Seeing, Targeting and Delivering with Upconverting Nanoparticles.

Efficient control over drug release is critical to increasing drug efficacy and avoiding side effects. An ideal drug delivery system would deliver drugs in the right amount, at the right location and at the right time noninvasively. This can be achieved using light-triggered delivery: light is noninvasive, spatially precise and safe if appropriate wavelengths are chosen. However, the use of light-controlled delivery systems has been limited to areas that are not too deep inside the body because ultraviolet (UV) or visible (Vis) light, the typical wavelengths used for photoreactions, have limited penetration and are toxic to biological tissues. The advent of upconverting nanoparticles (UCNPs) has made it possible to overcome this crucial challenge. UCNPs can convert near-infrared (NIR) radiation, which can penetrate deeper inside the body, to shorter wavelength NIR, Vis and UV radiation. UCNPs have been used as bright, in situ sources of light for on-demand drug release and bioimaging applications. These remote-controlled, NIR-triggered drug delivery systems are especially attractive in applications where a drug is required at a specific location and time such as in anesthetics, postwound healing, cardiothoracic surgery and cancer treatment. In this Perspective, we discuss recent progress and challenges as well as propose potential solutions and future directions, especially with regard to their translation to the clinic.

Photocleavable Hydrogel-Coated Upconverting Nanoparticles: A Multifunctional Theranostic Platform for NIR Imaging and On-Demand Macromolecular Delivery.

Lanthanide-doped upconverting nanoparticles (UCNPs) have emerged as excellent nanotransducers for converting longer wavelength near-infrared (NIR) light to shorter wavelengths spanning the ultraviolet (UV) to the visible (Vis) regions of the spectrum via a multiphoton absorption process, known as upconversion. Here, we report the development of NIR to UV-Vis-NIR UCNPs consisting of LiYF4:Yb(3+)/Tm(3+)@SiO2 individually coated with a 10 ± 2 nm layer of chitosan (CH) hydrogel cross-linked with a photocleavable cross-linker (PhL). We encapsulated fluorescent-bovine serum albumin (FITC-BSA) inside the gel. Under 980 nm excitation, the upconverted UV emission cleaves the PhL cross-links and instantaneously liberates the FITC-BSA under 2 cm thick tissue. The release is immediately arrested if the excitation source is switched off. The upconverted NIR light allows for the tracking of particles under the tissue. Nucleus pulposus (NP) cells cultured with UCNPs are viable both in the presence and in the absence of laser irradiation. Controlled drug delivery of large biomolecules and deep tissue imaging make this system an excellent theranostic platform for tissue engineering, biomapping, and cellular imaging applications.

Synthesis, characterization, and directional binding of anisotropic biohybrid microparticles for multiplexed biosensing.

Anisotropic microarchitectures with different physicochemical properties have been developed as advanced materials for challenging industrial and biomedical applications including switchable displays, multiplexed biosensors and bioassays, spatially-controlled drug delivery systems, and tissue engineering scaffolds. In this study, anisotropic biohybrid microparticles (MPs) spatio-selectively conjugated with two different antibodies (Abs) are first developed for fluorescence-based, multiplexed sensing of biological molecules. Poly(acrylamide-co-acrylic acid) is chemically modified with maleimide- or acetylene groups to introduce different targeting biological moieties into each compartment of anisotropic MPs. Modified polymer solutions containing two different fluorescent dyes are separately used for electrohydrodynamic co-jetting with side-by-side needle geometry. The anisotropic MPs are chemically stabilized by thermal imidization, followed by bioconjugation of two different sets of polyclonal Abs with two individual compartments via maleimide-thiol coupling reaction and Huisgen 1,3-dipolar cycloaddition. Finally, two compartments of the anisotropic biohybrid MPs are spatio-selectively associated with the respective monoclonal Ab-immobilized substrate in the presence of the antigen by sandwich-type immunocomplex formation, resulting in their ordered orientation due to the spatio-specific molecular interaction, as confirmed by confocal laser scanning microscopy. In conclusion, anisotropic biohybrid MPs capable of directional binding have great potential as a new fluorescence-based multiplexing biosensing system.

Controlled biohybrid nanoprobes with silver nanoparticle clusters for Raman imaging.

A new class of biohybrid nanoprobes has been developed for surface-enhanced Raman scattering-based bioimaging. Silver nanoparticle clusters were encapsulated in polymeric nanoparticles using electrohydrodynamic jetting, followed by stabilization and bioconjugation. Controlled SERS intensity with high sensitivity, chemical stability, and biocompatibility makes the SERS biohybrid nanoprobes useful for bioimaging.

Amniotic stromal stem cell-loaded hydrogel repairs cardiac tissue in infarcted rat hearts via paracrine mediators.

The use of stem cells to repair the heart after a myocardial infarction (MI) remains promising, yet clinical trials over the past 20 years suggest that cells fail to integrate into the native tissue, resulting in limited improvements in cardiac function. Here, we demonstrate the cardioprotective potential of a composite inserting human amniotic stromal mesenchymal stem cells (ASMCs) in a chitosan and hyaluronic acid (C/HA) based hydrogel in a rat MI model. Mechanical characterization of the C/HA platform indicated a swift elastic conversion at 40°C and a rapid sol-gel transition time at 37°C. Cell viability assay presented active and proliferating AMSCs in the C/HA. The ASMCs + C/HA injected composite significantly increased left ventricular ejection fraction, fractional shortening, and neovessel formation. The encapsulated AMSCs were abundantly detected in the infarcted myocardium 6 weeks post-administration and co-expressed cardiac proteins and notably proliferative markers. Proteomic profiling revealed that extracellular vesicles released from hypoxia preconditioned ASMCs contained proteins involved in cytoprotection, angiogenesis, cardiac differentiation and non-canonical Wnt-signaling. Independent activation of non-canonical Wnt-signaling pathways in ASMCs induced cardiogenesis. Despite a low injected cellular density at baseline, the encapsulated AMSCs were abundantly retained and increased cardiac function. Furthermore, the C/HA hydrogel provided an active milieu for the AMSCs to proliferate, co-express cardiac proteins, and induce new vessel formation. Hence, this novel composite of AMSCs + C/HA scaffold is a conceivable candidate that could restore cardiac function and reduce remodeling.

Thermally-Induced Actuations of Stimuli-Responsive, Bicompartmental Nanofibers for Decoupled Drug Release.

Stimuli-responsive anisotropic microstructures and nanostructures with different physicochemical properties in discrete compartments, have been developed as advanced materials for drug delivery systems, tissue engineering, regenerative medicine, and biosensing applications. Moreover, their stimuli-triggered actuations would be of great interest for the introduction of the functionality of drug delivery reservoirs and tissue engineering scaffolds. In this study, stimuli-responsive bicompartmental nanofibers (BCNFs), with completely different polymer compositions, were prepared through electrohydrodynamic co-jetting with side-by-side needle geometry. One compartment with thermo-responsiveness was composed of methacrylated poly(N-isopropylacrylamide-co-allylamine hydrochloride) (poly(NIPAM-co-AAh)), while the counter compartment was made of poly(ethylene glycol) dimethacrylates (PEGDMA). Both methacrylated poly(NIPAM-co-AAh) and PEGDMA in distinct compartments were chemically crosslinked in a solid phase by UV irradiation and swelled under aqueous conditions, because of the hydrophilicity of both poly(NIPAM-co-AAh) and PEGDMA. As the temperature increased, BCNFs maintained a clear interface between compartments and showed thermally-induced actuation at the nanoscale due to the collapsed poly(NIPAM-co-AAh) compartment under the PEGDMA compartment of identical dimensions. Different model drugs, bovine serum albumin, and dexamethasone phosphate were alternately loaded into each compartment and released at different rates depending on the temperature and molecular weight of the drugs. These BCNFs, as intelligent nanomaterials, have great potential as tissue engineering scaffolds and multi-modal drug delivery reservoirs with stimuli-triggered actuation and decoupled drug release.

Raman Spectroscopy for Inverted Papilloma: A Proof-of-Concept Study.

Inverted papillomas are tumors of the sinonasal tract with a propensity to recur. Raman spectroscopy can potentially identify inverted papillomas from other tissue based on biochemical signatures. A pilot study comparing Raman spectroscopy to histopathology for 3 types of sinonasal tissue was performed. Spectral data of biopsies from patients with normal sinonasal mucosa, chronic rhinosinusitis, and inverted papillomas are compared to histopathology using principal component analysis and linear discriminant analysis after data preprocessing. A total of 18 normal, 15 chronic rhinosinusitis, and 18 inverted papilloma specimens were evaluated. The model distinguished normal sinonasal mucosa, chronic rhinosinusitis, and inverted papilloma tissue with an overall accuracy of 90.2% (95% confidence interval, 0.86-0.94). In conclusion, Raman spectroscopy can distinguish inverted papilloma, normal sinonasal mucosa, and chronically rhinosinusitis tissue with acceptable accuracy.

Graphene oxide-stabilized perfluorocarbon emulsions for controlled oxygen delivery.

Perfluorocarbon (PFC) emulsions are capable of absorbing large quantities of oxygen. They are widely used as blood alternates for quick oxygenation of tissues. However, they are unsuitable for applications where sustained oxygen supply is desired over an extended period of time. Here, we have designed a new PFC oxygen delivery system that combines perfluorodecalin with graphene oxide (GO), where GO acts both as an emulsifier and a stabilizing agent. The resulting emulsions (PFC@GO) release oxygen at least one order of magnitude slower than emulsions prepared with other common surfactants. The release rate can be controlled by varying the thickness of the GO layer. Controlled release of oxygen make these emulsions excellent oxygen carriers for applications where sustained oxygen delivery is required e.g. in tissue regeneration and vascular wound healing.

Nano graphene oxide-wrapped gold nanostars as ultrasensitive and stable SERS nanoprobes.

We report a facile method to synthesize highly branched gold nanostars wrapped with nano graphene oxide (nGO) sheets with or without the addition of Raman dyes, as nanoprobes with high SERS activity. Both cysteamine and nGO are added to gold nanostars; the positively charged amino groups help self-assembly of nGO flakes around the nanostars. This increases the Raman signal of nGO by 5.3 folds compared to samples in which nGO is in contact with the nanostars but does not wrap them. We also prepare dye-based SERS nanoprobes by sandwiching a typical Raman reporter such as Rhodamine B (RhB), Crystal Violet (CV) and Rhodamine 6G (R6G) between the nanostars and the nGO coating. The Raman signals of RhB are 5.2 times larger when sandwiched between nGO and nanostars than if the molecules are just adsorbed on the nanostar surface, and similar enhancements are observed for the other dyes. In addition to improving SERS efficiency, the wrapping greatly improves the stability of the dye-based nanoprobes, showing a reproducible Raman signal of RhB for over a week in simulated body fluids at 37 °C. High SERS signal, facile fabrication method and excellent stability make these nanoprobes highly promising for SERS-based biosensing and bioimaging applications.

Real-time, non-invasive monitoring of hydrogel degradation using LiYF4:Yb(3+)/Tm(3+) NIR-to-NIR upconverting nanoparticles.

To design a biodegradable hydrogel as cell support, one should know its in vivo degradation rate. A technique commonly used to track gel degradation is fluorescence spectroscopy. However, the fluorescence from conventional fluorophores quickly decays, and the fluorophores are often moderately cytotoxic. Most importantly, they require ultraviolet or visible (UV-Vis) light as the excitation source, which cannot penetrate deeply through biological tissues. Lanthanide-doped upconverting nanoparticles (UCNPs) are exciting alternatives to conventional fluorophores because they can convert near-infrared (NIR) to UV-Vis-NIR light via a sequential multiphoton absorption process referred to as upconversion. NIR light can penetrate up to few cm inside tissues, thus making these UCNPs much better probes than conventional fluorophores for in vivo monitoring. Also, UCNPs have narrow emission bands, high photoluminescence (PL) signal-to-noise ratio, low cytotoxicity and good physical and chemical stability. Here, we show a nanocomposite system consisting of a biodegradable, in situ thermogelling injectable hydrogel made of chitosan and hyaluronic acid encapsulating silica-coated LiYF4:Yb(3+)/Tm(3+) UCNPs. We use these UCNPs as photoluminescent tags to monitor the gel degradation inside live, cultured intervertebral discs (IVDs) over a period of 3 weeks. PL spectroscopy and NIR imaging show that NIR-to-NIR upconversion of LiYF4:Yb(3+)/Tm(3+)@SiO2 UCNPs allows for tracking of the gel degradation in living tissues. Both in vitro and ex vivo release of UCNPs follow a similar trend during the first 5 days; after this time, ex vivo release becomes faster than in vitro, indicating a faster gel degradation ex vivo. Also, the amount of released UCNPs in vitro increases continuously up to 3 weeks, while it plateaus after 15 days inside the IVDs showing a homogenous distribution of UCNPs throughout the IVD tissue. This non-invasive optical method for real time, live tissue imaging holds great potential for tissue analysis, biomapping and bioimaging applications.

Tough, in-situ thermogelling, injectable hydrogels for biomedical applications.

Injectable hydrogels are extensively used in drug delivery and tissue engineering to administer drugs, genes, growth factors and live cells. We report a method to produce tough, in-situ thermogelling, non-toxic, injectable hydrogels made of chitosan and hyaluronic acid co-crosslinked with ß-glycerophophate and genipin. The gels are highly homogeneous and form within 32 min, i.e., faster than gels crosslinked with either genipin or ß-glycerophophate. The shear strength of co-crosslinked hydrogels is 3.5 kPa, higher than any chitosan-based gel reported. Chondrocytes and nucleus pulposus cells thrive inside the gels and produce large amounts of collagen II. Injection in rats shows that the gels form in-vivo within a short time and remain well localized for more than one week while the rats remain healthy and active. The excellent mechanical properties, fast in-situ gelation, good biocompatibility and the ability to encapsulate live cells at physiological conditions make these hydrogels ideal for tissue engineering, especially cartilage regeneration.

Fabrication and characterization of anisotropic nanofiber scaffolds for advanced drug delivery systems.

Stimuli-responsive, polymer-based nanostructures with anisotropic compartments are of great interest as advanced materials because they are capable of switching their shape via environmentally-triggered conformational changes, while maintaining discrete compartments. In this study, a new class of stimuli-responsive, anisotropic nanofiber scaffolds with physically and chemically distinct compartments was prepared via electrohydrodynamic cojetting with side-by-side needle geometry. These nanofibers have a thermally responsive, physically-crosslinked compartment, and a chemically-crosslinked compartment at the nanoscale. The thermally responsive compartment is composed of physically crosslinkable poly(N-isopropylacrylamide) poly(NIPAM) copolymers, and poly(NIPAM-co-stearyl acrylate) poly(NIPAM-co-SA), while the thermally-unresponsive compartment is composed of polyethylene glycol dimethacrylates. The two distinct compartments were physically crosslinked by the hydrophobic interaction of the stearyl chains of poly(NIPAM-co-SA) or chemically stabilized via ultraviolet irradiation, and were swollen in physiologically relevant buffers due to their hydrophilic polymer networks. Bicompartmental nanofibers with the physically-crosslinked network of the poly(NIPAM-co-SA) compartment showed a thermally-triggered shape change due to thermally-induced aggregation of poly(NIPAM-co-SA). Furthermore, when bovine serum albumin and dexamethasone phosphate were separately loaded into each compartment, the bicompartmental nanofibers with anisotropic actuation exhibited decoupled, controlled release profiles of both drugs in response to a temperature. A new class of multicompartmental nanofibers could be useful for advanced nanofiber scaffolds with two or more drugs released with different kinetics in response to environmental stimuli.