Optical Bioimaging and Therapy

Chapter 6 - Upconversion nanoparticles: a toolbox for biomedical applications

Here we report on photo-isomerization of azobenzene containing surfactants induced during irradiation with near-infrared (NIR) light in the presence of upconversion nanoparticles (UCNPs) acting as mediator. The surfactant molecule consists of charged head group and hydrophobic tail with azobenzene group incorporated in alkyl chain. The azobenzene group can be reversible photo-isomerized between two states: trans- and cis- by irradiation with light of an appropriate wavelength. The trans-cis photo-isomerization is induced by UV light, while cis-trans isomerization proceeds either thermally in darkness, or can be accelerated by exposure to illumination with a longer wavelength typically in a blue/green range. We present the application of lanthanide doped UCNPs to successfully switch azobenzene containing surfactants from cis to trans conformation in bulk solution using NIR light. Using Tm3+ or Er3+ as activator ions, the UCNPs provide emissions in the spectral range of 450 nm < λem < 480 nm (for Tm3+, three and four photon induced emission) or 525 nm < λem < 545 nm (for Er3+, two photon induced emission), respectively. Especially for UCNPs containing Tm3+ a good overlap of the emissions with the absorption bands of the azobenzene is present. Under illumination of the surfactant solution with NIR light (λex = 976 nm) in the presence of the Tm3+-doped UCNPs, the relaxation time of cis-trans photo-isomerization was increased by almost 13 times compared to thermally induced isomerization. The influence of thermal heating due to the irradiation using NIR light was shown to be minor for solvents not absorbing in NIR spectral range (e.g. CHCl3) in contrast to water, which shows a distinct absorption in the NIR.

The combination of organic dyes as photosensitizers with inorganic upconversion nanoparticles (UCNPs) has significantly enhanced upconversion luminescence when excited with near-infrared (NIR) light. Additionally, by employing surface functionalization techniques, a high signal-to-noise ratio can be achieved, resulting in exceptional resolution for bioimaging. The objective of our study was to develop a heptamethylcyanine derivative (IR61-BF) for dye-sensitized UCNPs, enhancing their luminescence efficiency in aqueous solutions. This was done by introducing a difluorobenzene group into the cyanine structure. Rare earth-doped UCNPs with a core-shell structure, NaYF4:YbTm@NaYF4:YbNd, were synthesized using a high-temperature decomposition method. In this structure, the Nd3+ ions function as efficient sensitizers by absorbing 808 nm NIR light. To further enhance the upconversion efficiency, the molecular structure of the NIR dye IR61 was optimized. By integrating a difluorinated phenyl group into the cyanine backbone, we significantly increased the fluorescence quantum yield of the dye and enhanced the dye sensitization effect of the UCNPs. Subsequently, the UCNPs were coated with an amphiphilic ligand, DSPE-PEG, and IR61-BF was incorporated into the hydrophobic region between the UCNPs, and a remarkable 167.1-fold enhancement in upconversion luminescence in the aqueous phase was achieved. Their biocompatibility for neuronal imaging was evaluated using NSC34, Neuro2a, and C6 glioma cells, while their potential for retrograde intra-neuronal delivery was confirmed by using a microfluidic model of cortical neurons. We demonstrated that the newly synthesized heptamethine cyanine derivative IR61-BF shows exceptional sensitization of UCNPs due to the incorporation of a difluorobenzene group, particularly in aqueous solutions.

Detection of Cu(2+) ions and study of their subcellular distribution in physiological processes are of considerable significance because of their potential environmental and biological applications. Some fluorescence based sensors have been developed for selective detection of Cu(2+) ions, based on organic fluorescent probes that specifically bind to Cu(2+) ions. However, these sensors are not suitable for detection in biological samples due to the short penetration depth of UV/visible light used to excite the fluorescent probes. The use of near-infrared (NIR) light can afford penetration depths of an order of magnitude greater than that of visible light, however, a material that can convert NIR light to visible light is required. A facile method has been developed for in-depth detection of Cu(2+) ions based on fluorescence upconversion. A mesoporous silica shell is coated on upconversion nanoparticles (UCNPs) and a Cu(2+) ion sensitive fluorescent probe, rhodamine B hydrazide, is incorporated into the mesoporous silica. Upon excitation by a NIR light, the UCNPs emit visible light to excite the Cu(2+)-sensitive fluorescent probe. Because of the unique optical properties of UCNPs and their ability to convert NIR light to visible light, this is a feasible method for sensitive and in-depth detection of Cu(2+) ions in a complex biological or environmental sample due to the low autofluorescence and the high penetration depth of NIR light.

Photodynamic therapy (PDT) has recently emerged as an effective, noninvasive, and economical treatment for diseases including cancers. Traditional PDT suffers mainly from having an insufficient number of photons penetrating the tissue and preferentially targeting cancerous tissues with photosensitizers. Therefore, it is necessary to construct a method for controllable singlet-oxygen (O2) generation (SOG) with high selectivity and accurate localization to provide more efficient PDTwith fewer side effects. Several research groups have developed novel selective PDT agents, such as peptide or protein conjugates, and photosensitizer encapsulated nanocarriers. In recent years, single-stranded oligonucleotides called aptamers have emerged as a novel class of molecules which rival antibodies in both therapeutic and diagnostic applications. Compared to antibodies, aptamers offer significant advantages, such as flexible design, synthetic accessibility, easy modification, chemical stability, and rapid tissue penetration. Therefore, aptamers can potentially endow traditional PDT with high selectivity and accurate localization. The formation of a G-quadruplex (GQ) structure is results from a type of DNA self-assembly mode. This structure can be stabilized by a central monovalent metal cation such as potassium or sodium ions and small-molecule ligands including porphyrins. Cationic porphyrins usually bind to G-quadruplexes through p–p interactions with G-quartets and through electrostatic interactions with the anionic phosphate groups on G-quadruplexes. According to previous studies, some photosensitizers are porphyrin derivatives and are broadly used in PDT, such as 5,10,15,20-tetrakis-(1-methyl-4-pyridyl)-21H, 23H-porphine (TMPyP4). Therefore, the G-quadruplex DNA sequence can be a carrier for photosensitizers with porphyrin molecular structures. By taking advantage of the loading function of the G-quadruplex structure and the recognition function of aptamers, the photosensitizer can be delivered to a target cell with high affinity and selectivity. Up to now, most photosensitizers have been activated by visible light. As a consequence, the shallow penetration depth of incident light has limited their otherwise wide applications. If the photosensitizers are linked to a visible-light generator and the generator is remotely controlled (turned on/off) by near-infrared (NIR) light, then the problem of limited depth penetration by visible light could be easily overcome. To solve the problem of limited depth penetration of current PDT techniques, we used upconversion nanoparticles (UCNPs), in particular, lanthanide-doped rare-earth nanocrystals. These nanomaterials are able to emit shorter-wavelength photons under excitation by NIR light, thus making them good visiblelight generators with the ability to be remotely controlled by NIR light. Furthermore, owing to the ladderlike arrangement of energy levels in lanthanide ions, UCNPs show high efficiency of photon upconversion with a distinct set of sharp emission peaks under moderate excitation densities, thereby enabling targeted bioimaging when functionalized with biomolecular recognition moieties. Herein, we report a specific aptamer-guided G-quadruplex DNA nanoplatform for targeted bioimaging and PDT, and it is capable of selective recognition and imaging of cancer cells, controllable and effective activation of the photosensitizer, and improvement of the therapeutic effect. In particular, a guanine-rich DNA segment is linked to an aptamer to form a bifunctional DNA sequence, termed a G4aptamer. The G4-aptamer not only loads the photosensitizer but also specifically recognizes target cells. As shown in Scheme 1, the G4-aptamer is bioconjugated to a UCNP, thus placing the photosensitizer TMPyP4 at position near the UCNP for energy transfer between the UCNP and TMPyP4. Once the nanoplatform is delivered into cancer cells, the UCNPs are excited by NIR light to emit visible light to image cancer cells and, in turn, to activate TMPyP4, which, finally, generates sufficient ROS to efficiently kill cancer cells. [*] Prof. Q. Yuan, Y. Wu, D. Lu, Dr. Z. Zhao, T. Liu, Prof. X. Zhang, Prof. W. Tan Molecular Science and Biomedicine Laboratory State Key Laboratory for Chemo/Bio-Sensing and Chemometrics College of Chemistry and Chemical Engineering, College of Biology, and Collaborative Research Center for Chemistry and Molecular Medicine Hunan University, Changsha 410082 (China) E-mail: tan@chem.ufl.edu Prof. Q. Yuan, J. Wang Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education) College of Chemistry and Molecular Sciences Wuhan University, Wuhan 430072 (China) [] These authors contributed equally to this work.

Due to their persistent luminescence, persistent luminescent (PersL) materials have attracted great interest. In the biomedical field, the use of persistent luminescent nanoparticles (PLNPs) eliminates the need for continuous in situ excitation, thereby avoiding interference from tissue autofluorescence and significantly improving the signal-to-noise ratio (SNR). Although persistent luminescence materials can emit light continuously, the luminescence intensity of small-sized nanoparticles in vivo decays quickly. Early persistent luminescent nanoparticles were mostly excited by ultraviolet (UV) or visible light and were administered for imaging purposes through ex vivo charging followed by injection into the body. Limited by the low in vivo penetration depth, UV light cannot secondary charge PLNPs that have decayed in vivo, and visible light does not penetrate deep enough to reach deep tissues, which greatly limits the imaging time of persistent luminescent materials. In order to address this issue, the development of PLNPs that can be activated by light sources with superior tissue penetration capabilities is essential. Near-infrared (NIR) light and X-rays are widely recognized as ideal excitation sources, making persistent luminescent materials stimulated by these two sources a prominent area of research in recent years. This review describes NIR and X-ray excitable persistent luminescence materials and their recent advances in bioimaging.

A biosensor is a device incorporating a biological sensing element either intimately connected to or integrated within a transducer, which is mainly determined by specific molecular recognition such as enzyme-substrate, antibody-antigen and so on. Currently, with the development of nanoscience and nanotechnology, more and more interest has been focused on using nanoparticles to fabricate biosensors (Alivisatos, 2004; Katz, 2004; Rosi 2005). Several kinds of biological sensors based on semiconductor quantum dots (QDs), gold nanoparticles (GNPs), carbon nanotubes (CNTs), fullerene, dendrimer nanoparticles have been presented. Semiconductor QDs are a new class of fluorescent materials for biosensor. In comparison with conventional organic dyes and fluorescent proteins, they have unique optoelectronic properties with size-tunable light emission, superior signal brightness, resistance to photobleaching and broad absorption spectra for simultaneous excitation of multiple fluorescence colors (Alivisatos, 1996). However, the use of semiconductor QDs for biosensor application still has some limitations (Jaiswal, 2004), for instance, the potential toxicity of QDs may pose risks to human health and the environment under certain conditions (Derfus, 2004). In addition, the absorption of UV and visible light by biological samples often induces autofluorescence, which interferes with fluorescent signals obtained from exogenous biomarkers. Moreover, if biological samples are prolonged exposure to UV radiation, it would cause the samples photo-damage and mutation. The drawbacks of QDs in biosensing application have prompted the development of upconverting nanoparticles (UCNs) emerged as another class of new biosensing materials. Usually, UCNs exhibit intense visible or near-infrared light excited by near-infrared light according to the anti-stokes law. The UCNs also show a sharp emission bandwidth, long lifetime, tunable emission, high photostability, low bio-toxicity and good biocompatibility, which are less harmful to biological samples and have greater penetration depth through biological samples than conventional ultraviolet excitation. Moreover, UCNs can be easily coupled to proteins or other biological macromolecular systems and used in a variety of

Psoriasis, with a global incidence rate of 2%-3%, afflicts patients with persistent erythema and scales and is often accompanied by comorbidities such as arthritis and depression, significantly impairing their quality of life. While emerging biologics and traditional medications as well as phototherapy have made certain progress in treating psoriasis, these therapies are hindered by significant drawbacks including high costs, suboptimal efficacy, and significant side effects. Photodynamic therapy (PDT) has great potential for psoriasis treatment, but its application is limited due to the weak penetration of short-wavelength light used for active photosensitizer (PS). Upconversion nanoparticles (UCNPs) offer a promising solution for traditional PDT as they could be excited by near-infrared (NIR) light to emit UV-visible light for reactive oxygen species generation, which effectively addresses penetration challenges. In this study, PS (ZnPc) was encapsulated on UCNPs coated with a porous silica shell (UCNPs-ZnPc). When topically applied to psoriatic mice skin, UCNPs-ZnPc was taken up by macrophages. Upon activation with 980 nm NIR light, psoriatic dermatitis was treated since the macrophage viability was reduced, and the overproliferation of psoriatic skin cells was suppressed. These results highlight the potential of UCNPs-mediated PDT as an innovative approach to managing psoriasis.

Upconversion nanoparticles (UCNs) are nanoparticles that are excited in the near infrared(NIR) region with emission in the visible or NIR regions. This makes these particlesattractive for use in biological imaging as the NIR light can penetrate the tissuebetter with minimal absorption/scattering. This paper discusses the study ofthe depth to which cells can be imaged using these nanoparticles. UCNs withNaYF4 nanocrystalsdoped with Yb3 + , Er3 + (visible emission)/Yb3 + ,Tm3 + (NIR emission) were synthesized and modified with silica enabling their dispersion in waterand conjugation of biomolecules to their surface. The size of the sample was characterizedusing transmission electron microscopy and the fluorescence measured using a fluorescencespectrometer at an excitation of 980 nm. Tissue phantoms were prepared by reportedmethods to mimic skin/muscle tissue and it was observed that the cells could be imaged upto a depth of 3 mm using the NIR emitting UCNs. Further, the depth of detectionwas evaluated for UCNs targeted to gap junctions formed between cardiac cells.

Optical bioimaging is an indispensable tool in modern biology and medicine, but the technique is susceptible to autofluorescence interference. Persistent nanophosphors provide an easy-to-perform and highly efficient means to eliminate tissue autofluorescence. However, direct synthesis of persistent nanophosphors with tunable properties to meet different bioimaging requirements remains largely unexplored. In this work, zinc gallogermanate (Zn1+xGa2-2xGexO4:Cr, 0 ≤ x ≤ 0.5, ZGGO:Cr) persistent luminescence nanoparticles with composition-dependent size and persistent luminescence are reported. The size of the ZGGO:Cr nanoparticles gradually increases with the increase of x in the chemical formula. Moreover, the intensity and decay time of persistent luminescence in ZGGO:Cr nanoparticles can also be fine-tuned by simply changing x in the formula. In vivo bioimaging tests demonstrate that ZGGO:Cr nanoparticles can efficiently eliminate tissue autofluorescence, and the nanoparticles also show good promise in long-term bioimaging as they can be easily reactivated in vivo. Furthermore, an aptamer-guided ZGGO:Cr bioprobe is constructed, and it displays excellent tumor-specific accumulation. The ZGGO:Cr nanoparticles are ideal for autofluorescence-free targeted bioimaging, indicating their great potential in monitoring cellular networks and construction of guiding systems for surgery.

Development of multicolor-emitting upconversion nanoparticles (UCNPs) is of significant importance for applications in optical encoding, anti-counterfeiting, display, and bioimaging. However, realizing the orthogonal three-primary color (TPC) upconversion luminescence in a single nanoparticle remains a huge challenge. Herein, we have rationally designed core-multishell-structured NaYF4 UCNPs through regulating the dopant concentration, composition of luminescent layers, and shell position and thickness, which are capable of emitting red, green, and blue luminescence with high color purity in response to ternary near-infrared quadrature excitations (1560/808/980 nm). Moreover, their high color purity is well retained with varying excitation power densities. This orthogonal TPC emissions property of such UCNPs endows them with great promise in the field of security. As a proof-of-concept, we have demonstrated the feasibility of combining such UCNPs with MnO2 nanosheets for information encryption and decryption. This work not only offers a new way to achieve TPC upconversion luminescence at a single nanoparticle level but also broadens the scope of application for security protection.

Potential application of functional porous TiO2 nanoparticles in light-controlled drug release and targeted drug delivery

Major medical advances in antibiotics for infectious diseases have dramatically improved the quality of life and greatly increased life expectancy. Nevertheless, the widespread and inappropriate exploitation of antibacterial agents has resulted in the emergence of multi-drug-resistant bacteria (MDR). Consequently, the study of new drugs for the treatment of diseases associated with multi-drug-resistant bacteria and the development of new treatments are urgently needed. Inspiringly, due to the advantages of a wide antimicrobial spectrum, fast sterilization, low resistance, and little damage to host tissues and normal flora, antibacterial photodynamic therapy (APDT), which is based on the interaction between light and a nontoxic photosensitizer (PS) concentrated at the lesion site to generate reactive oxygen species (ROS), has become one of the most promising antibacterial strategies. Recently, a burgeoning APDT based on a variety of upconversion nanoparticles (UCNPs) such as PS and near-infrared (NIR) light has been fully integrated in antibacterial applications and achieved excellent performances. Meanwhile, conjugated nanoparticles have been frequently reported in UCNP design, including surface-modified PS conjugates, antibiotic-PS conjugates, and dual or multiple antibacterial modal PS conjugates. This article provides an overview of the state-of-the-art design and bactericidal effects of UCNPs and their based APDTs. The first part discusses the design and mechanisms for UCNPs currently implemented in biomedicine. The second part focuses on the applications and antimicrobial effects of diverse APDT based on UCNPs in antibacterial-related infectious diseases.

Analysis of ROS dynamics based on dissolved oxygen sensing in upconversion nanoparticle-based photodynamic therapy.

The development of efficient red‐emitting persistent phosphor is still an ongoing challenge. In the search of persistent materials in red range, Pr 3+ is a good candidate owing to its transitions between 1 D 2 and 3 H 4 state at about 612 nm. In this paper, we investigated the red persistent properties of Pr 3+ ‐doped perovskite oxide ABO 3 , (A: K, Na and B: Nb), which can be elaborated as large single crystal. KN bO 3 :Pr 3+ appears to have weak photoluminescence and no persistent luminescence. However, the cationic substituted compounds K 1− x Na x NbO 3 :Pr 3+ ( x = 0, 0.4, 0.5, 0.7 0.9, 1) exhibit intense persistent luminescence, which increases steadily with increase in Na content. We correlated persistence behavior with the position of Metal‐to‐Metal Charge Transfer ( MMCT ) band, which plays crucial role in tuning of the trap depth. The MMCT band position decreases with the addition of Na contents and the thermoluminescence peak shifts toward higher temperature indicating the formation of deeper traps. This is in good agreement with the enhancement of the persistent luminescence suggesting that a proper tailoring of MMCT is needed to design efficient Pr 3+ ‐persistent phosphors with better performance. A detailed analysis on the trap depth, bandgap energy, and persistent luminescence properties is reported tuning the composition in the K 1− x Na x NbO 3 :Pr powder.