the photosensitive compounds are responsive. Here we describe a protocol for the use of upconversion nanoparticles (UCNs) as light transducers to convert deeply penetrating near-infrared (NIR) light to UV-visible wavelengths matching that of the absorption spectrum of photosensitive therapeutics. This allows the use of deep-penetrating and biologically friendly NIR light instead of low-penetrating and/or toxic visible or UV lights for photoactivation. In this protocol, we focus on two photoactivation applications: photodynamic therapy (PDT) and photoactivated control of gene expression. We describe how to prepare and characterize the UCNs, as well as how to check their function in biochemical assays and in cells. For both applications, the UCNs are coated with mesoporous silica for easy loading of the therapeutics. For PDT, the UCNs are coated with polyethylene glycol (PEG) for stabilization and folic acid for tumor targeting and then loaded with photosensitizers that would be expected to kill cells by singlet oxygen production; the nanoparticles are injected intravenously. For photoactivated control of gene expression, knockdown of essential tumor genes is achieved using UCNs loaded with caged nucleic acids, which are injected intratumorally. The whole process from nanoparticle synthesis to animal studies takes ∼36 d.

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.

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.

Photocontrolled nanoparticle delivery systems for biomedical applications.

"Smart" stimuli-responsive nanomaterials are becoming popular as targeted delivery systems because they allow the use of internal or external stimuli to achieve spatial or temporal control over the delivery process. Among the stimuli that have been used, light is of special interest because it is not only noninvasive but also controllable both spatially and temporally, thus allowing unprecedented control over the delivery of bioactive molecules such as nucleic acids, proteins, drugs, etc. This is particularly advantageous for biomedical applications where specificity and selectivity are highly desired. Several strategies have evolved under the umbrella of light based delivery systems and can be classified into three main groups. The first strategy involves "caging" of the bioactive molecule using photolabile groups, loading these caged molecules onto a carrier and then "uncaging" or activating them at the targeted site upon irradiation with light of a particular wavelength. The second strategy makes use of nanocarriers that themselves are made photoresponsive either through modification with photosensitive groups or through the attachment of photolinkers on the carrier surface. These nanoparticles upon irradiation dissociate, releasing the cargo encapsulated within, or the photolinkers attaching the cargo to the surface get cleaved, resulting in release. The third approach makes use of the surface plasmon resonance of noble metal based nanoparticles. Upon irradiation with light at the plasmon resonant frequency, the resulting thermal or nonthermal field enhancement effects facilitate the release of bioactive molecules loaded onto the nanoparticles. In addition, other materials, certain metal sulfides, graphene oxide, etc., also exhibit photothermal transduction that can be exploited for targeted delivery. These approaches, though effective, are constrained by their predominant use of UV or visible light to which most photolabile groups are sensitive. Near infrared (NIR) excitation is preferred because NIR light is safer and can penetrate deeper in biological tissues. However, most photolabile groups cannot be excited by NIR light directly. So light conversion from NIR to UV/visible is required. Nanomaterials that display upconversion or two-photon-excitation properties have been developed that can serve as nanotransducers, converting NIR to UV/visible light to which the aforementioned photoresponsive moieties are sensitive. This Account will review the existing light-based nanoparticle delivery systems, their applications, the limitations they face, and the technologies that have emerged in an effort to overcome these limitations.

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.