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.

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.

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.

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.

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.

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.

Crystallization Kinetics of an Amorphous Pharmaceutical Compound Using Fluorescence-Lifetime-Imaging Microscopy.

Pharmaceutical scientists are increasingly interested in amorphous drug formulations especially because of their higher dissolution rates. Consequently, the thorough characterization and analysis of these formulations are becoming more and more important for the pharmaceutical industry. Here, fluorescence-lifetime-imaging microscopy (FLIM) was used to monitor the crystallization of an amorphous pharmaceutical compound, indomethacin. Initially, we identified different solid indomethacin forms, amorphous and γ- and α-crystalline, on the basis of their time-resolved fluorescence. All of the studied indomethacin forms showed biexponential decays with characteristic fluorescence lifetimes and amplitudes. Using this information, the crystallization of amorphous indomethacin upon storage in 60 °C was monitored for 10 days with FLIM. The progress of crystallization was detected as lifetime changes both in the FLIM images and in the fluorescence-decay curves extracted from the images. The fluorescence-lifetime amplitudes were used for quantitative analysis of the crystallization process. We also demonstrated that the fluorescence-lifetime distribution of the sample changed during crystallization, and when the sample was not moved between measuring times, the lifetime distribution could also be used for the analysis of the reaction kinetics. Our results clearly show that FLIM is a sensitive and nondestructive method for monitoring solid-state transformations on the surfaces of fluorescent samples.

Understanding Dissolution and Crystallization with Imaging: A Surface Point of View.

The tendency for crystallization during storage and administration is the most considerable hurdle for poorly water-soluble drugs formulated in the amorphous form. There is a need to better detect often subtle and complex surface crystallization phenomena and understand their influence on the critical quality attribute of dissolution. In this study, the interplay between surface crystallization of the amorphous form during storage and dissolution testing, and its influence on dissolution behavior, is analyzed for the first time with multimodal nonlinear optical imaging (coherent anti-Stokes Raman scattering (CARS) and sum frequency generation (SFG)). Complementary analyses are provided with scanning electron microscopy, X-ray diffraction and infrared and Raman spectroscopies. Amorphous indomethacin tablets were prepared and subjected to two different storage conditions (30 °C/23% RH and 30 °C/75% RH) for various durations and then dissolution testing using a channel flow-through device. Trace levels of surface crystallinity previously imaged with nonlinear optics after 1 or 2 days of storage did not significantly decrease dissolution and supersaturation compared to the freshly prepared amorphous tablets while more extensive crystallization after longer storage times did. Multimodal nonlinear optical imaging of the tablet surfaces after 15 min of dissolution revealed complex crystallization behavior that was affected by both storage condition and time, with up to four crystalline polymorphs simultaneously observed. In addition to the well-known α- and γ-forms, the less reported metastable ε- and η-forms were also observed, with the ε-form being widely observed in samples that had retained significant surface amorphousness during storage. This form was also prepared in the pure form and further characterized. Overall, this study demonstrates the potential value of nonlinear optical imaging, together with more established solid-state analysis methods, to understand complex surface crystallization behavior and its influence on drug dissolution during the development of amorphous drugs and dosage forms.

Pectin and Mucin Enhance the Bioadhesion of Drug Loaded Nanofibrillated Cellulose Films.

PURPOSE: Bioadhesion is an important property of biological membranes, that can be utilized in pharmaceutical and biomedical applications. In this study, we have fabricated mucoadhesive drug releasing films with bio-based, non-toxic and biodegradable polymers that do not require chemical modifications. METHODS: Nanofibrillar cellulose and anionic type nanofibrillar cellulose were used as film forming materials with known mucoadhesive components mucin, pectin and chitosan as functional bioadhesion enhancers. Different polymer combinations were investigated to study the adhesiveness, solid state characteristics, film morphology, swelling, mechanical properties, drug release with the model compound metronidazole and in vitro cytotoxicity using TR146 cells to model buccal epithelium. RESULTS: SEM revealed lamellar structures within the films, which had a thickness ranging 40-240 µm depending on the film polymer composition. All bioadhesive components were non-toxic and showed high adhesiveness. Rapid drug release was observed, as 60-80% of the total amount of metronidazole was released in 30 min depending on the film formulation. CONCLUSIONS: The liquid molding used was a straightforward and simple method to produce drug releasing highly mucoadhesive films, which could be utilized in treating local oral diseases, such as periodontitis. All materials used were natural biodegradable polymers from renewable sources, which are generally regarded as safe.

Multimodal Nonlinear Optical Imaging for Sensitive Detection of Multiple Pharmaceutical Solid-State Forms and Surface Transformations.

Two nonlinear imaging modalities, coherent anti-Stokes Raman scattering (CARS) and sum-frequency generation (SFG), were successfully combined for sensitive multimodal imaging of multiple solid-state forms and their changes on drug tablet surfaces. Two imaging approaches were used and compared: (i) hyperspectral CARS combined with principal component analysis (PCA) and SFG imaging and (ii) simultaneous narrowband CARS and SFG imaging. Three different solid-state forms of indomethacin-the crystalline gamma and alpha forms, as well as the amorphous form-were clearly distinguished using both approaches. Simultaneous narrowband CARS and SFG imaging was faster, but hyperspectral CARS and SFG imaging has the potential to be applied to a wider variety of more complex samples. These methodologies were further used to follow crystallization of indomethacin on tablet surfaces under two storage conditions: 30 °C/23% RH and 30 °C/75% RH. Imaging with (sub)micron resolution showed that the approach allowed detection of very early stage surface crystallization. The surfaces progressively crystallized to predominantly (but not exclusively) the gamma form at lower humidity and the alpha form at higher humidity. Overall, this study suggests that multimodal nonlinear imaging is a highly sensitive, solid-state (and chemically) specific, rapid, and versatile imaging technique for understanding and hence controlling (surface) solid-state forms and their complex changes in pharmaceuticals.

Elucidation of Compression-Induced Surface Crystallization in Amorphous Tablets Using Sum Frequency Generation (SFG) Microscopy.

PURPOSE: To investigate the effect of compression on the crystallization behavior in amorphous tablets using sum frequency generation (SFG) microscopy imaging and more established analytical methods. METHOD: Tablets containing neat amorphous griseofulvin with/without excipients (silica, hydroxypropyl methylcellulose acetate succinate (HPMCAS), microcrystalline cellulose (MCC) and polyethylene glycol (PEG)) were prepared. They were analyzed upon preparation and storage using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, scanning electron microscopy (SEM) and SFG microscopy. RESULTS: Compression-induced crystallization occurred predominantly on the surface of the neat amorphous griseofulvin tablets, with minimal crystallinity being detected in the core of the tablets. The presence of various types of excipients was not able to mitigate the compression-induced surface crystallization of the amorphous griseofulvin tablets. However, the excipients affected the crystallization rate of amorphous griseofulvin in the core of the tablet upon compression and storage. CONCLUSIONS: SFG microscopy can be used in combination with ATR-FTIR spectroscopy and SEM to understand the crystallization behaviour of amorphous tablets upon compression and storage. When selecting excipients for amorphous formulations, it is important to consider the effect of the excipients on the physical stability of the amorphous formulations.

Indocyanine Green-Loaded Liposomes for Light-Triggered Drug Release.

Light-triggered drug delivery systems enable site-specific and time-controlled drug release. In previous work, we have achieved this with liposomes containing gold nanoparticles in the aqueous core. Gold nanoparticles absorb near-infrared light and release the energy as heat that increases the permeability of the liposomal bilayer, thus releasing the contents of the liposome. In this work, we replaced the gold nanoparticles with the clinically approved imaging agent indocyanine green (ICG). The ICG liposomes were stable at storage conditions (4-22 °C) and at body temperature, and fast near-infrared (IR) light-triggered drug release was achieved with optimized phospholipid composition and a 1:50 ICG-to-lipid molar ratio. Encapsulated small molecular calcein and FITC-dextran (up to 20 kDa) were completely released from the liposomes after light exposure for 15 s. Location of ICG in the PEG layer of the liposomes was simulated with molecular dynamics. ICG has important benefits as a light-triggering agent in liposomes: fast content release, improved stability, improved possibility of liposomal size control, regulatory approval to use in humans, and the possibility of imaging the in vivo location of the liposomes based on the fluorescence of ICG. Near-infrared light used as a triggering mechanism has good tissue penetration and safety. Thus, ICG liposomes are an attractive option for light-controlled and efficient delivery of small and large drug molecules.

Photothermally Triggered Lipid Bilayer Phase Transition and Drug Release from Gold Nanorod and Indocyanine Green Encapsulated Liposomes.

In light-activated liposomal drug delivery systems (DDSs), the light sensitivity can be obtained by a photothermal agent that converts light energy into heat. Excess heat increases the drug permeability of the lipid bilayer, and drug is released as a result. In this work, two near-IR responsive photothermal agents in a model drug delivery system are studied: either gold nanorods (GNRs) encapsulated inside the liposomes or indocyanine green (ICG) embedded into the lipid bilayer. The liposome system is exposed to light, and the heating effect is studied with fluorescent thermometers: laurdan and CdSe quantum dots (QDs). Both photothermal agents are shown to convert light into heat in an extent to cause a phase transition in the surrounding lipid bilayer. This phase transition is also proven with laurdan generalized polarization (GP). In addition to the heating results, we show that the model drug (calcein) is released from the liposomal cavity with both photothermal agents when the light power is sufficient to cause a phase transition in the lipid bilayer.

Fluorescence-suppressed time-resolved Raman spectroscopy of pharmaceuticals using complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector.

In this work, we utilize a short-wavelength, 532-nm picosecond pulsed laser coupled with a time-gated complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector to acquire Raman spectra of several drugs of interest. With this approach, we are able to reveal previously unseen Raman features and suppress the fluorescence background of these drugs. Compared to traditional Raman setups, the present time-resolved technique has two major improvements. First, it is possible to overcome the strong fluorescence background that usually interferes with the much weaker Raman spectra. Second, using the high photon energy excitation light source, we are able to generate a stronger Raman signal compared to traditional instruments. In addition, observations in the time domain can be performed, thus enabling new capabilities in the field of Raman and fluorescence spectroscopy. With this system, we demonstrate for the first time the possibility of recording fluorescence-suppressed Raman spectra of solid, amorphous and crystalline, and non-photoluminescent and photoluminescent drugs such as caffeine, ranitidine hydrochloride, and indomethacin (amorphous and crystalline forms). The raw data acquired by utilizing only the picosecond pulsed laser and a CMOS SPAD detector could be used for identifying the compounds directly without any data processing. Moreover, to validate the accuracy of this time-resolved technique, we present density functional theory (DFT) calculations for a widely used gastric acid inhibitor, ranitidine hydrochloride. The obtained time-resolved Raman peaks were identified based on the calculations and existing literature. Raman spectra using non-time-resolved setups with continuous-wave 785- and 532-nm excitation lasers were used as reference data. Overall, this demonstration of time-resolved Raman and fluorescence measurements with a CMOS SPAD detector shows promise in diverse areas, including fundamental chemical research, the pharmaceutical setting, process analytical technology (PAT), and the life sciences.

Interaction studies between indomethacin nanocrystals and PEO/PPO copolymer stabilizers.

PURPOSE: The lack of effective screening methods and systemic understanding of interaction mechanisms complicates the stabilizer selection process for nanocrystallization. This study focuses on the efficiency of stabilizers with various molecular compositions and structures to stabilize drug nanocrystals. METHODS: Five structurally different polymers were chosen as stabilizers for indomethacin nanocrystals. The affinity of polymers onto drug surfaces was measured using surface plasmon resonance (SPR) and contact angle techniques. Nanosuspensions were prepared using the wet-ball milling technique and their physico-chemical properties were thoroughly characterized. RESULTS: SPR and contact angle measurements correlated very well with each other and showed that the binding efficiency decreased in the order L64 > 17R4 > F68 ≈ T908 ≈ T1107, which is attributed to the reduced PPO/PEO ratio and different polymer structures. The electrostatic interactions between the protonated amine of poloxamines and ionized indomethacin enhanced neither the affinity nor the properties of nanosuspensions, such as particle size and physical stability. CONCLUSIONS: A good stabilizer should have high binding efficiency, full coverage, and optimal hydrophobic/hydrophilic balance. A high affinity combined with short PEO chains (L64, 17R4) caused poor physical stability of nanosuspensions, whereas moderate binding efficiencies (F68, T908, T1107) with longer PEO chains produced physically stable nanosuspensions.

Unravelling the relationship between degree of disorder and the dissolution behavior of milled glibenclamide.

Milling is an attractive method to prepare amorphous formulations as it does not require the use of solvents and is suitable for thermolabile drugs. One of the key critical quality attributes of milled amorphous formulations is their dissolution behavior. However, there are limited studies that have investigated the relationship between degree of disorder induced by milling and dissolution behavior. The main aim of this study was to identify the analytical technique used to characterize degree of disorder that correlates best with the recrystallization behavior during dissolution of milled glibenclamide samples. Solid state and surface changes during dissolution of milled glibenclamide samples were monitored in order to elucidate the processes that influence the dissolution behavior of milled glibenclamide samples. Glibenclamide was ball milled for different durations and analyzed using X-ray powder diffractometry (XRPD), Raman spectroscopy and differential scanning calorimetry (DSC). Recrystallization during dissolution of the milled amorphous materials was investigated using an in situ Raman setup. SEM was used to monitor the surfaces of the compacts during dissolution. XRPD, Raman spectroscopy and DSC indicated that glibenclamide was fully amorphous after milling for 30, 60, and 120 min, respectively. 'DSC amorphous' (i.e. fully amorphous according to the onset of crystallization obtained from DSC) glibenclamide samples experienced negligible recrystallization which had no effect on the dissolution profiles. Samples that were not 'DSC amorphous' experienced recrystallization which resulted in a decrease in dissolution rate. Unexpected elevated dissolution rate was observed initially during dissolution for samples milled for 15 to 45 min, and this was related to particle loss from surfaces of the disks during dissolution. In conclusion, the onset of crystallization obtained from DSC best predicts the recrystallization of glibenclamide during dissolution. Recrystallization and particle loss from the surface of the dissolution should be considered when interpreting the dissolution data of milled glibenclamide samples.

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.

Dissolution studies of poorly soluble drug nanosuspensions in non-sink conditions.

Sink conditions used in dissolution tests lead to rapid dissolution rates for nanosuspensions, causing difficulties in discriminating dissolution profiles between different formulations. Here, non-sink conditions were studied for the dissolution testing of poorly water-soluble drug nanosuspensions. A mathematical model for polydispersed particles was established to clarify dissolution mechanisms. The dissolution of nanosuspensions with either a monomodal or bimodal size distribution was simulated. In the experimental part, three different particle sizes of indomethacin nanosuspensions were prepared by the wet milling technique. The effects of the dissolution medium pH and agitation speed on dissolution rate were investigated. The dissolution profiles in sink and non-sink conditions were obtained by changing the ratio of sample amount to the saturation solubility. The results of the simulations and experiments indicated that when the sample amount was increased to the saturation solubility of drug, the slowest dissolution rate and the best discriminating dissolution profiles were obtained. Using sink conditions or too high amount of the sample will increase the dissolution rate and weaken the discrimination between dissolution profiles. Furthermore, the low solubility by choosing a proper pH of the dissolution medium was helpful in getting discriminating dissolution profiles, whereas the agitation speed appeared to have little influence on the dissolution profiles. This discriminatory method is simple to perform and can be potentially used in any nanoproduct development and quality control studies.

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.

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.