Lääkkeensaatto liposomeilla : Valoaktivoitu lääkkeen vapautus ja kohdennus silmänpohjaan

Biomolecules are emerging as the most important source of new therapeutic compounds. Commonly these molecules are fairly large and unstable in biological environment. Furthermore, the target sites are often located inside cells and specialized tissues. Nanoparticle based systems, including liposomes, have become the most studied method of biologics delivery. They increase drug stability in blood circulation and facilitates drug accumulation at the target site. However, often the amount of drug released remains insufficient. Lately, several stimuli-responsive nanoparticles have been developed for better control of the drug release. Among these are light triggered liposomes, which are the focus of this work. Liposomes consist of spherical bilayer forming lipids, phospholipids and sometimes additional stabilizers, such as cholesterol. The liposomes in this thesis were made with different manufacturing processes, among which the most common was the thin film hydration method. The size of the formed liposomes was reduced by extrusion, sonication or high pressure microfluidization. Light triggered release of cargo from the liposomes was achieved by encapsulating gold nanoparticles (AuNP) or indocyanine green (ICG), that convert light energy into heat. The produced heat affects the thermosensitive bilayer of the liposomes, making it more permeable for the drug molecules. The fluidity of the bilayer was analyzed to determine the optimal phospholipid composition. The size of the liposomes was measured by dynamic light scattering to evaluate the size reduction and uniformity. The stability of the different formulations was evaluated and compared with each other. Fluorescent molecules were used as model drug compounds to study the release properties of the liposomes in controlled in vitro and cell experiments. In this work, a large portfolio of methods and formulations was developed. By combining these properties into a single drug delivery system, efficient protection of the cargo and the healthy tissue, distribution to challenging target sites, controlled spatial and temporal drug release can be achieved. The next steps in this work involve evaluation of the optimized carrier with applicable disease models, analysis of the in vivo pharmacokinetics and more profound toxicological experiments. Even though many challenges remains to be solved, the beneficial qualities of the light triggered liposomes show great potential for treatment of posterior eye conditions, cancer and other diseases lacking in therapeutic efficacy.Biomolekyylit ovat nousseet yhdeksi tärkeimmistä terapeuttisista aineista. Yleisesti nämä yhdisteet ovat suhteellisen suuria ja epävakaita. Niiden kohteet elimistössä ovat solujen sisällä ja erikoistuneissa kudoksissa. Nanopartikkeleihin pohjautuvat menetelmät, kuten liposomit, ovat yleisin tapa biomolekyylien kuljettamiseen. Ne lisäävät lääkeen säilyvyyttä verenkierrossa ja mahdollistavat lääkkeen kerääntymisen kohteeseen. Valitettavasti kantajastaan vapautuneen lääkeaineen määrä on usein riittämätön. Viimeaikoina useita ulkoisella signaalilla aktivoitavia nanopartikkeleita on kehitetty. Näiden joukossa ovat valoaktivoitavat liposomit, jotka ovat tämän työn aiheena. Liposomit muodostuvat kaksoiskalvoksi järjestäytyneistä fosfolipideistä ja stabilointiaineista, kuten kolesterolista. Tässä työssä liposomeja on valmistettu eri menetelmin, mukaanlukien yleisin käytössä oleva ohutfilmihydraatiomenetelmä. Liposomien kokoa voidaan säätää ekstruusion, ultraäänen tai korkeapainemikrofluidisaatiolla. Valoaktivoitava lääkkeenvapautus aikaansaatiin pakkaamalla joko kultananopartikkeleita tai indosyaniini vihreää (ICG) liposomien sisään. Nämä aineet muuttavat valoenergiaa lämmöksi, mikä puolestaan aiheuttaa muutoksen lipidi kaksoiskalvossa mahdollistaen lääkkeen vapautumisen. Lukuisia menetelmiä ja lääkeformulaatioita kehitettiin tässä työssä. Ominaisuuksia yhdistelemällä lääke saatiin suojattua tehokkaasti, ja kontrolloitu sekä kohdennettu lääkevapautus saavutettiin. Jatkossa kehitettyjen lääkkeenkantajien soveltuvuus tautimalleissa todennetaan ja farmakokinetiikka sekä turvallisuus analysoidaan eläinkoikeilla. Huolimatta lukuisista haasteista, valoaktivoitavien liposomien hyvät ominaisuudet lupaavat edistystä silmänpohjan, syöpien ja muiden vaikeasti hoidettavien tautien parantamisessa

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

Funding Information: The flow cytometry analysis was performed at the HiLife Flow Cytometry Unit, University of Helsinki. We thank the DDCB Faculty of Pharmacy Unit, hosted by the University of Helsinki and supported by HiLIFE and Biocenter Finland, for providing access to Varioskan LUX and Cytation 5. We also thank Sina Bahrpeyma and Joonatan Haapalainen for their technical assistance with the QuasiVivo system, and Shirin Tavakoli and Niklas Johansson for conjugating the DSPE-HA. Ti.L. acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC CoG, grant agreement No 101001016). Ta.L. acknowledges funding from Phospholipid Research Center (#TLA-2019-068/1-1), Orion Research Foundation (#9-8214-9) and Academy of Finland (#330656). Open access funded by Helsinki University Library. The images were drawn and photographed by E.M. Funding Information: The flow cytometry analysis was performed at the HiLife Flow Cytometry Unit, University of Helsinki. We thank the DDCB Faculty of Pharmacy Unit, hosted by the University of Helsinki and supported by HiLIFE and Biocenter Finland, for providing access to Varioskan LUX and Cytation 5. We also thank Sina Bahrpeyma and Joonatan Haapalainen for their technical assistance with the QuasiVivo system, and Shirin Tavakoli and Niklas Johansson for conjugating the DSPE-HA. Ti.L. acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (ERC CoG, grant agreement No 101001016). Ta.L. acknowledges funding from Phospholipid Research Center (#TLA-2019-068/1-1), Orion Research Foundation (#9-8214-9) and Academy of Finland (#330656). Open access funded by Helsinki University Library. The images were drawn and photographed by E.M. Publisher Copyright: © 2023, The Author(s).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.Peer reviewe

Nanoparticle release from anionic nanocellulose hydrogel matrix

Nanocellulose hydrogels have been shown to be excellent platforms for sustained delivery of drug molecules. In this study, we examine the suitability of anionic nanocellulose hydrogels for the sustained release of various nanoparticles. Systems releasing nanoparticles could produce applications especially for therapeutic nanocarriers, whose life-times in vivo might be limited. Micelles, liposomes and DNA origami nanostructures were incorporated into the nanocellulose hydrogels, and their release rates were measured. Two different hydrogel qualities (with 1% and 2% mass of fiber content) were used for each nanoparticle formulation. We showed that the drug release rates depend on nanoparticle size, shape, and charge. Smaller particles with neutral charge were released faster from 1% hydrogels than from 2% hydrogels. Nanoparticles with cationic labeling were retained in both hydrogels, whereas for the neutral nanoparticles, we were able to determine the cut-off size for released particles for both hydrogels. Rod-shaped DNA origami were released rapidly even though their length was above the cut-off size of spherical particles, indicating that their smaller radial dimension facilitates their fast release. Based on our results, anionic nanocellulose hydrogels are versatile platforms for the sustained release of the chosen model nanoparticles (liposomes, micelles, and DNA origami). Alternatively, for the tightly bound nanoparticles, this could lead to nanoparticle reservoirs within hydrogels, which could act as immobilized drug release systems.Peer reviewe

Liposomal sunitinib for ocular drug delivery : A potential treatment for choroidal neovascularization

Choroidal neovascularization (CNV) is a prevalent vision-threatening vascular disorder in aging population. CNV is associated with several diseases in the posterior segment of the eye such as age-related macular degeneration (AMD). In this study we developed sunitinib-loaded liposomes to block the neovascularization signalling pathway through inhibition of tyrosine kinase of vascular endothelial growth factor receptors (VEGFRs). Liposomal sunitinib formulations were prepared by thin film hydration method and studied for their encapsulation efficiency (EE), loading capacity (LC) and drug release profile in buffer andvitreous. Our finding showed that the liposomes (mean size 104 nm) could effectively entrap sunitinib (EE approximate to 95%) at relatively high loading capacity (LC approximate to 5%) and release sunitinib over at least 3 days. Intravitreal sunitinib-loaded liposomes revealed inhibitory effect on established neovascularization in laser-induced CNV mouse model while the intravitreal injection of sunitinib solubilized with cyclodextrin was inefficient in management of neovascularization. Accordingly, liposomal sunitinib is a promising drug delivery system that should be further studied to inhibit the CNV related to AMD.Peer reviewe

The effect of light sensitizer localization on the stability of indocyanine green liposomes

Light triggered drug delivery systems offer attractive possibilities for sophisticated therapy, providing both temporal and spatial control of drug release. We have developed light triggered liposomes with clinically approved indocyanine green (ICG) as the light sensitizing compound. Amphiphilic ICG can be localized in different compartments of the liposomes, but the effect of its presence, on both triggered release and long term stability, has not been studied. In this work, we report that ICG localization has a significant effect on the properties of the liposomes. Polyethylene glycol (PEG) coating of the liposomes leads to binding and stabilization of the ICG molecules on the surface of the lipid bilayer. This formulation showed both good storage stability in buffer solution (at +4-37 degrees C) and adequate stability in serum and vitreous (at +37 degrees C). The combination of ICG within the lipid bilayer and PEG coating lead to poor stability at elevated temperatures of +22 degrees C and +37 degrees C. The mechanisms of the increased instability due to ICG insertion in the lipid bilayer was elucidated with molecular dynamics simulations. Significant PEG insertion into the bilayer was induced in the presence of ICG in the lipid bilayer. Finally, feasibility of freeze-drying as a long term storage method for the ICG liposomes was demonstrated. Overall, this is the first detailed study on the interactions of lipid bilayer, light sensitizer (ICG) and PEG coating on the liposome stability. The localization of the light triggering agent significantly alters the structure of the liposomes and it is important to consider these aspects in triggered drug delivery system design.Peer reviewe

Imaging, quantitation and kinetic modelling of intravitreal nanomaterials

In this study, the intravitreal pharmacokinetics of nanomaterials were investigated in vivo in rats and rabbits. Impact of particle size and shape (spherical, longitudinal) on ocular particle distribution and elimination was investigated with fundus camera, optical coherence tomography and ocular fluorophotometry. Differently sized particles showed prolonged ocular retention and remarkable differences in vitreal elimination, but size dependence was consistent, suggesting that other features have influence on their vitreal kinetics. We also demonstrate that liposomes are eliminated from the rabbit vitreous mainly via the anterior route. Simulation of drug concentrations after injection of intravitreal particles shows the importance of synchronized particle retention and drug release rate for efficient drug delivery. In conclusion, we provide kinetic insights in intravitreally administered nanoparticles to improve retinal drug delivery.Peer reviewe

Design and synthesis of lipid-mimetic cationic iridium complexes and their liposomal formulation for in vitro and in vivo application in luminescent bioimaging

Two iridium [Ir(NC)(2)(NN)](+) complexes with the diimine NN ligand containing a long polymethylene hydrophobic chain were synthesized and characterized by using NMR and ESI mass-spectrometry: NN - 2-(1-hexadecyl-1H-imidazol-2-yl)pyridine, NC - methyl-2-phenylquinoline-4-carboxylate (Ir1) and 2-phenylquinoline-4-carboxylic acid (Ir2). These complexes were used to prepare the luminescent PEGylated DPPC liposomes (DPPC/DSPE-PEG2000/Ir-complex = 95/4.5/1 mol%) using a thin film hydration method. The narrowly dispersed liposomes had diameters of about 110 nm. The photophysics of the complexes and labeled liposomes were carefully studied. Ir1 and Ir2 give red emission (lambda(em) = 667 and 605 nm) with a lifetime in the microsecond domain and quantum yields of 4.8% and 10.0% in degassed solution. Incorporation of the complexes into the liposome lipid bilayer results in shielding of the emitters from interaction with molecular oxygen and partial suppression of excited state nonradiative relaxation due to the effect of the relatively rigid bilayer matrix. Delivery of labeled liposomes to the cultured ARPE-19 cells demonstrated the usefulness of Ir1 and Ir2 in cellular imaging. Labeled liposomes were then injected intravitreally into rat eyes and imaged successfully with optical coherence tomography and funduscopy. In conclusion, iridium complexes enabled the successful labeling and imaging of liposomes in cells and animals.Peer reviewe

Light-Activated Liposomes Coated with Hyaluronic Acid as a Potential Drug Delivery System

Light-activated liposomes permit site and time-specific drug delivery to ocular and systemic targets. We combined a light activation technology based on indocyanine green with a hyaluronic acid (HA) coating by synthesizing HA–lipid conjugates. HA is an endogenous vitreal polysaccharide and a potential targeting moiety to cluster of differentiation 44 (CD44)-expressing cells. Light-activated drug release from 100 nm HA-coated liposomes was functional in buffer, plasma, and vitreous samples. The HA-coating improved stability in plasma compared to polyethylene glycol (PEG)-coated liposomes. Liposomal protein coronas on HA- and PEG-coated liposomes after dynamic exposure to undiluted human plasma and porcine vitreous samples were hydrophilic and negatively charged, thicker in plasma (~5 nm hard, ~10 nm soft coronas) than in vitreous (~2 nm hard, ~3 nm soft coronas) samples. Their compositions were dependent on liposome formulation and surface charge in plasma but not in vitreous samples. Compared to the PEG coating, the HA-coated liposomes bound more proteins in vitreous samples and enriched proteins related to collagen interactions, possibly explaining their slightly reduced vitreal mobility. The properties of the most abundant proteins did not correlate with liposome size or charge, but included proteins with surfactant and immune system functions in plasma and vitreous samples. The HA-coated light-activated liposomes are a functional and promising alternative for intravenous and ocular drug delivery

Diffusion and Protein Corona Formation of Lipid-Based Nanoparticles in the Vitreous Humor : Profiling and Pharmacokinetic Considerations

The vitreous humor is the first barrier encountered by intravitreally injected nanoparticles. Lipid-based nanoparticles in the vitreous are studied by evaluating their diffusion with single-particle tracking technology and by characterizing their protein coronae with surface plasmon resonance and high-resolution proteomics. Single-particle tracking results indicate that the vitreal mobility of the formulations is dependent on their charge. Anionic and neutral formulations are mobile, whereas larger (>200 nm) neutral particles have restricted diffusion, and cationic particles are immobilized in the vitreous. PEGylation increases the mobility of cationic and larger neutral formulations but does not affect anionic and smaller neutral particles. Convection has a significant role in the pharmacokinetics of nanoparticles, whereas diffusion drives the transport of antibodies. Surface plasmon resonance studies determine that the vitreal corona of anionic formulations is sparse. Proteomics data reveals 76 differentially abundant proteins, whose enrichment is specific to either the hard or the soft corona. PEGylation does not affect protein enrichment. This suggests that protein-specific rather than formulation-specific factors are drivers of protein adsorption on nanoparticles in the vitreous. In summary, our findings contribute to understanding the pharmacokinetics of nanoparticles in the vitreous and help advance the development of nanoparticle-based treatments for eye diseases.Peer reviewe

Exploring Light-Sensitive Nanocarriers for Simultaneous Triggered Antibiotic Release and Disruption of Biofilms Upon Generation of Laser-Induced Vapor Nanobubbles

Impaired penetration of antibiotics through bacterial biofilms is one of the reasons for failure of antimicrobial therapy. Hindered drug diffusion is caused on the one hand by interactions with the sticky biofilm matrix and on the other hand by the fact that bacterial cells are organized in densely packed clusters of cells. Binding interactions with the biofilm matrix can be avoided by encapsulating the antibiotics into nanocarriers, while interfering with the integrity of the dense cell clusters can enhance drug transport deep into the biofilm. Vapor nanobubbles (VNB), generated from laser irradiated nanoparticles, are a recently reported effective way to loosen up the biofilm structure in order to enhance drug transport and efficacy. In the present study, we explored if the disruptive force of VNB can be used simultaneously to interfere with the biofilm structure and trigger antibiotic release from light-responsive nanocarriers. The antibiotic tobramycin was incorporated in two types of light-responsive nanocarriers—liposomes functionalized with gold nanoparticles (Lip-AuNP) and graphene quantum dots (GQD)—and their efficacy was evaluated on Pseudomonas aeruginosa biofilms. Even though the anti-biofilm efficacy of tobramycin was improved by liposomal encapsulation, electrostatic functionalization with 70 nm AuNP unfortunately resulted in premature leakage of tobramycin in a matter of hours. Laser-irradiation consequently did not further improve P. aeruginosa biofilm eradication. Adsorption of tobramycin to GQD, on the other hand, did result in a stable formulation with high encapsulation efficiency, without burst release of tobramycin from the nanocarriers. However, even though laser-induced VNB formation from GQD resulted in biofilm disruption, an enhanced anti-biofilm effect was not achieved due to tobramycin not being efficiently released from GQD. Even though this study was unsuccessful in designing suitable nanocarriers for simultaneous biofilm disruption and light-triggered release of tobramycin, it provides insights into the difficulties and challenges that need to be considered for future developments in this regard