Far-red light-triggered cargo release from liposomes b ound to a photosensitizer-cellulose nanofiber hydrogel.

In our research we used the anionic nanofibrillar cellulose (ANFC) as a platform for far-red light-induced release of cargo from liposomes. In contrast to previous works, where photosensitizers are usually in the liposomal bilayers, we used a cellulose-binding dye. Our phthalocyanine derivative has been shown to bind very strongly to cellulose and cellulose nanofiber hydrogels, allowing us to place it outside of the liposomes. Both the sensitizer and cationic liposomes bind strongly to the ANFC after mixing, making the system easy to fabricate. Upon light activation, the photosensitizer generates reactive oxygen species (ROS) within the ANFC hydrogel, where the reactive oxygen species oxidize unsaturated lipids in the liposomal membrane, which makes the liposomes more permeable, resulting in on-demand cargo release. We were able to achieve ca. 70 % release of model hydrophilic cargo molecule calcein from the hydrogels with a relatively low dose of light (262 J/cm2) while employing the straightforward fabrication techniques. Our system was remarkably responsive to the far-red light (730 nm), enabling deep tissue penetration. Therefore, this very promising novel cellulose-immobilized photosensitizer liposomal platform could be used as a controlled drug delivery system, which can have applications in externally activated coatings or implants.

Development of Robust Cationic Light-Activated Thermosensitive Liposomes: Choosing the Right Lipids.

Extensive research has been conducted on cationic light-activated thermosensitive liposomes (CLTSLs) as a means for site-specific and controlled drug release; however, less attention has been given to the stability of these nanoparticles. Selecting the appropriate lipids is crucial for the development of a stable and responsive system. In this study, we investigated the impact of various lipids on the physical properties of cationic light-activated liposomes. Incorporating poly(ethylene glycol) PEG molecules resulted in uniform liposomes with low polydispersity index, while the addition of unsaturated lipid (DOTAP) resulted in extremely leaky liposomes, with almost 80% release in just 10 min of incubation at body temperature. Conversely, the inclusion of cholesterol in the formulation increased liposome stability too much and decreased their sensitivity to stimuli-responsive release, with only 14% release after 2 min of light exposure. To achieve stable and functional CLTSL, we substituted an equivalent amount of unsaturated lipid with a saturated lipid (DPTAP), resulting in stable liposomes at body temperature that were highly responsive to light, releasing 90% of their content in 10 s of light exposure. We also conducted two atomistic molecular dynamics simulations using lipid compositions with saturated and unsaturated lipids to investigate the effect of lipid composition on the dynamical properties of the liposomal lipid bilayer. Our findings suggest that the nature of lipids used to prepare liposomes significantly affects their properties, especially when the drug loading needs to be stable but triggered drug release properties are required at the same time. Selecting the appropriate lipids in the right amount is therefore essential for the preparation of liposomes with desirable properties.

Triplet sensitization enables bidirectional isomerization of diazocine with 130 nm redshift in excitation wavelengths.

Diazocines are bridged azobenzenes with phenyl rings connected by a CH2-CH2 group. Despite this rather small structural difference, diazocine exhibits improved properties over azobenzene as a photoswitch and most importantly, its Z configuration is more stable than the E isomer. Herein, we reveal yet another unique feature of this emerging class of photoswitches. In striking contrast to azobenzenes and other photochromes, diazocine can be selectively switched in E → Z direction and most intriguingly from its thermodynamically stable Z to metastable E isomer upon successive excitation of two different triplet sensitizers present in solution at the same time. This approach leads to extraordinary large redshift of excitation wavelengths to perform isomerization i.e. from 400 nm blue to 530 nm green light (Z → E) and from 530 nm green to 740 nm far-red one (E → Z), which falls in the near-infrared window in biological tissue. Therefore, this work opens up of potential avenues for utilizing diazocines for example in photopharmacology, smart materials, light energy harvesting/storage devices, and out-of-equilibrium systems.

Unraveling an Alternative Mechanism in Polymer Self-Assemblies: An Order-Order Transition with Unusual Molecular Interactions between Hydrophilic and Hydrophobic Polymer Blocks.

Polymer self-assembly leading to cooling-induced hydrogel formation is relatively rare for synthetic polymers and typically relies on H-bonding between repeat units. Here, we describe a non-H-bonding mechanism for a cooling-induced reversible order-order (sphere-to-worm) transition and related thermogelation of solutions of polymer self-assemblies. A multitude of complementary analytical tools allowed us to reveal that a significant fraction of the hydrophobic and hydrophilic repeat units of the underlying block copolymer is in close proximity in the gel state. This unusual interaction between hydrophilic and hydrophobic blocks reduces the mobility of the hydrophilic block significantly by condensing the hydrophilic block onto the hydrophobic micelle core, thereby affecting the micelle packing parameter. This triggers the order-order transition from well-defined spherical micelles to long worm-like micelles, which ultimately results in the inverse thermogelation. Molecular dynamics modeling indicates that this unexpected condensation of the hydrophilic corona onto the hydrophobic core is due to particular interactions between amide groups in the hydrophilic repeat units and phenyl rings in the hydrophobic ones. Consequently, changes in the structure of the hydrophilic blocks affecting the strength of the interaction could be used to control macromolecular self-assembly, thus allowing for the tuning of gel characteristics such as strength, persistence, and gelation kinetics. We believe that this mechanism might be a relevant interaction pattern for other polymeric materials as well as their interaction in and with biological environments. For example, controlling the gel characteristics could be considered important for applications in drug delivery or biofabrication.

Establishing a simple perfusion cell culture system for light-activated liposomes.

The off-target effects of light-activated or targeted liposomes are difficult to distinguish in traditional well plate experiments. Additionally, the absence of fluid flow in traditional cell models can lead to overestimation of nanoparticle uptake. In this paper, we established a perfusion cell culture platform to study light-activated liposomes and determined the effect of flow on the liposomal cell uptake. The optimal cell culturing parameters for the A549 cells under flow conditions were determined by monitoring cell viability. To determine optimal liposome treatment times, particle uptake was measured with flow cytometry. The suitability of commercial QuasiVivo flow-chambers for near-infrared light activation was assessed with a calcein release study. The chamber material did not hinder the light activation and subsequent calcein release from the liposomes. Furthermore, our results show that the standard cell culturing techniques are not directly translatable to flow cultures. For non-coated liposomes, the uptake was hindered by flow. Interestingly, hyaluronic acid coating diminished the uptake differences between the flow and static conditions. The study demonstrates that flow affects the liposomal uptake by lung cancer cell line A549. The flow also complicates the cell attachment of A549 cells. Moreover, we show that the QuasiVivo platform is suitable for light-activation studies.

Intracellular Dynamics of Extracellular Vesicles by Segmented Trajectory Analysis.

The analysis of nanoparticle (NP) dynamics in live cell studies by video tracking provides detailed information on their interactions and trafficking in the cells. Although the video analysis is not yet routinely used in NP studies, the equipment suitable for the experiments is already available in most laboratories. Here, we compare trajectory patterns, diffusion coefficients, and particle velocities of NPs in A549 cells with a rather simple experimental setup consisting of a fluorescence microscope and openly available trajectory analysis software. The studied NPs include commercial fluorescent polymeric particles and two subpopulations of PC-3 cell-derived extracellular vesicles (EVs). As bioderived natural nanoparticles, the fluorescence intensities of the EVs limited the recording speed. Therefore, we studied the effect of the recording frame rate and analysis parameters to the trajectory results with bright fluorescent commercial NPs. We show that the trajectory classification and the apparent particle velocities are affected by the recording frame rate, while the diffusion constants stay comparable. The NP trajectory patterns were similar for all NP types and resembled intracellular vesicular transport. Interestingly, the EV movements were faster than the commercial NPs, which contrasts with their physical sizes and may indicate a greater role of the motor proteins in their intracellular transports.

Correction: Addressing challenges in the removal of unbound dye from passively labelled extracellular vesicles.

[This corrects the article DOI: 10.1039/D1NA00755F.].

Phototoxicity of BODIPY in long-term imaging can be reduced by intramolecular motion.

For long-term live-cell fluorescence imaging and biosensing, it is crucial to work with a dye that has high fluorescence quantum yield and photostability without being detrimental to the cells. In this paper, we demonstrate that neutral boron-dipyrromethene (BODIPY)-based molecular rotors have great properties for high-light-dosage demanding live-cell fluorescence imaging applications that require repetitive illuminations. In molecular rotors, an intramolecular rotation (IMR) allows an alternative route for the decay of the singlet excited state (S1) via the formation of an intramolecular charge transfer state (CT). The occurrence of IMR reduces the probability of the formation of a triplet state (T1) which could further react with molecular oxygen (3O2) to form cytotoxic reactive oxygen species, e.g., singlet oxygen (1O2). We demonstrate that the oxygen-related nature of the phototoxicity for BODIPY derivatives can be significantly reduced if a neutral molecular rotor is used as a probe. The studied neutral molecular rotor probe shows remarkably lower phototoxicity when compared with both the non-rotating BODIPY derivative and the cationic BODIPY-based molecular rotor in different light dosages and dye concentrations. It is also evident that the charge and localization of the fluorescent probe are as significant as the IMR in terms of the phototoxicity in a long-term live-cell imaging.

Microfluidic oxygen tolerability screening of nanocarriers for triplet fusion photon upconversion.

The full potential of triplet fusion photon upconversion (TF-UC) of providing high-energy photons locally with low-energy excitation is limited in biomedicine and life sciences by its oxygen sensitivity. This hampers the applicability of TF-UC systems in sensors, imaging, optogenetics and drug release. Despite the advances in improving the oxygen tolerability of TF-UC systems, the evaluation of oxygen tolerability is based on comparing the performance at completely deoxygenated (0% oxygen) and ambient (20-21%) conditions, leaving the physiological oxygen levels (0.3-13.5%) neglected. This oversight is not deliberate and is only the result of the lack of simple and predictable methods to obtain and maintain these physiological oxygen levels in an optical setup. Herein, we demonstrate the use of microfluidic chips made of oxygen depleting materials to study the oxygen tolerability of four different micellar nanocarriers made of FDA-approved materials with various oxygen scavenging capabilities by screening their TF-UC performance over physiological oxygen levels. All nanocarriers were capable of efficient TF-UC even in ambient conditions. However, utilizing oxygen scavengers in the oil phase of the nanocarrier improves the oxygen tolerability considerably. For example, at the mean tumour oxygen level (1.4%), nanocarriers made of surfactants and oil phase both capable of oxygen scavenging retained remarkably 80% of their TF-UC emission. This microfluidic concept enables faster, simpler and more realistic evaluation of, not only TF-UC, but any micro or nanoscale oxygen-sensitive system and facilitates their development and implementation in biomedical and life science applications.

Azobenzene Photoswitching with Near-Infrared Light Mediated by Molecular Oxygen.

Efficient photoisomerization between the cis and the trans states of azobenzenes using low-energy light is desirable for a range of applications in, e.g., photobiology yet challenging to accomplish directly with modified azobenzenes. Herein, we utilize molecular iodine as a photocatalyst to induce indirect cis-to-trans isomerization of 4,4'-dimethoxyazobenzene with 770 nm near-infrared light, showing robustness during more than 1000 cycles in ambient conditions. Intriguingly, the catalysis is mediated by molecular oxygen, and we demonstrate that other singlet-oxygen-generating photosensitizers besides iodine, i.e., palladium phthalocyanine, catalyze the isomerization as well. Thus, we envision that the approach can be further improved by employing other catalysts with suitable photoelectrochemical properties. Further studies are needed to explore the applicability of the approach with other azobenzene derivatives.

Angiogenic Effects and Crosstalk of Adipose-Derived Mesenchymal Stem/Stromal Cells and Their Extracellular Vesicles with Endothelial Cells.

Adipose-derived mesenchymal stem/stromal cells (ASCs) are an adult stem cell population able to self-renew and differentiate into numerous cell lineages. ASCs provide a promising future for therapeutic angiogenesis due to their ability to promote blood vessel formation. Specifically, their ability to differentiate into endothelial cells (ECs) and pericyte-like cells and to secrete angiogenesis-promoting growth factors and extracellular vesicles (EVs) makes them an ideal option in cell therapy and in regenerative medicine in conditions including tissue ischemia. In recent angiogenesis research, ASCs have often been co-cultured with an endothelial cell (EC) type in order to form mature vessel-like networks in specific culture conditions. In this review, we introduce co-culture systems and co-transplantation studies between ASCs and ECs. In co-cultures, the cells communicate via direct cell-cell contact or via paracrine signaling. Most often, ASCs are found in the perivascular niche lining the vessels, where they stabilize the vascular structures and express common pericyte surface proteins. In co-cultures, ASCs modulate endothelial cells and induce angiogenesis by promoting tube formation, partly via secretion of EVs. In vivo co-transplantation of ASCs and ECs showed improved formation of functional vessels over a single cell type transplantation. Adipose tissue as a cell source for both mesenchymal stem cells and ECs for co-transplantation serves as a prominent option for therapeutic angiogenesis and blood perfusion in vivo.

Expanding excitation wavelengths for azobenzene photoswitching into the near-infrared range via endothermic triplet energy transfer.

Developing azobenzene photoswitches capable of selective and efficient photoisomerization by long-wavelength excitation is an enduring challenge. Herein, rapid isomerization from the Z- to E-state of two ortho-functionalized bistable azobenzenes with near-unity photoconversion efficiency was driven by triplet energy transfer upon red and near-infrared (up to 770 nm) excitation of porphyrin photosensitizers in catalytic micromolar concentrations. We show that the process of triplet-sensitized isomerization is efficient even when the sensitizer triplet energy is substantially lower (>200 meV) than that of the azobenzene used. This makes the approach applicable for a wide variety of sensitizer-azobenzene combinations and enables the expansion of excitation wavelengths into the near-infrared spectral range. Therefore, indirect excitation via endothermic triplet energy transfer provides efficient and precise means for photoswitching upon 770 nm near-infared light illumination with no chemical modification of the azobenzene chromophore, a desirable feature in photocontrollable biomaterials.

Comparing the taste-modifying properties of nanocellulose and carboxymethyl cellulose.

The taste-modifying properties of nanofibrillar cellulose (NFC) and carboxymethyl cellulose (CMC) are compared for the first time. The samples were prepared in the form of gels, with and without added sweet and bitter taste components. As viscosity itself is known to affect taste perception, the viscosities of NFC and CMC samples were set to the same level as shear rates commonly found in the oral cavity. A trained panel of 10 assessors evaluated the bitterness and sweetness of the samples. Further, the assessors were given an opportunity to describe the samples in free words. The taste-modifying capacities of the thickening agents were at the same level when sweet compounds were added. However, CMC was better able to reduce the bitterness of quinine hydrochloride than NFC, which did not show any bitterness-reduction ability with the compound. This was unexpected, as our previous studies of NFC showed fairly high binding capacity with quinine. The open-ended responses revealed that the NFC-containing samples had an astringent sensation, while certain assessors observed a sensation of saltiness in the CMC samples. This may explain the inability of NFC to mask the bitterness of quinine hydrochloride, as astringency may act as a bitterness enhancer, while saltiness may suppress it. Both thickening agents were perceived as slightly bitter. Our study reveals the need for further assessment of the orosensory properties of NFC, particularly the magnitude and origin of its astringency, before it can be fully utilized in food industry applications.

Drug diffusivities in nanofibrillar cellulose hydrogel by combined time-resolved Raman and fluorescence spectroscopy.

Hydrogels, natural and synthetic origin, are actively studied for their use for implants and payload carriers. These biomaterials for delivery systems have enormous potential in basic biomedical research, drug development, and long-term delivery of biologics. Nanofibrillated cellulose (NFC) hydrogels, both natural and anionic (ANFC) ones, allow drug loading for immediate and controlled release via the slow drug dissolution of solid drug crystals into hydrogel and its subsequent release. This property makes NFC originated hydrogels an interesting non-toxic and non-human origin material as drug reservoir for long-term controlled release formulation or implant for patient care. A compelling tool for studying NFC hydrogels is Raman spectroscopy, which enables to resolve the chemical structures of different molecules in a high-water content like hydrogels, since Raman spectroscopy is insensitive to water molecules. That offers real time investigation of label-free drugs and their release in high-water-content media. Despite the huge potential of Raman spectroscopy in bio-pharmaceutical applications, the strong fluorescence background of many drug samples masking the faint Raman signal has restricted the widespread use of it. In this study we used a Raman spectrometer capable of suppressing the unpleasant fluorescence background by combining a pulsed laser and time-resolved complementary metal-oxide-semiconductor (CMOS) single-photon avalanche diode (SPAD) line sensor for the label-free investigation of Metronidazole and Vitamin C diffusivities in ANFC. The results show the possibility to modulate the ANFC-based implants and drug delivery systems, when the release rate needs to be set to a desired value. More importantly, the now developed label free real-time method is universal and can be adapted to any hydrogel/drug combination for producing reliable drug diffusion coefficient data in complex and heterogeneous systems, where traditional sampling-based methods are cumbersome to use. The wide temporal range of the time-resolved CMOS SPAD sensors makes it possible to capture also the fluorescence decay of samples, giving rise to a combined time-resolved Raman and fluorescence spectroscopy, which provides additional information on the chemical, functional and structural changes in samples.

Addressing challenges in the removal of unbound dye from passively labelled extracellular vesicles.

Studies of extracellular vesicles (EVs), their trafficking and characterization often employ fluorescent labelling. Unfortunately, little attention has been paid thus far to a thorough evaluation of the purification of EVs after labelling, although the presence of an unbound dye may severely compromise the results or even lead to wrong conclusions on EV functionality. Here, we systematically studied five dyes for passive EV labelling and meticulously compared five typical purification methods: ultracentrifugation (UC), ultracentrifugation with discontinuous density gradient (UCG), ultrafiltration (UF), size exclusion chromatography (SEC), and anion exchange chromatography (AEC). A general methodology for evaluation of EV purification efficiency after the labelling was developed and tested to select the purification methods for the chosen dyes. Firstly, we found that some methods initially lead to high EV losses even in the absence of the dye. Secondly, the suitable purification method needs to be found for each particular dye and depends on the physical and chemical properties of the dye. Thirdly, we demonstrated that the developed parameter E rp (relative purification efficiency) is a useful tool for the pre-screening of the suitable dye-purification method combinations. Additionally, it was also shown that the labelled EVs properly purified from the unbound dye may show significantly reduced contrast and visibility in the target application, e.g. in the live cell fluorescence lifetime imaging.

Taste compound - Nanocellulose interaction assessment by fluorescence indicator displacement assay.

Interactions between taste compounds and nanofibrillar cellulose were studied. For this, a new fluorescent indicator displacement method was developed. Two fluorescent indicators, namely, Calcofluor white and Congo red, were chosen because of their specific binding to cellulose and intrinsic fluorescence. Seven taste compounds with different structures were successfully measured together with nanofibrillar cellulose (NFC) and ranked according to their binding constants. The most pronounced interactions were found between quinine and NFC (1.4 × 104 M-1), whereas sucrose, aspartame and glutamic acid did not bind at all. Naringin showed moderate binding while stevioside and caffeine exhibited low binding. The comparison with microcrystalline cellulose indicates that the larger surface area of nanofibrillated cellulose enables stronger binding between the binder and macromolecules. The developed method can be further utilized to study interactions of different compound classes with nanocellulose materials in food, pharmaceutical and dye applications, using a conventional plate reader in a high-throughput manner.

Surface Stabilization and Dissolution Rate Improvement of Amorphous Compacts with Thin Polymer Coatings: Can We Have It All?

The distinction between surface and bulk crystallization of amorphous pharmaceuticals, as well as the importance of surface crystallization for pharmaceutical performance, is becoming increasingly evident. An emerging strategy in stabilizing the amorphous drug form is to utilize thin coatings at the surface. While the physical stability of systems coated with pharmaceutical polymers has recently been studied, the effect on dissolution performance as a function of storage time, as a further necessary step toward the success of these formulations, has not been previously studied. Furthermore, the effect of coating thickness has not been elucidated. This study investigated the effect of these polymer-coating parameters on the interplay between amorphous surface crystallization and drug dissolution for the first time. The study utilized simple tablet-like coated dosage forms, comprising a continuous amorphous drug core and thin polymer coating (hundreds of nanometers to a micrometer thick). Monitoring included analysis of both the solid-state of the model drug (with SEM, XRD, and ATR FTIR spectroscopy) and dissolution performance (and associated morphology and solid-state changes) after different storage times. Stabilization of the amorphous form (dependent on the coating thickness) and maintenance of early-stage intrinsic dissolution rates characteristic for the unaged amorphous drug were achieved. However, dissolution in the latter stages was likely inhibited by the presence of a polymer at the surface. Overall, this study introduced a versatile coated system for studying the dissolution of thin-coated amorphous dosage forms suitable for different drugs and coating agents. It demonstrated the importance of multiple factors that need to be taken into consideration when aiming to achieve both physical stability and improved release during the shelf life of amorphous formulations.

Linking volatile and non-volatile compounds to sensory profiles and consumer liking of wild edible Nordic mushrooms.

Current information on the links between the chemistry and hedonic liking of edible mushrooms is scarce. In this study, 84 consumers evaluated the appearance, odor, taste, texture and overall liking of samples of Nordic edible wild mushroom species. Subsequently, multivariate models on the effects of non-volatile compounds, odor-contributing volatile compounds, sensory attributes and hedonic likings were created. The non-volatile compounds were measured with quantitative NMR. The five studied mushroom species were different in their sugar and acid contents. Three consumer clusters were found with species*cluster interactions. Correlations with sensory attributes and chemical components were found, and the multivariate models indicated predictor attributes for each consumer cluster. The results indicate that the sensory properties could be correlated to both volatile and non-volatile compounds, there are consumer clusters with differing likings as regards mushrooms, and these clusters are heterogenic groups with no simple factors such as age explaining their liking scores.

Endothermic and Exothermic Energy Transfer Made Equally Efficient for Triplet-Triplet Annihilation Upconversion.

Expanding the anti-Stokes shift for triplet-triplet annihilation upconversion (TTA-UC) systems with high quantum yields without compromising power density thresholds (Ith) remains a critical challenge in photonics. Our studies reveal that such expansion is possible by using a highly endothermic TTA-UC pair with an enthalpy difference of +80 meV even in a polymer matrix 1000 times more viscous than toluene. Carrying out efficient endothermic triplet-triplet energy transfer (TET) requires suppression of the reverse annihilator-to-sensitizer TET, which was achieved by using sensitizers with high molar extinction coefficients and long triplet state lifetimes as well as optimized annihilator concentrations. Under these conditions, the sensitizer-to-annihilator forward TET becomes effectively entropy driven, yielding upconversion quantum yields comparable to those achieved with the exothermic TTA-UC pair but with larger anti-Stokes shifts and even lower Ith, a previously unattained achievement.

Effects of nanofibrillated cellulose hydrogels on adipose tissue extract and hepatocellular carcinoma cell spheroids in freeze-drying.

The aim of this study was to evaluate the effects of two nanofibrillated cellulose (NFC) hydrogels on two human derivatives during freeze-drying. Native NFC hydrogel is a suitable platform to culture 3D cell spheroids and a hydrogel processed further, called anionic NFC (ANFC) hydrogel, is an excellent platform for controlled release of proteins. Moreover, it has been shown to be compatible with freeze-drying when correct lyoprotectants are implemented. Freeze-drying is a method, where substance is first frozen, and then vacuum dried trough sublimation of water in order to achieve dry matter without the loss of the original three-dimensional structures. The first chosen human derivative was adipose tissue extract (ATE) which is a cell-free growth factor-rich preparation capable of promoting growth of regenerative cells. The release of growth factors from the freeze-dried mixture of ATE and ANFC was compared to that of non-freeze-dried control mixtures. The release profiles remained at the same level after freeze-drying. The second derivative was hepatocellular carcinoma (HepG2) cell spheroids which were evaluated before and after freeze-drying. The 3D structure of the HepG2 cell spheroids was preserved and the spheroids retained 18% of their metabolic activity after rehydration. However, the freeze-dried and rehydrated HepG2 cell spheroids did not proliferate and the cell membrane was damaged by fusion and formation of crystals.