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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.

On the surface of the eye, both the cornea and conjunctiva are restricting ocular absorption of topically applied drugs, but barrier contributions of these two membranes have not been systemically compared. Herein, we studied permeability of 32 small molecular drug compounds across an isolated porcine cornea and built a quantitative structure-property relationship (QSPR) model for the permeability. Corneal drug permeability (data obtained for 25 drug molecules) showed a 52-fold range in permeability (0.09-4.70x10(-6) cm/s) and the most important molecular descriptors in predicting the permeability were hydrogen bond donor, polar surface area and halogen ratio. Corneal permeability values were compared to their conjunctival drug permeability values. Ocular drug bioavailability and systemic absorption via conjunctiva were predicted for this drug set with pharmacokinetic calculations. Drug bioavailability in the aqueous humour was simulated to be

Mucus layer covers the ocular surface, and soluble mucins are also present in the tear fluid. After topical ocular drug administration, the drugs and formulations may interact with mucus layer that may act as a barrier in ocular drug delivery. In this mini-review, we illustrate the mucin composition of the ocular surface and discuss the influence of mucus layer on ocular drug absorption. Based on the current knowledge the role of mucus barrier in drug delivery is still undefined. Furthermore, interactions with mucus may prolong the retention of drug formulations on the ocular surface. Mucus may decrease or increase ocular bioavailability depending on the magnitude of its role as barrier or retention site, respectively. Mechanistic studies are needed to clarify the role of mucin in ocular drug delivery.

The RNA-binding protein, HuR, modulates mRNA processing and gene expression of several stress response proteins i.e. Hsp70 and p53 that have been postulated to be involved in the pathogenesis of glaucoma, a chronic optic neuropathy leading to irreversible blindness. We evaluated HuR protein expression in retinas and optic nerves of glaucomatous rats and human primary open angle glaucoma patients and its possible impact on stress response mechanisms. We found that the cytoplasmic content of HuR was reduced more extensively in glaucomatous retinas than in optic nerves and this was linked with a declined cytoplasmic Hsp70 level and p53 nuclear translocation. In the optic nerve, the p53 content was decreased as a feature of reactive gliosis. Based on our findings, we conclude that the alteration in the HuR content, observed both in rat glaucoma model and human glaucoma samples, affects post-transcriptionally the expression of genes crucial for maintaining cell homeostasis; therefore, we postulate that HuR may be involved in the pathogenesis of glaucoma.

The eye is protected by several tissues that limit the permeability and entry of potentially harmful substances, but also hamper the delivery of drugs in the treatment of ocular diseases. Active transport across the ocular barriers may affect drug distribution, but the impact of drug transporters on ocular drug delivery is not well known. We have collected and critically reviewed the literature for ocular expression and activity of known drug transporters. The review concentrates on drug transporters that have been functionally characterized in ocular tissues or primary cells and on transporters for which there is available expression data at the protein level. Species differences are highlighted, since these may explain observed inconsistencies in the influence of specific transporters on drug disposition. There is variable evidence about the pharmacokinetic role of transporters in ocular tissues. The strongest evidence for the role of active transport is available for the blood-retinal barrier. We explored the role of active transport in the cornea and blood retinal barrier with pharmacokinetic simulations. The simulations show that the active transport is important only in the case of specific parameter combinations.

Alzheimer’s disease (AD) may disturb functions of the blood-brain barrier and change the disposition of drugs to the brain. This study assessed the disease-induced changes in drug transporters in the brain capillaries of transgenic AD mice. Eighteen drug transporters and four tight junction-associated proteins were analyzed by RT-qPCR in cortex, hippocampus and cerebellum tissue samples of 12–16-month-old APdE9, Tg2576 and APP/PS1 transgenic mice and their healthy age-matched controls. In addition, microvessel fractions enriched from 1-3-month-old APdE9 mice were analyzed using RT-qPCR and Western blotting. Brain transport of methotrexate in APdE9 mice was assessed by in vivo microdialysis. The expression profiles of studied genes were similar in brain tissues of AD and control mice. Instead, in the microvessel fraction in APdE9 mice, >2-fold alterations were detected in the expressions of 11 genes but none at the protein level. In control mice strains, >5-fold changes between different brain regions were identified for Slc15a2, Slc22a3 and occludin. Methotrexate distribution into hippocampus of APdE9 mice was faster than in controls. The expression profile of mice carrying presenilin and amyloid precursor protein mutations is comparable to controls, but clear regional differences exist in the expression of drug transporters in brain.

Drug delivery to the posterior eye segment is an important challenge in ophthalmology, because many diseases affect the retina and choroid leading to impaired vision or blindness. Currently, intravitreal injections are the method of choice to administer drugs to the retina, but this approach is applicable only in selected cases (e.g. anti-VEGF antibodies and soluble receptors). There are two basic approaches that can be adopted to improve retinal drug delivery: prolonged and/or retina targeted delivery of intravitreal drugs and use of other routes of drug administration, such as periocular, suprachoroidal, sub-retinal, systemic, or topical. Properties of the administration route, drug and delivery system determine the efficacy and safety of these approaches. Pharmacokinetic and pharmacodynamic factors determine the required dosing rates and doses that are needed for drug action. In addition, tolerability factors limit the use of many materials in ocular drug delivery. This review article provides a critical discussion of retinal drug delivery, particularly from the pharmacokinetic point of view. This article does not include an extensive review of drug delivery technologies, because they have already been reviewed several times recently. Instead, we aim to provide a systematic and quantitative view on the pharmacokinetic factors in drug delivery to the posterior eye segment. This review is based on the literature and unpublished data from the authors' laboratory., published version, peerReviewed

Conjunctiva occupies most of the ocular surface area, and conjunctival permeability affects ocular and systemic drug absorption of topical ocular medications. Therefore, the aim of this study was to obtain a computational in silico model for structure-based prediction of conjunctival drug permeability. This was done by employing cassette dosing and quantitative structure-property relationship (QSPR) approach. Permeability studies were performed ex vivo across fresh porcine conjunctiva and simultaneous dosing of a cassette mixture composed of 32 clinically relevant drug molecules with wide chemical space. The apparent permeability values were obtained using drug concentrations that were quantified with liquid chromatography tandem-mass spectrometry. The experimental data were utilized for building a QSPR model for conjunctival permeability predictions. The conjunctival permeability values presented a 17-fold range (0.63-10.74 × 10−6 cm/s). The final QSPR had a Q2 value of 0.62 and predicted the external test set with a mean fold error of 1.34. The polar surface area, hydrogen bond donor, and halogen ratio were the most relevant descriptors for defining conjunctival permeability. This work presents for the first time a predictive QSPR model of conjunctival drug permeability and a comprehensive description on conjunctival isolation from the porcine eye. The model can be used for developing new ocular drugs., final draft, peerReviewed

Intracellular limiting steps and molecules involved in internalization and intracellular routing of non-viral gene delivery systems are still poorly understood. In this study, the intracellular kinetics of three different gene delivery systems calcium phosphate precipitates (CaP), polyethyleneimine (PEI) and N-[1-(2,3-dioleyl)propyl]-N,N,N-trimethylammonium chloride (DOTAP)) were quantified at cellular, nuclear, transcriptional and translational levels by using qRT-PCR. Additionally, a role of cell surface glycosaminoglycans (GAGs) was evaluated by performing the aforementioned studies in cells devoid of GAGs (pgsB-618) and cells lacking heparan sulphate (HS). The obtained data showed that the intracellular kinetics was dependent on the type of gene carrier and the weakest intracellular step varied between the carriers; rapid elimination of cell-associated pDNA in CaP, nuclear uptake in DOTAP and transcriptional and translational events in PEI mediated transfections. Overall, neither the amount of cell- nor nuclear associated pDNA correlated with transgene expression but the mRNA expression of the transgene correlated well with the expression at protein level. The nuclear uptake of pDNA in all cases was rapid and efficient thus indicating that the post-nuclear processes including transcription and translation steps have a critical role in defining the efficiency of non-viral gene delivery systems. Our study demonstrated that cell-surface GAGs are not essential for cell surface binding and internalization of gene delivery complexes, but they are able to define the intracellular routing of the complexes by leading them to pathways with high pDNA elimination.

Four inhibitors of human carbonic anhydrase II (hCA II) were designed based on the previously reported subnanomolar 1,3-oxazole-based sulfonamide inhibitors of the enzyme to incorporate primary and secondary amine functionality in the carboxamide side chain. The new hydrophilic compounds were found to inhibit the target isoform in sub-nanomolar to low nanomolar range with a good degree of selectivity to several other hCA isoforms. The hydrophilic character of these compounds is advantageous for intraocular residence time but not for corneal permeability which generally requires that a drug be sufficiently lipophilic. Two of the four compounds investigated, however, were found to exert comparable efficacy as 1% eye drops in PBS to that of the clinically used 2% dorzolamide (Trusopt®) eye drops. This indicated that the absorption of the compounds may occur via alternative route across conjunctiva and sclera.

1. Currently available in vitro blood-brain barrier models all have recognized restrictions. In addition to leakiness, inconsistent data about P-glycoprotein mediated efflux limit the attractiveness of the primary bovine brain microvessel endothelial cells (BBMECs). Therefore, we re-evaluated the role of P-glycoprotein mediated efflux with two culture conditions in BBMECs for prediction of drug permeability of potential P-glycoprotein substrates. 2. BBMECs were monocultured on filters on petri dishes and on filter inserts, and expression and localization of P-glycoprotein were compared by using western blot and confocal microscopy, respectively. The functionality of P-glycoprotein was assessed by using cellular uptake, calcein-AM and bidirectional transport assays. 3. P-glycoprotein expression was higher in BBMECs cultured on filter inserts decreasing the permeability of digoxin and paclitaxel, but not the permeability of vinblastine. However, the monocultured BBMECs were not able to demonstrate efflux in the bidirectional transport assays. Under certain culture conditions, occludin may not be correctly located, perhaps explaining in part the leakiness of BBMECs. 4. In conclusion, BBMECs, despite possessing a functional P-glycoprotein, under certain culture conditions may not be a suitable in vitro model for the bidirectional transport assays and for predicting the permeability of drugs and xenobiotics that are potential P-glycoprotein substrates.

In this study, avidin-biotin technology was combined with a multifunctional drug carrier modality i.e. liposomes to achieve an active and versatile targeting approach. The anti-cancer drug doxorubicin (DOX) was modified with direct biotinylation (B-DOX) (Allart et al., 2003), or encapsulated in biotinylated sterically stabilized pH-sensitive liposomes (BL-DOX), and targeted to the lentiviral vector transduced cells expressing an avidin fusion protein on the cell membrane (Lehtolainen et al., 2003; Lesch et al., 2009). The direct biotinylation of doxorubicin improved cell internalization in rat glioma (BT4C) cells expressing avidin fusion protein receptor but cell toxicity was reduced by 78-fold due to impaired nuclear localization. In contrast, liposomal formulations restored the biological activity of the DOX in several cell lines. However, mainly due to uptake via non-specific pathways the active targeting of BL-DOX was negligible in both in vitro and in vivo studies. Active targeting with multifunctional drug carrier systems is challenging and further studies will be needed to optimize the properties of targeted drug carrier and receptor expression systems.

Induced pluripotent stem cells (iPSCs) are becoming an important source of pre-clinical models for research focusing on neurodegeneration. They offer the possibility for better understanding of common and divergent pathogenic mechanisms of brain diseases. Moreover, iPSCs provide a unique opportunity to develop personalized therapeutic strategies, as well as explore early pathogenic mechanisms, since they rely on the use of patients’ own cells that are otherwise accessible only post-mortem, when neuronal death-related cellular pathways and processes are advanced and adaptive. Neurodegenerative diseases are in majority of unknown cause, but mutations in specific genes can lead to familial forms of these diseases. For example, mutations in the superoxide dismutase 1 gene lead to the motor neuron disease amyotrophic lateral sclerosis (ALS), while mutations in the SNCA gene encoding for alpha-synuclein protein lead to familial Parkinson’s disease (PD). The generations of libraries of familial human ALS iPSC lines have been described, and the iPSCs rapidly became useful models for studying cell autonomous and non-cell autonomous mechanisms of the disease. Here we report the generation of a comprehensive library of iPSC lines of familial PD and an associated synucleinopathy, multiple system atrophy (MSA). In addition, we provide examples of relevant neural cell types these iPSC can be differentiated into, and which could be used to further explore early disease mechanisms. These human cellular models will be a valuable resource for identifying common and divergent mechanisms leading to neurodegeneration in PD and MSA.

Nanomedicines are widely studied for intracellular delivery of cancer drugs. However, the relationship between intracellular drug concentrations and drug responses are poorly understood. In this study, cellular and nuclear concentrations of doxorubicin were quantified with LC/MS after cell exposure with free and liposomal doxorubicin (pH-sensitive and pegylated liposomes). Cellular uptake of pegylated liposomes was low (∼3-fold extracellular concentrations) compared with doxorubicin in free form and pH-sensitive liposomes (up to 280-fold extracellular concentrations) in rat glioma (BT4C) and renal clear cell carcinoma (Caki-2) cells. However, after the cell exposure with pegylated liposomes, intracellular doxorubicin was distributed into the nuclear compartment in both cell types. Despite high drug concentrations in the nuclei, Caki-2 cells showed strong resistance toward doxorubicin. A model was successfully built to describe PK/PD relationship between drug concentrations in nucleus and cytotoxic responses in BT4C cells. This model is the first step to link target site concentration of doxorubicin into its effect and can be a useful part of more comprehensive future in vivo PK/PD models.

Detailed understanding of the uptake mechanisms and intracellular processing of nonviral gene delivery systems will allow design of more effective carriers. This work gets insight into the intracellular kinetics of pDNA delivered by polyethyleneimine (PEI), cationic lipid DOTAP and calcium phosphate (CaP) precipitates. Amount of cell- and nuclear-associated pDNA was quantified by qRT-PCR at multiple time points after transfection. Moreover, the impact of specific endocytic pathways on the cell entry and intracellular kinetics of pDNA was studied by inhibition (blockage) of either clathrin- or dynamin-mediated endocytosis by using both genetically manipulated cell lines and chemical inhibitors of endocytosis. Quantitative analysis of defined kinetic parameters revealed that neither cellular nor nuclear uptake of pDNA correlated with transgene expression, emphasizing the importance of the post-nuclear processes in overall transfection efficacy. Changes in transgene expression observed upon blockage of endocytosis was carrier dependent and correlated relatively well with the changes at the cellular and nuclear uptake levels but not with the amount of cell-associated pDNA. Due to low specificity of chemical inhibitors and activation of alternative endocytosis pathways after genetic blockage of endocytosis neither of these methods is optimal for studying the role of endocytosis. Therefore, one should be careful when interpreting the obtained results from such studies and not to trust the data obtained only from one method.

Several neuromuscular diseases involve dysfunction of neuromuscular junctions (NMJs), yet there are no patient-specific human models for electrophysiological characterization of NMJ. We seeded cells of neurally-induced embryoid body-like spheres derived from induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) lines as monolayers without basic fibroblast factor (bFGF) and observed differentiation of neuronal as well as spontaneously contracting, multinucleated skeletal myotubes. The myotubes showed striation, immunoreactivity for myosin heavy chain, actin bundles typical for myo-oriented cells, and generated spontaneous and evoked action potentials (APs). The myogenic differentiation was associated with expression of MyoD1, myogenin and type I ryanodine receptor. Neurons formed end plate like structures with strong binding of α-bungarotoxin, a marker of nicotinic acetylcholine receptors highly expressed in the postsynaptic membrane of NMJs, and expressed SMI-32, a motoneuron marker, as well as SV2, a marker for synapses. Pharmacological stimulation of cholinergic receptors resulted in strong depolarization of myotube membrane and raised Ca(2+) concentration in sarcoplasm, while electrical stimulation evoked Ca(2+) transients in myotubes. Stimulation of motoneurons with N-Methyl-D-aspartate resulted in reproducible APs in myotubes and end plates displayed typical mEPPs and tonic activity depolarizing myotubes of about 10 mV. We conclude that simultaneous differentiation of neurons and myotubes from patient-specific iPSCs or ESCs results also in the development of functional NMJs. Our human model of NMJ may serve as an important tool to investigate normal development, mechanisms of diseases and novel drug targets involving NMJ dysfunction and degeneration.

Background Polyethylenimine (PEI) polyplexes mediate efficient gene transfer only at high +/− charge ratios at which free noncomplexed PEI is present. The excess of PEI gives polyplexes a positive surface charge that plays a role in polyplex binding on the cell membrane. Although positively charged PEI polyplexes are known to interact with anionic cell-surface glycosaminoglycans (GAGs), the exact role of free PEI in such interactions is unclear. Methods Chinese hamster ovary wild-type cells and mutants lacking cell-surface GAGs were transfected with marker genes using PEI polyplexes with and without free PEI. The total amount of cell-associated plasmid DNA (pDNA) delivered by polyplexes was determined by quantitative real-time PCR and transgene expression was determined using β-galactosidase and luciferase assays. Results Transfection activity of polyplexes without free PEI in cells expressing cell-surface GAGs was low even though pDNA was delivered to cells. In the absence of cell-surface GAGs, polyplexes without free PEI had high transfection efficacy. This indicates that the cell-surface GAGs inhibit transfection by purified polyplexes. PEI polyplexes with free carrier mediated transfection in both normal and GAG-deficient cells because free PEI overcomes the inhibitory effect of cell-surface GAGs on transfection. The intracellular elimination of pDNA was faster in the presence of GAGs and, despite improved transfection, free PEI reduced pDNA association with the cells. Conclusions Free PEI is essential for minimizing the undesirable binding of polyplexes to cell-surface GAGs that have a negative impact on transfection. The same mechanism may be important in transfections with other polyplexes that require high charge ratios for transfection. Copyright © 2011 John Wiley & Sons, Ltd.

Bacterial adhesion to the cell surface is a crucial step before infection can take place. Inhibition of bacterial binding offers a novel preventive approach against infections. Cranberry (Vaccinium macrocarpon Ait.) juice has been found to have antiadhesive activity against different bacteria. Streptococcus pneumoniae is an important pathogen and the most common cause for pneumonia, meningitis, and otitis media. In this study the inhibitory activity of cranberry (Vaccinium oxycoccos L.), bilberry (Vaccinium myrtillus L.) and crowberry (Empetrum nigrum and Empetrum hermaphroditum L.) juice fractions against pneumococcal binding was tested using human bronchial cells (Calu-3) as an adhesion model. In addition, the antimicrobial activity of the berry juice fractions was tested. It was found that the studied berry juice fractions had antiadhesion activity and cranberry juice was the most active. The adhesion inhibition activity of cranberry juice was nearly 90% at a concentration of 8.7 mg/g of soluble solids. The antimicrobial activity of the studied berry juice fractions was found to be remarkable; pneumococcal growth was inhibited totally at a concentration of ∼86 mg/g. Both antiadhesion and antimicrobial activities were reduced after solid-phase extraction of the berry juices, which may suggest molecular synergistic effects of the berry juice molecules against S. pneumoniae. The findings indicate that cranberry, bilberry and crowberry juices have potential against pneumococcal infections.

Background Here we report on studies that probe whether the intracellular kinetics of plasmid DNA (pDNA) and cell surface glycosaminoglycans (GAGs) are modified during the cell cycle in a way that can be correlated with changes in gene transfer efficiency with poly(ethyleneimine) (PEI) and poly-L-lysine (PLL) polyplexes. Methods Synchronized D407 retinal cells were transfected with PEI and PLL polyplexes using a luciferase reporter. The free and/or loosely complexed nuclear pDNA was determined by real-time PCR, and compared with transgene expression, the rate of pinocytosis by FITC-dextran uptake and the content of cell surface GAGs. Results The amount of free and/or loosely complexed nuclear pDNA between cell cycle phases varied ∼4–20 times (G1 < S < G2/M). Both carriers delivered pDNA in a similar way into the nucleus (PLL vs. PEI ⩽ 3.5-fold), but PEI was ∼10–100 times more efficient in gene expression than PLL (G1 < G2/M < S). The rate of pinocytosis increased up to 70-fold from G1 to middle S phase. Cell surface heparan and chondroitin sulfate increased 50–80%, and hyaluronan decreased 50% when the cells went from G1 through S to G2/M. Conclusions The data obtained indicates that no single parameter (pinocytosis, cell surface GAGs, nuclear uptake) solely accounts for the differential pDNA uptake or expression during cell cycle, and that the main difference in PLL- and PEI-mediated transfections seems to be at the nuclear level. Copyright © 2007 John Wiley & Sons, Ltd.

Improved functional recovery after spinal cord injury by transplantation of induced pluripotent stem cell-derived neural stem/progenitor cells (iPSC-NPCs) has been reported. However, beneficial effects of iPSC-based therapy have so far been produced mostly using genetically immunodeficient rodents. Because of the long time required for generation and characterization of iPSCs, the use of autologous iPSCs for treating patients with acute spinal cord injury (SCI) is not feasible. Therefore, it is of utmost importance to investigate the effect of iPSC-based therapy on functional recovery after SCI using pharmacologically immunosuppressed, immunocompetent animal models. Here we studied the functional outcome following subacute transplantation of human iPSC-derived NPCs into contused mouse spinal cord when tacrolimus was used as an immunosuppressive agent. We show that human iPSC-derived NPCs transplanted into pharmacologically immunosuppressed C57BL/6J mice exhibited poor long-term survival and failed to improve functional recovery after SCI as measured by Basso Mouse Scale (BMS) for locomotion and CatWalk gait analysis when compared to vehicle-treated animals. The scarce effect of iPSC-based therapy observed in the current study may be attributable to insufficient immunosuppressive effect, provided by monotherapy with tacrolimus in combination with immunogenicity of transplanted cells and complex microenvironment of the injured spinal cord. Our results highlight the importance of extensive preclinical studies of transplanted cells before the clinical application of iPSC-based cell therapy is achieved.

The anticancer drug doxorubicin and its liposomal formulations are in clinical use, doxorubicin also during pregnancy. However, little is known about how doxorubicin and its liposomal formulations are taken up by placental cells and whether they can cross human placenta. We therefore investigated quantitative cellular uptake and toxicity of doxorubicin and its two liposomal formulations, pH-sensitive liposomal doxorubicin (L-DOX) and commercially available pegylated liposomal doxorubicin (PL-DOX), in human placental choriocarcinoma (BeWo) cells. PL-DOX showed significantly lower cellular uptake and toxicity compared with doxorubicin and L-DOX. In preliminary studies with human placental perfusion, PL-DOX did not cross the placenta at all in 4h, whereas doxorubicin and L-DOX crossed the placenta at low levels (max 12% of the dose). Furthermore, PL-DOX did not accumulate in placental tissue while doxorubicin did (up to 70% of the dose). Surface pegylation probably explains the low placental cell and tissue uptake of PL-DOX. Formulation of doxorubicin thus seems to enable a decrease of fetal exposure.

Glycosaminoglycans (GAGs) are negatively charged polysaccharides that are found, e.g. on cell surface. GAGs have been reported to influence gene transfection. We have previously reported that cationic lipid-mediated gene transfection can be improved by combining a small polyethylenimine (PEI) with cationic lipids. In the present study, we examined if GAGs have any effect on the synergism of small PEIs and other cationic carriers. We used wild-type CHO (GAG+) and pgsB-618 cells (GAG-). Transfection efficiency was studied using lacZ and GFP reporter genes. We found that GAGs decreased the overall level of transgene expression in a reagent-dependent manner, but the synergism caused by low-MW PEIs was less affected. There were no major differences between cell lines in cellular uptake or intracellular localization of plasmids when measured with flow cytometry and confocal microscopy, respectively. In conclusion, cell-surface GAGs interfere with transfection efficiency of different cationic reagents, but that is not necessarily related to the synergy of small PEIs and cationic lipids.

We investigated the permeation of liposomal and polymeric gene delivery systems through neural retina into retinal pigment epithelium (RPE) and determined the roles of various factors in permeation and subsequent uptake of the delivery systems by RPE. Anterior parts and vitreous of fresh bovine eyes were removed. Retina was left intact or peeled away. Complexes of ethidium monoazide (EMA)-labeled plasmid DNA and cationic carriers (polyethyleneimine, poly-L-lysine, DOTAP liposomes) were pipetted on the retina or RPE. Two hours later the neural retina was removed, if present, and the RPE cells were detached. Contaminants were removed by sucrose centrifugation, and the RPE cells were analyzed for DNA uptake by flow cytometry. Cellular uptake of FITC-dextrans (molecular weight [mw] 20 000, 500 000 and 2 000 000), FITC-poly-L-lysine (mw 20 000), FITC-labeled oligonucleotide (15-mer), and naked EMA-labeled plasmid DNA was determined after pipetting the solutions on the RPE or neural retina. Location of the fluorescent materials in the retina was visualized with fluorescence microscopy. Neural retina decreased the cellular uptake of DNA complexes by an order of magnitude, the uptake of FITC-dextrans slightly, whereas delivery of polycationic FITC-poly-L-lysine to RPE was almost completely inhibibited. Neural retina decreased the cellular uptake of FITC-oligonucleotides, while the uptake of uncomplexed plasmid was always negligible. conclusions from FACS and fluorescence microscopy were similar: delivery of polymeric and liposomal DNA complexes into RPE are limited by the neural retina. This is due to the size and positive charge of the complexes.

Background Cationic polymers and liposomes are used to wrap DNA into complexes that promote its cellular uptake. The mechanisms of the uptake and the intracellular fate of these complexes are obscure, as are reasons for an unpredictable and sometimes poor efficiency of the transgene expression. Polyanionic glycosaminoglycans (GAGs) on the cell surface interact with the cationic DNA complexes and influence transfection. Methods The quantities of heparan sulfate (HS), chondroitin sulfate (CS) and hyaluronan (HA) on the cell surface of mutated Chinese hamster ovary (CHO) cells and manipulated (chlorate, xyloside, chondroitinase ABC or Streptomyces hyaluronidase) smooth muscle cells were correlated with the uptake of four different DNA complexes, and the expression of the transgene. Results Two CHO mutants, without cell-surface HS and CS, showed a 1.5–6-fold increase in cellular association of DNA, and 3–25-fold increase of transgene expression, as compared with the wild type. A CHO mutant with a 5.7-fold increase of cell-surface CS, but devoid of HS, showed enhanced DNA association, but 20–40% reduction in its expression. The removal of HS, CS, or HA from the cell surface of smooth muscle cells had a minor or insignificant effect on the cell association and transfection of the carriers and only polyethyleneimine showed increased association and expression of the transgene. Conclusions The uptake of DNA complexes varies depending on carrier, cell type and amounts of cell-surface HS, CS, and HA, whereas all GAGs inhibit the transgene expression. This implies that cell-surface GAGs probably direct complexes into intracellular compartments that do not support transcription. Copyright © 2004 John Wiley & Sons, Ltd.

Low-molecular-weight PEIs and cationic liposomes can be combined resulting in a synergistic increase in transfection efficiency as we have reported earlier. Here, we have further investigated the potential mechanisms of this synergy. Complex morphology, complex sizes and DNA condensation were studied using transmission electron microscopy, light scattering methods and ethidium bromide exclusion, respectively. Cellular uptake, transfection efficiency, and effect of proton pump inhibitor bafilomycin A1 were examined in cell cultures. The cellular uptake of DNA was negligible with PEI2K-DNA complexes, whereas the uptake of the PEI2K-DNA-Dosper or the Dosper-DNA complexes was maximally about 40%. The number of transfected cells was two times higher with PEI2K-DNA-Dosper complexes than with Dosper-DNA complexes. The PEI2K-DNA-Dosper combination was slightly less sensitive to bafilomycin A1 than the PEI25K-DNA or Dosper-DNA complexes. There were no differences between PEI2K and PEI25K in DNA condensation. Dosper condensed DNA slightly more in PEI2K complexes. The PEI25K-DNA complexes were much smaller (250 nm) than the PEI2K-DNA complexes (0.5-12 micro m) which were also rather polydisperse. It is suggested that two independent mechanisms would lead to synergistic transfection efficiency: (1) Dosper improves the cellular uptake of PEI2K-DNA complexes, and (2) PEI2K improves a transfer of the complexes from lysosomes to nucleus.

Purpose. To investigate the role of vitreous in nonviral gene delivery into retinal pigment epithelial (RPE) cells.

The influence of shape, molecular weight and pegylation of linear, grafted, dendritic and branched poly- l -lysines on their DNA delivery properties were investigated. DNA binding, condensation, complex size and morphology, cell uptake and transfection efficiency were determined. Most polylysines condense DNA, linear polymers being more efficient than most dendritic ones. At low molecular weights of PLL DNA binding and condensation were less efficient, particularly with dendrimers. Pegylation did not decrease DNA condensation of PLLs at less than 60% (fraction of Mw) of PEG. Pegylation stabilized the complexes sterically, but did not protect them from interaction with polyanionic chondroitin sulfate. Cell uptake of polylysine/DNA complexes was high and pegylation increased the transfection efficacy. However, overall transfection level of polylysines is low possibly due to inadequate escape of the complexes from endosomes or poor release of DNA from the complexes. Physicochemical and biological structure–property relationships of poly- l -lysines were demonstrated, but no clear correlations between the tested physicochemical determinants (size of complexes, zeta-potentials, condensation of DNA and the shape of complexes) and biological activities were seen. Transfection activity may be ultimately determined by intracellular factors and/or still unknown features of DNA complexation with the carriers.

Understanding mechanisms of endocytosis and trafficking of nanoparticles through endothelial and epithelial barriers leads potentially to improved efficacy of nanoparticulate drug delivery systems. Detailed characterizations of cell models with respect to endocytic pathway expression and activity (endocytic profiling) should facilitate data interpretation. We performed endocytic profiling of CaCo-2 and hCMEC/D3 cell lines, widely used as human intestinal and blood-brain barrier permeability models, respectively, during cell differentiation. Furthermore, we compared endocytic profiles of cell lines with those of primary cells. Expression of genes involved in specific endocytic pathways was analyzed at mRNA levels by quantitative real time PCR. Where possible, the respective protein levels were analyzed by Western blotting, and endocytic activities of cells were analyzed by flow cytometry. We showed that differentiated CaCo-2 cells formed tight, well polarized monolayers with reduced endocytic activity accompanied by reduced mRNA expression of most of the endocytosis-related genes. In contrast, hCMEC/D3 cells formed a leaky, less polarized barrier, and in vitro differentiation had little effect on either the expression of endocytosis-related genes or endocytic activity of these cells. Endocytic profiling of in vitro models and comparison with primary cells is an important measure to avoid misleading conclusions in nanoparticle permeation studies.

It has been shown that extracellular glycosaminoglycans (GAGs) limit the gene transfer by cationic lipids and polymers. The purpose of this study was to clarify how interactions with anionic GAGs (hyaluronic acid and heparan sulfate) modify the cellular uptake and distribution of lipoplexes and polyplexes. Experiments on cellular DNA uptake and GFP reporter gene expression showed that decreased gene expression can rarely be explained by lower cellular uptake. In most cases, the cellular uptake is not changed by GAG binding to the lipoplexes or polyplexes. Reporter gene expression is decreased or blocked by heparan sulfate, but it is increased by hyaluronic acid; this suggests that intracellular factors are involved. Confocal microscopy experiments demonstrated that extracellular heparan sulfate and hyaluronic acid are taken into cells both with free and DNA-associated carriers. We conclude that extracellular GAGs may alter both the cellular uptake and the intracellular behavior of the DNA complexes.

Transplantation of human neural progenitor cells (hNPCs) is a promising therapeutic approach for various diseases of the central nervous system (CNS). Reliable testing of hNPC transplantation in animal models of neurological diseases requires that these cells can be produced in sufficient amounts, show consistent homogeneity as a neural cell population, and be reliably labeled for in vivo tracking. In addition, the cells should be characterized as being at the optimal state of differentiation favoring successful engraftment. Here, we show that high numbers of purified hNPCs can be produced from human embryonic stem cells (hESCs) by manually selecting specifically sized and shaped spheres followed by fluorescence-activated cell sorting based on the relative cell size. In addition, we report that labeling of hNPCs with ultra-small superparamagnetic iron oxide (USPIO) particles does not affect the cellular morphology or growth. More importantly, we show that the transduction with lentiviral vector encoding green fluorescent protein (GFP) decreases the neurality of the cell population. We conclude that our cost-effective protocol of generating hNPCs is widely applicable for preclinical studies on CNS disorders. This improved method of producing large quantities of high-purity hNPCs maybe useful also when generating hNPCs from human induced pluripotent stem (hiPS) cell lines. However, caution should be used when lenti-GFP transduction is applied for hNPC labeling.

Chondrogenic differentiation of human embryonic (hESCs) or induced pluripotent stem cells (hiPSCs) has been achieved in embryoid bodies (EBs) by adding selected growth factors to the medium. Also chondrocyte-secreted factors have been considered to promote the chondrogenic differentiation. Hence, we studied whether co-culture with primary chondrocytes can induce hESCs or hiPSCs to differentiate into chondrocyte lineage. Co-culture of hESCs or hiPSCs was established in a transwell insert system in feeder-free culture conditions, while hESCs or hiPSCs grown alone in the wells were used as controls. After 3-week co-culture with weekly replenished chondrocytes, the chondrogenically committed cells (hCCCs) were evaluated by morphology, immunocytochemistry, quantitative real-time RT-PCR, and analysis of chondrogenic, osteogenic and adipogenic differentiation markers. The expressions of chondrocyte- and pluripotency-associated genes were frequently measured during the monolayer expansion of hCCCs from passage 1 to 10. Human CCCs displayed morphology similar to chondrocytes, and expressed chondrocyte-associated genes, which were declined following passaging, similarly to passaged chondrocytes. They also formed a chondrogenic cell pellet, and differentiated into chondrocytic cells, which secreted abundant extracellular matrix. Human CCCs also proliferated rapidly. However, they did not show osteogenic or adipogenic differentiation capacity. Our results show that co-culture of hESCs or hiPSCs with primary chondrocytes could induce specific chondrogenic differentiation.

To understand more about the lower generations of poly(amido amine) dendrimer (PAMAM) as a non-viral vector for antisense (ANS) therapy, a 21-mer epidermal growth factor receptor (EGFR) ANS was delivered by generation five of PAMAM in T47D breast carcinoma cells in culture. The semi-quantitative polymerase chain reaction (PCR) and western blot analysis were used to quantify the expression of EGFR mRNA and protein, respectively. The results showed that PAMAM G5/ANS nanoparticles were able to decrease the level of EGFR mRNA more than 40% even at the lower dendrimer primary amine to the antisense phosphate groups (N/P) ratio of 0.5. But, only the data of western blot analysis at the higher N/P ratios of 10 and 20 showed a decrease of the protein expression level similar to the mRNA expression level. Moreover, PAMAM dendrimer had a positive effect on the EGFR ANS action to inhibit the EGFR mRNA and protein expression. Further studies revealed that PAMAM G5 dendrimer as such inhibits the expression of EGFR in a concentration-dependent manner. Since PAMAM as such was able to inhibit the mRNA expression of p53 gene, we speculated that the effect of PAMAM G5 on the EGFR is a kind of its non-selective effect on the transcription and/or translation machinery of the cell. Key words: Poly(amido amine) dendrimer (PAMAM) dendrimer, epidermal growth factor receptor (EGFR) antisense, epidermal growth factor, RNAi, polyamidoamine dendrimer, toxicogenomics, gene delivery.

The low transfection efficacy of non-viral gene delivery systems limits the therapeutic application of these vectors. Besides the inefficient release of the complexes or pDNA from endolysosomes into the cytoplasm or poor nuclear uptake, the nuclear and post-nuclear processing might unfavorably affect the transgene expression. Positively charged amphiphilic 1,4-dihydropyridine (1,4-DHP) derivatives were earlier proposed as a promising tool for the delivery of DNA into target cells in vitro and in vivo. However, the structure/activity relationship of these carriers is poorly understood as yet. In this work we studied the intracellular processing of complexes, composed of three structurally related 1,4-DHP derivatives, in a retinal pigment epithelial (ARPE-19) cell line. The pre- and post-nuclear processing of the complexes was quantified on the nuclear, mRNA and transgene expression level. Here we show that the interaction of 1,4-DHP complexes with the cell membrane temporarily increases the permeability of the ARPE-19 cell membrane for small molecular compounds. However, the main mechanism for internalization of 1,4-DHP complexes is endocytosis. We found that all examined derivatives are able to destabilize endosomal membranes by lipid exchange upon acidification. In addition, the buffering capacity of some of the compounds may contribute to the endosomal escape of the complexes as well through the proton sponge effect. Previously we reported that cellular uptake of 1,4-DHP complexes does not correlate with transgene expression. In this study we surprisingly revealed that there is no correlation between the amount of plasmids taken up by the cell and the amount of plasmids found in the cell nucleus. Furthermore, it was found that a high amount of plasmid in the nucleus does not ensure high mRNA expression, likely due to remaining interactions of the carrier with the plasmids. Neither did the expression of mRNA always result in the production of a functional protein, possibly due to the interaction of free carrier with intracellular components which are involved in the post-translational modification of protein and folding process. Overall, our data suggest that succeeding of both the pre- and the post-nuclear intracellular processes is equally essential for successful transgene expression.

Background Interaction of cell-surface glycosaminoglycans (GAGs) with non-viral vectors seems to be an important factor which modifies the intracellular destination of the gene complexes. Intracellular kinetics of polyamidoamine (PAMAM) dendrimer as a non-viral vector in cellular uptake, intranuclear delivery and transgene expression of plasmid DNA with regard to the cell-surface GAGs has not been investigated until now. Methods The physicochemical properties of the PAMAM–pDNA complexes were characterized by photon correlation spectroscopy, atomic force microscopy, zeta measurement and agarose gel electrophoresis. The transfection efficiency and toxicity of the complexes at different nitrogen to phosphate (N:P) ratios were determined using various in vitro cell models such as human embryonic kidney cells, chinese hamster ovary cells and its mutants lacking cell-surface GAGs or heparan sulphate proteoglycans (HSPGs). Cellular uptake, nuclear uptake and transfection efficiency of the complexes were determined using flow cytometry and optimized cell-nuclei isolation with quantitative real-time PCR and luciferase assay. Results Physicochemical studies showed that PAMAM dendrimer binds pDNA efficiently, forms small complexes with high positive zeta potential and transfects cells properly at N:P ratios around 5 and higher. The cytotoxicity could be a problem at N:Ps higher than 10. GAGs elimination caused nearly one order of magnitude higher pDNA nuclear uptake and more than 2.6-fold higher transfection efficiency than CHO parent cells. However, neither AUC of nuclear uptake, nor AUC of transfection affected significantly by only cell-surface HSPGs elimination and interesting data related to the effect of GAGs on intranuclear pDNA using PAMAM as delivery vector have been reported in this study. Conclusion Presented data shows that the rate-limiting step of PAMAM–pDNA complexes transfection is located after delivery to the cell nucleus and GAGs are regarded as an inhibitor of the intranuclear delivery step, while slightly promotes transgene expression.

Polyethylenimine (PEI) polyplexes mediate efficient gene transfer only at high +/- charge ratios at which free noncomplexed PEI is present. The excess of PEI gives polyplexes a positive surface charge that plays a role in polyplex binding on the cell membrane. Although positively charged PEI polyplexes are known to interact with anionic cell-surface glycosaminoglycans (GAGs), the exact role of free PEI in such interactions is unclear.Chinese hamster ovary wild-type cells and mutants lacking cell-surface GAGs were transfected with marker genes using PEI polyplexes with and without free PEI. The total amount of cell-associated plasmid DNA (pDNA) delivered by polyplexes was determined by quantitative real-time PCR and transgene expression was determined using β-galactosidase and luciferase assays.Transfection activity of polyplexes without free PEI in cells expressing cell-surface GAGs was low even though pDNA was delivered to cells. In the absence of cell-surface GAGs, polyplexes without free PEI had high transfection efficacy. This indicates that the cell-surface GAGs inhibit transfection by purified polyplexes. PEI polyplexes with free carrier mediated transfection in both normal and GAG-deficient cells because free PEI overcomes the inhibitory effect of cell-surface GAGs on transfection. The intracellular elimination of pDNA was faster in the presence of GAGs and, despite improved transfection, free PEI reduced pDNA association with the cells.Free PEI is essential for minimizing the undesirable binding of polyplexes to cell-surface GAGs that have a negative impact on transfection. The same mechanism may be important in transfections with other polyplexes that require high charge ratios for transfection.

Bacterial adhesion to the cell surface is a crucial step before infection can take place. Inhibition of bacterial binding offers a novel preventive approach against infections. Cranberry (Vaccinium macrocarpon Ait.) juice has been found to have antiadhesive activity against different bacteria. Streptococcus pneumoniae is an important pathogen and the most common cause for pneumonia, meningitis, and otitis media. In this study the inhibitory activity of cranberry (Vaccinium oxycoccos L.), bilberry (Vaccinium myrtillus L.) and crowberry (Empetrum nigrum and Empetrum hermaphroditum L.) juice fractions against pneumococcal binding was tested using human bronchial cells (Calu-3) as an adhesion model. In addition, the antimicrobial activity of the berry juice fractions was tested. It was found that the studied berry juice fractions had antiadhesion activity and cranberry juice was the most active. The adhesion inhibition activity of cranberry juice was nearly 90% at a concentration of 8.7 mg/g of soluble solids. The antimicrobial activity of the studied berry juice fractions was found to be remarkable; pneumococcal growth was inhibited totally at a concentration of ∼86 mg/g. Both antiadhesion and antimicrobial activities were reduced after solid-phase extraction of the berry juices, which may suggest molecular synergistic effects of the berry juice molecules against S. pneumoniae. The findings indicate that cranberry, bilberry and crowberry juices have potential against pneumococcal infections.

Background Cell and tissue-specific DNA delivery can be achieved by derivatizing vehicles with a targeting ligand for certain receptor. CXCR4 is a receptor of chemokine stromal cell-derived factor (SDF)-1 and viral protein viral macrophage inflammatory protein (vMIP)-II. It is expressed on some types of stem and cancer cells. The present study aimed to design and characterize the group of CXCR4 targeted peptides for receptor-mediated gene delivery. We focused on bifunctional peptide carriers: two derived from N-terminal sequences of SDF-1 and one from vMIP-II. Methods Three synthetic chemokine-derived peptides, designated long CDP (KPVSLSYRSPSRFFESH-K9-biotin), short CDP (KPVSLSYR-K9-biotin) and viral CDP (D-LGASWHRPDK-K9-biotin), were evaluated for gene delivery to CXCR4 positive and negative cells. Oligolysine K9-biotin was used as a control. The Lys 8 moiety binds DNA electrostatically, whereas C-terminal lysine was modified with biotin to study intracellular uptake of the peptides. Complex formation with DNA was monitored by ethidium bromide fluorescence quenching. Results All peptides condensed plasmid DNA. Gene delivery by CDP/DNA complexes is glycerol-dependent and the level of luciferase expression with signal modified carriers was comparable with the efficacy of polyethylenimine (PEI) in CXCR4 expressing cell lines (A172, HeLa) and was ten- to 50-fold higher compared to unmodified peptide. By contrast, CDP transfection efficacy on CXCR4-negative cells (chinese hamster ovary) was much lower than in PEI. Intracellular uptake analysis of biotin-labeled peptides indicated that CDPs entered cells more efficiently than oligolysine. Conclusions The small, bifunctional peptides reported in the present study may be useful in gene delivery to (and gene therapy of) the different tumors and other cells expressing CXCR4. Copyright © 2009 John Wiley & Sons, Ltd.

The intracellular limiting steps in non-viral gene delivery are still unclear. The purpose of this study was to quantize intracellular DNA release and elimination kinetics after transfection with various non-viral carriers (calcium phosphate precipitates, branched poly(ethyleneimine), poly-L-lysine, DOTAP, DOTAP/DOPE) and to correlate these factors with transgene expression. Intracellular kinetics of DNA was determined by novel quantitative method based on qRT-PCR and DNase treatment. Intracellular elimination of DNA after calcium phosphate transfection was rapid (half-life of 0.37 h) whereas the amount of DNA in the cells was stable for at least 136 h after poly(ethyleneimine) mediated transfection. Intracellular elimination half-lives for DNA delivered by other carrier systems ranged from 9 to 27 h. Calcium phosphate precipitates are not able to protect DNA, which explains the short elimination half-life. In the case of other carriers DNA is after complex removal mostly carrier bound but after 24 h the major fraction of DNA is in the released or loosened state. Overall, neither total nor released amount of intracellular DNA correlates with the transgene expression.

A major problem with the pharmaceutical use of nonviral gene delivery systems arises from their limited characterization due to their size and heterogeneity. In this study, we provide a more intuitive view of their structure and behavior employing an empirically based phase diagram approach. Complexes formed between plasmid DNA and four cationic carriers (a monovalent lipid, the same monovalent lipid combined with a helper lipid, polylysine, and a branched form of polyethyleneimine), at both positive and negative nitrogen/phosphorous ratios, are characterized employing dynamic light scattering, circular dichroism, and extrinsic dye fluorescence as methods sensitive to various aspects of the structure of the complexes. These measurements were performed as a function of pH and ionic strength to perturb the electrostatic contacts that are key to complex formation. Using a multidimensional eigenvalue approach, the data are presented in the form of a colored, five dimensional diagram. The resultant eight empirical phase diagrams display three to five variably resolved phases. In contrast, the phase diagram of the plasmid alone showed only two to three such phases. Each state is assigned to a particular form of the complex in terms of their size, extent of collapse and conformation of the associated DNA component. The utility of this approach is then briefly discussed.

The enhancing effect of cell-permeable peptides and polycations to virus-mediated gene transfer was investigated by transducing different cell lines with virus-polycation complexes. In addition, blocking of intracellular polyamine production with defluoromethylornithine (DFMO) was used to intensify heparan sulfate proteoglycan (HSPG) transport from cell membrane to cytoplasm. With this approach we tested whether the accelerated HSPG transport could also improve the transport of virus-polycation or virus-peptide complex.

Nonviral gene delivery methods with naked plasmids and various plasmid carrier complexes have been used for intravascular, intramuscular and periadventitial gene delivery to cardiovascular system. Efficacy, homogenity and quality of the nonviral gene delivery complexes can be significantly affected by the way they are produced. This chapter presents basic methods to produce nonviral gene delivery complexes and describes common models to test their properties in cardiovascular applications in vivo.

Nonviral gene delivery methods with naked plasmids and various plasmid carrier complexes have been used for intravascular, intramuscular and periadventitial gene delivery to cardiovascular system. Efficacy, homogenity and quality of the nonviral gene delivery complexes can be significantly affected by the way they are produced. This chapter presents basic methods to produce nonviral gene delivery complexes and describes common models to test their properties in cardiovascular applications in vivo.

Purpose: The purpose of this study was to investigate the extent, duration, and direction of transgene expression after nonviral gene transfer to differentiated retinal pigment epithelial (RPE) cells. Methods: Polarized human RPE cells (ARPE-19) were transfected with nonviral vectors [DOTAP/DOPE with and without protamine sulfate (PS), DOTAP, PEI (polyethyleneimine), DHP-12] using secreted alkaline phosphatase (SEAP) as a reporter gene. Cellular uptake was studied by flow cytometry. Results: Up to 80-fold differences were observed in the peak reporter gene expression. The highest peak levels and the longest lifetime of SEAP expression (> 69 days) were obtained with DOTAP/DOPE/PS/pDNA complexes. With PEI, higher expression was seen to the apical side than to the basolateral side. Conclusions: In contrast to most differentiated epithelial cells, the differentiated RPE cells can be transfected at high and prolonged levels with selected lipoplexes.

Complexes of DNA with cationic lipids and cationic polymers are frequently used for gene transfer. Extracellular interactions of the complexes with anionic glycosaminoglycans (GAGs) may interfere with gene transfer. Interactions of GAGs with carrier DNA complexes have been studied using tests for DNA relaxation (ethidium bromide intercalation), DNA release (electrophoresis), and transfection (pCMVbGal transfer into RAA smooth muscle cells). Several cationic lipid formulations (DOTAP, DOTAP/Chol, DOTAP/DOPE, DOTMA/DOPE, DOGS) and cationic polymers (fractured dendrimer, polyethylene imines 25 and 800 kDa, polylysines 20 and 200 kDa) were tested. Polycations condensed DNA more effectively than monovalent lipids. Hyaluronic acid did not release or relax DNA in any complex, but it inhibited transfection by some polyvalent systems (PEI, dendrimers, DOGS). Gene transfer by other carriers was not affected by hyaluronic acid. Sulfated GAGs (heparan sulfate, chondroitin sulfates B and C) completely blocked transfection, except in the case of liposomes with DOPE. Sulfated GAGs relaxed and released DNA from some complexes, but these events were not prerequisites for the inhibition of transfection. Furthermore, preliminary results suggest that cell surface GAGs, particularly heparan sulfate, inhibit gene transfer by cationic lipids and polymers.

To investigate the role of vitreous in nonviral gene delivery into retinal pigment epithelial (RPE) cells.Human RPE cell line D407 was cultured in six-well plates. Bovine vitreous, hyaluronan, or DMEM was added on the cells. Complexes of DNA and cationic carriers (polyethyleneimine, poly-L-lysine, DOTAP liposomes) were pipetted onto the vitreous, hyaluronan, or DMEM. Cellular uptake of DNA was studied with ethidium monoazide DNA and gene expression with GFP-plasmid complexes. FITC-dextrans and FITC-polylysines were used to probe the effects of the size and cationic charge on permeation in the vitreous in a similar experimental setup. Fluorescent cells were analyzed by flow cytometry.Vitreous decreased the cellular uptake of DNA complexes 2-30 times, and GFP expression was also impaired. In hyaluronan solutions the cellular uptake of the complexes was also decreased significantly in most cases. In vitreous, cellular uptake of all FITC-dextrans decreased slightly, and uptake of poly-L-lysines was decreased substantially, whereas in hyaluronan solutions the effects were mild or nonexistent.Polymeric and liposomal gene delivery is substantially limited by the vitreous. This is probably because of the size and charge of the retinal gene delivery after intravitreal injections.

Double-charged 1,4-dihydropyridine (1,4-DHP) amphiphiles have been shown to condense DNA and efficiently transfect it into cells in vitro [Hyvonen et al., Biochim. Biophys. Acta 1509 (2000) 451]. Alkyl chain length and buffering capacity at endosomal pH range (5.0-7.4) affected complexation and transfection activity. In this study we examined how those chemical modifications of amphiphile-DNA complexes (amphiplexes) affect their interactions with extracellular polyanions (glycosaminoglycans, albumin) and lipid bilayers, their cellular uptake and intracellular distribution. To evaluate cellular uptake, CV1-P cells were incubated with labeled DNA-amphiphile complexes and analyzed by flow cytometry. Confocal laser fluorescence microscopy was used to investigate the intracellular distribution of amphiplexes. The results showed that biophysical properties of compounds can be changed by slight structural modifications. These factors determine the intracellular kinetics and transfection efficacy of the compounds. Some extracellular glycosaminoglycans and serum interfere with 1,4-DHP-amphiphile-mediated transfection by destabilizing the amphiplexes. Neither high cellular uptake, membrane destabilizing activity nor buffering capacity alone is adequate for high transfection efficacy. The activity results from complex interplay of various factors that determine intracellular kinetics and, consequently, transfection.

Different lipids and cationic polymers were tested in vitro for their ability to transfect rabbit aortic smooth muscle cells and human endothelial cells with lacZ marker gene. Toxicity of the complexes was evaluated with MTT assay. Selected plasmid-polymer complexes with different charge ratios were then tested for in vivo gene transfer efficiency using adventitial gene transfer by placing a silastic gene delivery reservoir (collar) around the carotid artery. Transfection efficiency was determined by X-gal staining 3 days after the gene transfer. Based on in vitro experiments, fractured polyamidoamine dendrimers and polyethylenimines (PEI) were selected for in vivo experiments. Fractured dendrimers (generation 6, +/- charge ratio of 3) had the highest in vivo gene transfer efficiency (4.4% +/- 1.7). PEI with molecular size of 25 kDa (+/- charge ratio 4) was also effective (2.8% +/- 1.8) in this model. PEI of 800 kDa showed a constant but modest gene transfer efficiency (1.8% +/- 0.1) with all charge ratios. A low level gene transfer was also detected with naked DNA (0.5% +/- 0.3). No signs of inflammation were seen in any of the study groups. We show here that in vitro cell culture experiments can be used to identify efficient in vivo gene transfer methods for arterial gene therapy, but the charge ratios for each complex must be optimized in vivo. It is concluded that fractured dendrimer and PEI are efficient gene delivery vehicles and can be used for arterial gene therapy via adventitial gene delivery route.