Solujen kapselointi hydrogeeleihin pitkäaikaista proteiinituotantoa ja kudosteknologisia sovelluksia varten

Cell therapy is defined as cell transplantation into the patient to treat a certain disease state. Therapies utilizing cells can be divided into two main categories, (1) tissue regeneration or engineering and (2) drug delivery. In tissue engineering, the transplanted cells are used to regenerate the functions of a diseased tissue. In drug delivery, the transplanted cells are used as biological factories that produce therapeutic molecules inside the body. For successful cell therapy applications, cells usually must be combined with biomaterials and bioactive factors to mimic the growth environment in vivo. The properties of these scaffolds are important for outcomes of the treatments, because the local environment determines the functionality of the cells. Thus, research on cell-biomaterial interactions is essential for the progress of cell based therapies. Hydrogels are promising cell therapy materials, because their structure resembles the natural tissue environment; they consist of long polymer chains with high water content and elastic properties, thereby enabling cellular functionality. The aim of this study was to investigate hydrogels for cell therapy applications. Firstly, we encapsulated human retinal pigment epithelial cell line (ARPE-19) genetically engineered to secrete an anti-angiogenic protein (1) into alginate-poly-L-lysine-alginate (APA) microcapsules and (2) into a composite hydrogel of cross-linked collagen and interpenetrating hyaluronic acid (HA). A custom-made cell encapsulation device was designed, built and optimized, and pharmacokinetic/pharmacodynamic (PK/PD) model was developed to investigate the intravitreal drug delivery of the anti-angiogenic protein by the encapsulated cells. Secondly, chondrocytes were encapsulated into the cross-linked collagen/HA hydrogel supplemented with transforming growth factor β1 (TGFβ1). Using the cell encapsulation device, cell microcapsules of symmetrical shape and narrow size distribution were produced. The encapsulated ARPE-19 cells remained viable and functional for at least five months. The cross-linked collagen-HA hydrogel was shown to be a suitable encapsulation matrix for ARPE-19 cells; the cells maintained viability and secreted the anti-angiogenic protein at a constant rate for at least 50 days. Moreover, the hydrogel composition could be modified to adjust the properties of the gel structure without compromising cell viability. This approach is suggested to have potential in the treatment of retinal neovascularization. The developed PK/PD model could be used to predict drug levels and therapeutic responses after intravitreal anti-angiogenic drug delivery. The simulations may augment the design of in vivo experiments. The collagen/HA matrix with TGFβ1 was suitable for chondrocyte encapsulation. The hydrogel supported viability and phenotypic cell stability. This hydrogel is strong, stable and biodegradable, and it can be delivered non-invasively as injection. Overall, it is potentially a useful delivery vehicle of chondrocytes for cartilage tissue engineering. In conclusion, ARPE-19 cells maintain viability in different hydrogels for prolonged periods and secrete the therapeutic transgene product constantly, supporting the suitability of ARPE-19 cells for cell therapy. The cross-linked collagen/HA hydrogel appears to be a potential matrix for cell therapy. It is an injectable system that supports functionality of cells, and it is applicable in drug delivery and tissue engineering.Soluterapialla tarkoitetaan sairauden hoitamista transplantoimalla soluja potilaaseen. Soluja hyödyntävät terapiat voidaan jakaa kahteen pääluokkaan: (1) kudosteknologiaan eli kudosten uudismuodostukseen ja (2) lääkeaineiden kuljettamiseen. Kudosteknologiassa transplantoituja soluja käytetään regeneroimaan sairaita kudoksia. Lääkeaineiden kuljetuksessa transplantoituja soluja käytetään biologisina tehtaina , jotka tuottavat terapeuttisia molekyylejä kehossa. Soluterapiassa solujen luonnollista in vivo -elinympäristöä pyritään yleensä jäljittelemään yhdistämällä solut biomateriaalien ja bioaktiivisten molekyylien kanssa. Käytettyjen materiaalien ominaisuudet ovat tärkeitä, koska paikallinen ympäristö määrittää solujen toiminnallisuuden. Tämän takia solujen ja biomateriaalien välisten vuorovaikutusten tutkiminen on keskeistä soluterapioiden kehitykselle. Hydrogeelit ovat lupaavia materiaaleja soluterapian sovelluksiin, koska niiden rakenne muistuttaa luonnollista kudosympäristöä. Hydrogeelit koostuvat pitkistä polymeeriketjuista, joiden korkea vesipitoisuus ja elastiset ominaisuudet mahdollistavat solujen toiminnallisuuden. Tämän tutkimuksen tavoite oli tutkia hydrogeelejä erilaisissa soluterapian sovelluksissa. Ihmisen verkkokalvon pigmenttiepiteelisolulinja (ARPE-19) muunneltiin geneettisesti tuottamaan verisuonten kasvua estävää eli anti-angiogeneettistä proteiinia. Näitä soluja kapseloitiin (1) alginaatti-poly-L-lysiini-alginaatti (APA) mikrokapseleihin ja (2) ristisidotun kollageenin ja hyaluronihapon (HA) muodostamaan komposiitihydrogeeliin. Solujen mikrokapselointia varten suunniteltiin, rakennettiin ja optimointiin erityisvalmisteinen kapselointilaite. Kapseloitujen solujen lasiaisensisäistä lääkeaineen kuljetusta tutkittiin kehittämällä farmakokineettinen/farmakodynaaminen (PK/PD) simulaatiomalli. Lisäksi rustosoluja eli kondrosyyttejä kapseloitiin ristisidottuun kollageeni/HA hydrogeeliin, jota täydennettiin transforming growth factor β1 (TGFβ1) -kasvutekijällä. Kehitetyllä kapselointilaitteella oli mahdollista valmistaa symmetrisen muotoisia ja tasakokoisia mikrokapseleita. APA-mikrokapseleihin kapseloidut ARPE-19 solut säilyivät elävinä ja toiminnallisina ainakin viiden kuukauden ajan. Ristisidotun kollageeni/HA-geelin osoitettiin olevan sopiva kapselointimateriaali ARPE-19 soluille: solut pysyivät elävinä ja erittivät terapeuttista anti-angiogeneettistä proteiinia tasaisella nopeudella ainakin 50 päivän ajan. Lisäksi hydrogeelin koostumusta oli mahdollista muunnella geelirakenteen säätelemiseksi vaikuttamatta soluelävyyteen. Kollageeni/HA-geeliin kapseloituja, geneettisesti muunneltuja ARPE-19 soluja voidaan mahdollisesti käyttää verkkokalvon neovaskularisaation eli verisuonten uudismuodostuksen hoitoon. Kehitetyn PK/PD mallin avulla oli mahdollista ennustaa anti-angiogeneettisten lääkeaineiden lasiaisensisäisestä annosta seuraavia lääkeainepitoisuuksia ja terapeuttisia vasteita. Simulaatioita voidaan tulevaisuudessa käyttää apuna in vivo -kokeiden suunnittelussa. TGFβ1-kasvutekijää sisältävä kollageeni/HA-hydrogeeli oli sopiva materiaali kondrosyyttien kapselointiin. Tämä hydrogeeli on luja, kestävä ja biohajoava, ja se voidaan kuljettaa kehoon non-invasiivisesti injektoimalla. Hydrogeeli on mahdollisesti käyttökelpoinen kondrosyyttien kasvualustana ruston kudosteknologiassa. Yhteenvetona voidaan todeta, että ARPE-19 solut säilyttävät elävyytensä ja erittävät terapeuttista transgeenin tuotetta tasaisesti erilaisissa hydrogeeleissä pitkiä ajanjaksoja, mikä osoittaa ARPE-19 solujen sopivuuden soluterapian sovelluksiin. Lisäksi ristisidottu kollageeni/HA hydrogeeli vaikuttaa soveltuvalta materiaalilta soluterapiaan. Tämä injektoitava materiaali tukee solujen toiminnallisuutta, ja se soveltuu sekä lääkeaineiden kuljetukseen että kudosteknologiaan

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

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.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-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

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

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