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(N^C)2(N^N)]+ complexes with the diimine N^N ligand containing a long polymethylene hydrophobic chain were synthesized and characterized by using NMR and ESI mass-spectrometry: N^N - 2-(1-hexadecyl-1H-imidazol-2-yl)pyridine, N^C - 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 (λ 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.

Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment.

Cell therapy combined with biomaterial scaffolds is used to treat cartilage defects. We hypothesized that chondrogenic differentiation bone marrow-derived mesenchymal stem cells (BM-MSCs) in three-dimensional biomaterial scaffolds would initiate cartilaginous matrix deposition and prepare the construct for cartilage regeneration in situ. The chondrogenic capability of human BM-MSCs was first verified in a pellet culture. The BM-MSCs were then either seeded onto a composite scaffold rhCo-PLA combining polylactide and collagen type II (C2) or type III (C3), or commercial collagen type I/III membrane (CG). The BM-MSCs were either cultured in a proliferation medium or chondrogenic culture medium. Adult human chondrocytes (ACs) served as controls. After 3, 14, and 28 days, the constructs were analyzed with quantitative polymerase chain reaction and confocal microscopy and sulfated glycosaminoglycans (GAGs) were measured. The differentiated BM-MSCs entered a hypertrophic state by Day 14 of culture. The ACs showed dedifferentiation with no expression of chondrogenic genes and low amount of GAG. The CG membrane induced the highest expression levels of hypertrophic genes. The two different collagen types in composite scaffolds yielded similar results. Regardless of the biomaterial scaffold, culturing BM-MSCs in chondrogenic differentiation medium resulted in chondrocyte hypertrophy. Thus, caution for cell fate is required when designing cell-biomaterial constructs for cartilage regeneration.

Characterization of CDNF-Secreting ARPE-19 Cell Clones for Encapsulated Cell Therapy.

Cerebral dopamine neurotrophic factor (CDNF) shows beneficial effects in rodent models of Parkinson's and Alzheimer's disease. The brain is a challenging target for protein therapy due to its exclusive blood-brain barrier. Hence, the therapeutic protein should be delivered directly to the brain parenchyma. Implantation of encapsulated mammalian cells that constantly secrete CDNF is a potential approach for targeted and long-term protein delivery to the brain. In this study, we generated several CDNF-secreting cell clones derived from human retinal pigment epithelial cell line ARPE-19, and studied CDNF secretion from the clones maintained as monolayers and in polymeric microcapsules. The secretion of wild type (wt) CDNF transgene was low and the majority of the produced protein remained intracellular, locating mainly to the endoplasmic reticulum (ER). The secretion of wtCDNF decreased to even lower levels when the clones were in a non-dividing state, as in the microcapsules. Both codon optimization and deletion of the putative ER-retrieval signal (four last amino acids: KTEL) improved CDNF secretion. More importantly, the secretion of KTEL-deleted CDNF remained constant in the non-dividing clones. Thus, cells expressing KTEL-deleted CDNF, in contrast to wtCDNF, can be considered for cell encapsulation applications if the KTEL-deleted CDNF is proven to be biologically active in vivo.

Light-Triggered Cellular Delivery of Oligonucleotides.

The major challenge in the therapeutic applicability of oligonucleotide-based drugs is the development of efficient and safe delivery systems. The carriers should be non-toxic and stable in vivo, but interact with the target cells and release the loaded oligonucleotides intracellularly. We approached this challenge by developing a light-triggered liposomal delivery system for oligonucleotides based on a non-cationic and thermosensitive liposome with indocyanine green (ICG) as photosensitizer. The liposomes had efficient release properties, as 90% of the encapsulated oligonucleotides were released after 1-minute light exposure. Cell studies using an enhanced green fluorescent protein (EGFP)-based splicing assay with HeLa cells showed light-activated transfection with up to 70%⁻80% efficacy. Moreover, free ICG and oligonucleotides in solution transfected cells upon light induction with similar efficacy as the liposomal system. The light-triggered delivery induced moderate cytotoxicity (25%⁻35% reduction in cell viability) 1⁻2 days after transfection, but the cell growth returned to control levels in 4 days. In conclusion, the ICG-based light-triggered delivery is a promising method for oligonucleotides, and it can be used as a platform for further optimization and development.

Light activated liposomes: Functionality and prospects in ocular drug delivery.

Ocular drug delivery, especially to the retina and choroid, is a major challenge in drug development. Liposome technology may be useful in ophthalmology in enabling new routes of delivery, prolongation of drug action and intracellular drug delivery, but drug release from the liposomes should be controlled. For that purpose, light activation may be an approach to release drug at specified time and site in the eye. Technical advances have been made in the field of light activated drug release, particularly indocyanine green loaded liposomes are a promising approach with safe materials and effective light triggered release of small and large molecules. This review discusses the liposomal drug delivery with light activated systems in the context of ophthalmic drug delivery challenges.

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.

Light induced cytosolic drug delivery from liposomes with gold nanoparticles.

Externally triggered drug release at defined targets allows site- and time-controlled drug treatment regimens. We have developed liposomal drug carriers with encapsulated gold nanoparticles for triggered drug release. Light energy is converted to heat in the gold nanoparticles and released to the lipid bilayers. Localized temperature increase renders liposomal bilayers to be leaky and triggers drug release. The aim of this study was to develop a drug releasing system capable of releasing its cargo to cell cytosol upon triggering with visible and near infrared light signals. The liposomes were formulated using either heat-sensitive or heat- and pH-sensitive lipid compositions with star or rod shaped gold nanoparticles. Encapsulated fluorescent probe, calcein, was released from the liposomes after exposure to the light. In addition, the pH-sensitive formulations showed a faster drug release in acidic conditions than in neutral conditions. The liposomes were internalized into human retinal pigment epithelial cells (ARPE-19) and human umbilical vein endothelial cells (HUVECs) and did not show any cellular toxicity. The light induced cytosolic delivery of calcein from the gold nanoparticle containing liposomes was shown, whereas no cytosolic release was seen without light induction or without gold nanoparticles in the liposomes. The light activated liposome formulations showed a controlled content release to the cellular cytosol at a specific location and time. Triggering with visual and near infrared light allows good tissue penetration and safety, and the pH-sensitive liposomes may enable selective drug release in the intracellular acidic compartments (endosomes, lysosomes). Thus, light activated liposomes with gold nanoparticles are an attractive option for time- and site-specific drug delivery into the target cells.

Encapsulated cells for long-term secretion of soluble VEGF receptor 1: Material optimization and simulation of ocular drug response.

Anti-angiogenic therapies with vascular endothelial growth factor (VEGF) inhibiting factors are effective treatment options for neovascular diseases of the retina, but these proteins can only be delivered as intravitreal (IVT) injections. To sustain a therapeutic drug level in the retina, VEGF inhibitors have to be delivered frequently, every 4-8weeks, causing inconvenience for the patients and expenses for the healthcare system. The aim of this study was to investigate cell encapsulation as a delivery system for prolonged anti-angiogenic treatment of retinal neovascularization. Genetically engineered ARPE-19 cells secreting soluble vascular endothelial growth factor receptor 1 (sVEGFR1) were encapsulated in a hydrogel of cross-linked collagen and interpenetrating hyaluronic acid (HA). The system was optimized in terms of matrix composition and cell density, and long-term cell viability and protein secretion measurements were performed. sVEGFR1 ARPE-19 cells in the optimized hydrogel remained viable and secreted sVEGFR1 at a constant rate for at least 50days. Based on pharmacokinetic/pharmacodynamic (PK/PD) modeling, delivery of sVEGFR1 from this cell encapsulation system is expected to lead only to modest VEGF inhibition, but improvements of the protein structure and/or secretion rate should result in strong and prolonged therapeutic effect. In conclusion, the hydrogel matrix herein supported the survival and protein secretion from the encapsulated cells. The PK/PD simulation is a convenient approach to predict the efficiency of the cell encapsulation system before in vivo experiments.

Xeno-free chondrogenesis of bone marrow mesenchymal stromal cells: towards clinical-grade chondrocyte production.

Current cell-based cartilage therapies relay on articular cartilage-derived autologous chondrocytes as a cell source, which possesses disadvantages, such as, donor site morbidity and dedifferentiation of chondrocytes during in vitro expansion. Due to these and other limitations, novel cell sources and production strategies are needed. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are a fascinating alternative, but they are not spontaneously capable of producing hyaline cartilage-like repair tissue in vivo. In vitro pre-differentiation of BM-MSCs could be used to produce chondrocytes for clinical applications. However, clinically compatible defined and xeno-free differentiation protocol is lacking. Hence, this study aimed to develop such chondrogenic differentiation medium for human BM-MSCs. We assessed the feasibility of the medium using three human BM-MSCs donors and validated the method by comparing BM-MSCs to three other cell types holding potential for articular cartilage repair. The effectiveness of the method was compared to conventional serum-free and commercially available chondrogenic differentiation media. The results show that the defined xeno-free differentiation medium is at least as efficient as conventionally used serum-free chondrogenic medium and performed significantly better on all cell types tested compared to the commercially available chondrogenic medium.

High-throughput in vitro drug release and pharmacokinetic simulation as a tool for drug delivery system development: application to intravitreal ocular administration.

In vitro estimation of release kinetics from drug delivery systems is needed in formulation development. Cost-effective methods of assessment for delivery systems are needed particularly in the case of biologicals and drug administration routes that are difficult to screen in vivo (e.g. intraocular drug delivery). As a proof-of-concept, we demonstrate here a practical high-throughput methodology to investigate in vitro drug release and predict resulting drug concentrations in the eye after intravitreal administration. 96-well plate based assay aided with robotic sampling was used to study release of eight model drugs of varying physicochemical properties (dexamethasone, vancomycin, alpha-lactalbumin, lysozyme, myoglobin, albumin, lactoferrin, human IgG) from twelve alginate microsphere formulations. The amount of drug released over a period of time was assessed by photometric and fluorescence methods. In vitro drug release rates obtained were used in pharmacokinetic simulations using one-compartment model of the vitreal cavity with anatomical volume of distribution and clearance estimates based on the literature precedence. An integrated approach of drug release screening and pharmacokinetic simulations can prove to be a useful methodology in guiding formulation development for ocular delivery in animal models. In general, the methodology has the potential to be a cost-effective tool for early stage drug delivery system discovery and development.

An injectable, in situ forming type II collagen/hyaluronic acid hydrogel vehicle for chondrocyte delivery in cartilage tissue engineering.

In this study, chondrocytes were encapsulated into an injectable, in situ forming type II collagen/hyaluronic acid (HA) hydrogel cross-linked with poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4SPEG) and supplemented with the transforming growth factor ß1 (TGFß1). The chondrocyte-hydrogel constructs were cultured in vitro for 7 days and studied for cell viability and proliferation, morphology, glycosaminoglycan production, and gene expression. Type II collagen/HA/4SPEG formed a strong and stable hydrogel, and the chondrocytes remained viable during the encapsulation process and for the 7-day culture period. In addition, the encapsulated cells showed spherical morphology characteristic for chondrocytic phenotype. The cells were able to produce glycosaminoglycans into their extracellular matrix, and the gene expression of type II collagen and aggrecan, genes specific for differentiated chondrocytes, increased over time. The results indicate that the studied composite hydrogel with incorporated chondrogenic growth factor TGFß1 is able to maintain chondrocyte viability and characteristics, and thus, it can be regarded as potential injectable cell delivery vehicle for cartilage tissue engineering.

A laboratory-scale device for the straightforward production of uniform, small sized cell microcapsules with long-term cell viability.

Microencapsulated and genetically engineered cells may be used for prolonged delivery of therapeutically active proteins. The objective of this study was to develop a simple, inexpensive and flexible laboratory-scale device for the production of cell microcapsules, especially capsules of small diameter (<300 µm). Many microencapsulation devices are expensive, difficult to assemble and to use, and often more suitable for large-scale experiments. However, the simplicity and low price of the encapsulation system should not limit the quality of capsules and reproducibility of the process: for successful in vitro and in vivo experiments it is important to be able to produce uniform, spherical microcapsules without deformities with high reproducibility. In addition, an advantage of the present procedure compared to other similar, co-axial laminar gas flow systems is the possibility to produce also small microcapsules, less than 200 µm in diameter, with narrow size distribution. First, design, optimization and reproducibility testing of this custom-built device were carried out. Second, microencapsulated retinal pigment epithelial cells (ARPE-19) capable of secreting soluble vascular endothelial growth factor receptor 1 (sVEGFR1) were engineered. The cells remained viable in alginate-poly-L-lysine-alginate microcapsules and secreted sVEGFR1 for prolonged periods.

The cerebellar GABAAR α6-R100Q polymorphism alters ligand binding in outbred Sprague-Dawley rats in a similar manner as in selectively bred AT and ANT rats.

The alcohol-tolerant AT and alcohol-nontolerant ANT rat lines have been selectively bred for innate sensitivity to ethanol-induced motor impairment. The cerebellar GABAA receptor (GABAAR) α6 subunit alleles α6-100R and α6-100Q are segregated in the AT and ANT rats, respectively. This α6 polymorphism might explain various differences in pharmacological properties and density of GABAARs between the rat lines. In the present study, we have used nonselected outbred Sprague-Dawley rats homozygous for the α6-100RR (RR) and α6-100QQ (QQ) genotypes to show that these RR and QQ rats display similar differences between genotypes as AT and ANT rat lines. The genotypes differed in their affinity for [3H]Ro 15-4513 and classic benzodiazepines (BZs) to cerebellar "diazepam-insensitive" (DZ-IS) binding sites, in density of cerebellar [3H]muscimol binding and in the antagonizing effect of furosemide on GABA-induced inhibition of [3H]EBOB binding. The results suggest the involvement of α6-R100Q polymorphism in these line differences and in the differences previously found between AT and ANT rats. In addition, the α6-R100Q polymorphism induces striking differences in [3H]Ro 15-4513 binding kinetics to recombinant α6ß3γ2s receptors and cerebellar DZ-IS sites. Association of [3H]Ro 15-4513 binding was ∼10-fold faster and dissociation was ∼3-4-fold faster in DZ-IS α6ßγ2 receptors containing the α6-100Q allele, with a resulting change of ∼2.5-fold in equilibrium dissociation constant (KD). The results indicate that in addition to the central role of the homologous α6-100R/Q (α1-101H) residue in BZ binding and efficacy, this critical BZ binding site residue has a major impact on BZ binding kinetics.

AMPAR signaling mediating GABA(A)R delta subunit up-regulation in cultured mouse cerebellar granule cells.

Depolarization of cerebellar granule cells in culture leads to up-regulation of the GABA(A) receptor delta subunit expression. To determine the signaling molecules involved, we examined the effects of protein kinase inhibitors and cyclic AMP-elevating compounds on basal and AMPAR agonist-induced delta mRNA expression in cerebellar granule cells. Treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 or with pituitary adenylate activating polypeptide increased delta subunit expression by 70%. Selective activation of AMPA receptors with CPW-399 also increased delta mRNA expression (2-4-fold). CPW-399 induction of delta subunit mRNA was reduced by prior treatment with either the MEK1/2 inhibitor U0126 or protein kinase A (PKA) inhibitors KT 5720 and H89. These effects were additive and combined treatment with U0126 and H89 completely prevented induction of delta subunit expression above basal levels. These results suggest that the role of JNK and ERK1/2/PKA on maintainence of delta subunit expression is diammetrically opposite. While JNK activity negatively regulates delta subunit mRNA expression in unstimulated neurons, activity of ERK1/2 and PKA are required for full induction of GABA(A) receptor delta subunit expression in response to AMPA receptor stimulation.

AMPA receptors serum-dependently mediate GABAA receptor alpha1 and alpha6 subunit down-regulation in cultured mouse cerebellar granule cells.

Depolarization of cultured mouse cerebellar granule cells with potassium or kainate results in developmentally arrested state that includes down-regulation of GABA(A) receptor alpha1, alpha6 and beta2 subunit expression. These subunits are normally strongly expressed in cerebellar granule cells from second postnatal week throughout the adulthood. In the present study we demonstrate that selective activation of AMPA subtype of glutamate receptors down-regulates alpha1 and alpha6 subunit mRNA expression. Removal of AMPA agonist from culture medium restores expression of these subunits indicating reversibility of the down-regulation. In serum-free culture medium AMPA receptor activation did not down-regulate alpha1 or alpha6 subunit expression. Furthermore, the down-regulation was strongly attenuated when the cells were cultured in the presence of dialysed fetal calf serum. The results indicate that down-regulation of GABA(A) receptor alpha1 and alpha6 subunits by AMPA receptor activation is dependent on the presence of low molecular weight compounds present in fetal calf serum. In order to study mouse cerebellar granule cell maturation and/or regulation of GABA(A) receptor subunit expression in culture, the experiments should be performed in the absence of fetal calf serum.