The race to the pole: how high-aspect ratio shape and heterogeneous environments limit phagocytosis of filamentous Escherichia coli bacteria by macrophages.

While bioengineers ask how the shape of diagnostic and therapeutic particles impacts their pharmacological efficiency, biodistribution, and toxicity, microbiologists suggested that morphological adaptations enable pathogens to perhaps evade the immune response. Here, a shape-dependent process is described that limits phagocytosis of filamentous Escherichia coli bacteria by macrophages: successful uptake requires access to one of the terminal bacterial filament poles. By exploiting micropatterned surfaces, we further demonstrate that microenvironmental heterogeneities can slow or inhibit phagocytosis. A comparison to existing literature reveals a common shape-controlled uptake mechanism for both high-aspect ratio filamentous bacteria and engineered particles.

Porous polysulfone coatings for enhanced drug delivery.

The synthesis of a porous polysulfone (PSU) coating for use in drug delivery applications is presented. PSU can serve as a functional surface coating for drug delivery vehicles, such as intraocular biomicrorobots. The coatings can be applied using spin coating or dip coating. The porosity is introduced by selectively dissolving calcium carbonate nanoparticles embedded in the bulk polymer. The network of pores thus formed increases by a factor of thirty the amount of Rhodamine B (model drug) that can be loaded and by a factor of fifteen the amount that can be released. The films do not affect cell viability and exhibit poor cell adhesion. The straightforward synthesis and predictability of porosity enables the tuning of the amount of drug that can be loaded.

Alkali treatment of microrough titanium surfaces affects macrophage/monocyte adhesion, platelet activation and architecture of blood clot formation.

Titanium implants are most commonly used for bone augmentation and replacement due to their favorable osseointegration properties. Here, hyperhydrophilic sand-blasted and acid-etched (SBA) titanium surfaces were produced by alkali treatment and their responses to partially heparinized whole human blood were analyzed. Blood clot formation, platelet activation and activation of the complement system was analyzed revealing that exposure time between blood and the material surface is crucial as increasing exposure time results in higher amount of activated platelets, more blood clots formed and stronger complement activation. In contrast, the number of macrophages/monocytes found on alkali-treated surfaces was significantly reduced as compared to untreated SBA Ti surfaces. Interestingly, when comparing untreated to modified SBA Ti surfaces very different blood clots formed on their surfaces. On untreated Ti surfaces blood clots remain thin (below 15 mm), patchy and non-structured lacking large fibrin fiber networks whereas blood clots on differentiated surfaces assemble in an organized and layered architecture of more than 30 mm thickness. Close to the material surface most nucleated cells adhere, above large amounts of non-nucleated platelets remain entrapped within a dense fibrin fiber network providing a continuous cover of the entire surface. These findings might indicate that, combined with findings of previous in vivo studies demonstrating that alkali-treated SBA Ti surfaces perform better in terms of osseointegration, a continuous and structured layer of blood components on the blood-facing surface supports later tissue integration of an endosseous implant.

Cellular uptake and intracellular pathways of PLL-g-PEG-DNA nanoparticles.

Polycationic molecules form condensates with DNA and are used for gene therapy as an alternative to viral vectors. As clinical efficacy corresponds to cellular uptake, intracellular stability of the condensates, and bioavailability of the DNA, it is crucial to analyze uptake mechanisms and trafficking pathways. Here, a detailed study of uptake, stability, and localization of PLL-g-PEG-DNA nanoparticles within COS-7 cells is presented, using FACS analysis to assess the involvement of different uptake mechanisms, colocalization studies with markers indicative for different endocytotic pathways, and immunofluorescence staining to analyze colocalization with intracellular compartments. PLL-g-PEG-DNA nanoparticles were internalized in an energy-dependent manner after 2 h and accumulated in the perinuclear region after >6 h. The nanoparticles were found to be stable within the cytoplasm for at least 24 h and did not colocalize with the endosomal pathway. Nanoparticle uptake was approximately 50% inhibited by genistein, an inhibitor of the caveolae-mediated pathway. However, genistein did not inhibit gene expression, and PLL-g-PEG-DNA nanoparticles were not colocalized with caveolin-1 indicating that caveolae-mediated endocytosis is not decisive for DNA delivery. Clathrin-mediated endocytosis and macropinocytosis pathways were reduced by 17 and 24%, respectively, in the presence of the respective inhibitors. When cells were transfected in the presence of double and triple inhibitors, transfection efficiencies were increasingly reduced by 40 and 70%, respectively; however, no differences were found between the different uptake mechanisms. These findings suggest that PLL-g-PEG-DNA nanoparticles enter by several pathways and might therefore be an efficient and versatile tool to deliver therapeutic DNA.

Molecular design and characterization of the neuron-microelectrode array interface.

Electrophysiological activities of neuronal networks can be recorded on microelectrode arrays (MEAs). This technique requires tight coupling between MEA-surfaces and cells. Therefore, this study investigated the interface between DRG neurons and MEA-surface materials after adsorption of neurite promoting proteins: laminin-111, fibronectin, L1Ig6 and poly-l-lysine. Moreover, substrate-induced effects on neuronal networks with time were analyzed. The thickness of adsorbed protein layers was found between approximately 1 nm for poly-l-lysine and approximately 80 nm for laminin-111 on platinum, gold and silicon nitride. The neuron-to-substrate interface was characterized by Scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and SEM after in situ focused-ion-beam milling demonstrating that the ventral cell membrane adhered inhomogeneously to laminin-111 or L1Ig6 surfaces. Tight areas of 20-30 nm and distant areas <1 microm alternated and even tightest areas did not correlate with the physical thickness of the protein layers. This study illustrates the difficulties to predict cell-to-material interfaces that contribute substantially to the success of in vitro or in vivo systems. Moreover, focused ion beam (FIB)/SEM is explored as a new technique to analyze such interfaces.

Activation of cell-survival transcription factor NFkappaB in L1Ig6-stimulated endothelial cells.

Ligation of the integrin alpha(v)beta(3) in endothelial cells has been shown to be important for their survival. Such ligation induces signalling events merging into the Raf-Ras-ERK cascade that eventually induces activation of nuclear factor kappa B (NFkappaB), leading to its phosphorylation and nuclear translocation and thus inhibiting apoptosis. Here, the recombinant sixth immunoglobulin-like domain of cell adhesion molecules L1 (L1Ig6), a ligand for integrin alpha(v)beta(3), was explored as a component of vascular implant surfaces to initiate the NFkappaB-cell survival pathway. This supposition was supported. Specifically, NFkappaB-p65 was expressed in human umbilical vein endothelial cells (HUVECs) and when stimulated on L1Ig6, the phosphorylated form was found in the nucleus in over 60% of the cells. NFkappaB was not translocated into the nucleus on a number of other extracellular matrix substrates examined or when fibroblasts were cultured on L1Ig6. NFkappaB phosphorylation and nuclear translocation could be inhibited by blocking ligation of alpha(v)beta(3) by L1Ig6 with an antibody recognizing alpha(v)beta(3), with a cyclic RGD peptide, and with soluble L1Ig6. Moreover, blocking of alpha(v)beta(3) interaction with L1Ig6 was correlated with induction of apoptosis. Thus, these experiments demonstrate that L1Ig6 may be useful as alpha(v)beta(3) ligand for the induction of endothelial survival pathways mediated by NFkappaB-p65.

Mechanical properties, proteolytic degradability and biological modifications affect angiogenic process extension into native and modified fibrin matrices in vitro.

During initial stages of wound healing, fibrin clots provide a three-dimensional scaffold that induces cell infiltration and regeneration. Here, L1Ig6, a ligand for alphavbeta3 integrin was covalently incorporated within fibrin matrices to explore it as a matrix-immobilized angiogenic factor. Incorporation at concentrations greater than 1 microg/ml reduced the fibrin crosslink density, as reflected by measurements of elastic modulus and swelling. The influence of crosslink density on endothelial cell process extension was characterized by modulating factor XIII concentrations in the coagulation mixture. At low incorporated concentrations of L1Ig6, it was possible to compensate gel elastic modulus via increased factor XIII, but not at high concentrations of L1Ig6. Similar findings were found when matrix swelling was analyzed. Fibrin crosslink density strongly influenced endothelial cell process extension, fewer and shorter processes were observed at high crosslink density. Matrix metalloproteinases (MMPs) were required for process extension and zymography and Western blots identified MMP-2 but not MMP-9. The amount of active MMP-2 increased for endothelial cells cultured in native and L1Ig6-modified matrices or when stimulated with VEGF-A165. The data indicate that distinct matrix properties can be tailored such that they become biologically stimulating and respond to cellular proteolytic activities, being a prerequisite for potential use of such matrices in biomedical applications.

Human preterm amnion cells cultured in 3-dimensional collagen I and fibrin matrices for tissue engineering purposes.

OBJECTIVE: In this study, human preterm amnion cells were investigated in 3-dimensional (3D) cell-matrix culture systems in an attempt to design therapeutic strategies for preterm premature rupture of the membranes. STUDY DESIGN: Three-dimensional collagen I and fibrin cell-containing biomatrices were created to mimic the architecture of native amnion. Amnion mesenchymal cells were embedded in 3D matrices, and epithelial cells were placed on top of these matrices. Cell viability and morphology were visualized by DiI-ac-LDL, F-actin, and nuclear staining. Proteolytic activity of matrix metalloproteinases (MMPs) was investigated using gelatine zymography. RESULTS: Preterm amnion epithelial and mesenchymal cells cultured in collagen I and fibrin matrices assume cell morphologies similar to those observed in vivo. Mesenchymal cells were capable of remodelling collagen I, as seen by extensive volume contraction, by 40% at day 1 and 80% at day 5. Matrix contraction was independent of the presence of epithelial cells, and could not be inhibited by GM6001 and/or aprotinin. No contraction was observed in fibrin matrices over 8 days. The migratory response of mesenchymal cells cultured in 3D fibrin matrices supplemented with fibronectin was associated with specific activated MMP-9. CONCLUSION: Three-dimensional fibrin matrices might be useful in amnion cell tissue engineering, including cell-matrix transplantation.

Non-viral gene delivery for local and controlled DNA release.

Non-viral DNA delivery systems show important advantages vs. viral systems that are usually associated with an immunological response and safety risks. In this study, disulfide cross-linked peptide-DNA condensates were investigated for local gene delivery. Two different 21 amino acid peptides were designed to have a DNA binding sequence in combination with a transglutaminase substrate site or a nuclear localization site. The peptides were used in different ratios to each other to form stable cross-linked DNA-peptide condensates with a mean diameter of 164 nm and a size distribution from 43 to 204 nm. Such aggregates showed similar stability compared to condensates formed between DNA and high molecular weight poly-L-lysine (PLL). Peptide-DNA condensates were covalently immobilized into fibrin matrices by the activity of factor XIII and were used for gene delivery in vitro. After internalization, reduction of the cross-linked peptide-DNA condensates yielded increased transfection efficiencies into different cell types cultured in 2D sandwich assays, and comparable values for HUVECs cultured in a 3D fibrin matrix, as compared to PLL-DNA condensates. Cell viability 24 h after transfection remained above 95%. The target was to develop a transfection system based on small peptides that can be covalently cross-linked into fibrin-matrices where DNA-release takes place upon cellular degradation of the matrix. This approach provides an interesting tool in non-viral gene delivery.

Patterning of supported lipid bilayers and proteins using material selective nitrodopamine-mPEG.

We present a generic patterning process by which biomolecules in a passivated background are patterned directly from physiological buffer to microfabricated surfaces without the need for further processing. First, nitrodopamine-mPEG is self-assembled to selectively render TiO2 patterns non-fouling to biomolecule adsorption on hydrophilic and adhesive glass surfaces. After the controlled TiO2 passivation, the biomolecules can be directly adsorbed from solution in a single step creating large scale micropatterned and highly homogeneous arrays of biomolecules with very high pattern definition. We demonstrate the formation of fluid supported lipid bilayers (SLBs) down to the single µm-level limited only by the photolithographic process. Non-specific adsorption of lipid vesicles to the TiO2 background was found to be almost completely suppressed. The SLB patterns can be further selectively functionalized with retained mobility, which we demonstrate through biotin-streptavidin coupling. We envision this single step patterning approach to be very beneficial for membrane-based biosensors and for pattering of cells on a passivated background with complex, sub-cellular geometries; in each application the adherent areas have a tunable mobility of interaction sites controlled by the fluidity of the membrane.

Comparative assessment of the stability of nonfouling poly(2-methyl-2-oxazoline) and poly(ethylene glycol) surface films: an in vitro cell culture study.

Poly(ethylene glycol) (PEG) has been the most frequently reported and commercially used polymer for surface coatings to convey nonfouling properties. PEGylated surfaces are known to exhibit limited chemical stability, particularly due to oxidative degradation, which limits long-term applications. In view of excellent anti-adhesive properties in the brush conformation and resistance to oxidative degradation, poly(2-methyl-2-oxazoline) (PMOXA) has been proposed recently as an alternative to PEG. In this study, the authors systematically compare the (bio)chemical stability of PEG- and PMOXA-based polymer brush monolayer thin films when exposed to cultures of human umbilical vein endothelial cells (HUVECs) and human foreskin fibroblasts (HFFs). To this end, the authors used cell-adhesive protein micropatterns in a background of the nonfouling PEG and PMOXA brushes, respectively, and monitored the outgrowth of HUVECs and HFFs for up to 21 days and 1.5 months. Our results demonstrate that cellular micropatterns spaced by PMOXA brushes are significantly more stable under serum containing cell culture conditions in terms of confinement of cells to the adhesive patterns, when compared to corresponding micropatterns generated by PEG brushes. Moreover, homogeneous PEG and PMOXA-based brush monolayers on Nb2O5 surfaces were investigated after immersion in endothelial cell medium using ellipsometry and x-ray photoelectron spectroscopy.

Macrophages lift off surface-bound bacteria using a filopodium-lamellipodium hook-and-shovel mechanism.

To clear pathogens from host tissues or biomaterial surfaces, phagocytes have to break the adhesive bacteria-substrate interactions. Here we analysed the mechanobiological process that enables macrophages to lift-off and phagocytose surface-bound Escherichia coli (E. coli). In this opsonin-independent process, macrophage filopodia hold on to the E. coli fimbriae long enough to induce a local protrusion of a lamellipodium. Specific contacts between the macrophage and E. coli are formed via the glycoprotein CD48 on filopodia and the adhesin FimH on type 1 fimbriae (hook). We show that bacterial detachment from surfaces occurrs after a lamellipodium has protruded underneath the bacterium (shovel), thereby breaking the multiple bacterium-surface interactions. After lift-off, the bacterium is engulfed by a phagocytic cup. Force activated catch bonds enable the long-term survival of the filopodium-fimbrium interactions while soluble mannose inhibitors and CD48 antibodies suppress the contact formation and thereby inhibit subsequent E. coli phagocytosis.

Influence of micro and submicro poly(lactic-glycolic acid) fibers on sensory neural cell locomotion and neurite growth.

For successful peripheral nerve regeneration, a complex interplay of growth factors, topographical guidance structure by cells and extracellular matrix proteins, are needed. Aligned fibrous biomaterials with a wide variety in fiber diameter have been used successfully to support neuronal guidance. To better understand the importance of size of the topographical features, we investigated the directionality of neuronal migration of sensory ND7/23 cells on aligned electrospun poly(lactic-glycolic acid) PLGA fibers in the range of micrometer and submicrometer diameters by time-lapse microscopy. Cell trajectories of single ND7/23 cells were found to significantly follow topographies of PLGA fibers with micrometer dimensions in contrast to PLGA fibers within the submicrometer range, where cell body movement was observed to be independent of fibrous structures. Moreover, neurite alignment of ND7/23 cells on various topographies was assessed. PLGA fibers with micrometer dimensions significantly aligned 83.3% of all neurites after 1 day of differentiation compared to similar submicrometer structures, which orientated 25.8% of all neurites. Interestingly, after 7 days of differentiation ND7/23 cells on submicrometer PLGA fibers increased their alignment of neurites to 52.5%. Together, aligned PLGA fibers with micrometer dimensions showed a superior influence on directionality of neuronal migration and neurite outgrowth of sensory ND7/23 cells, indicating that electrospun micro-PLGA fibers might represent a potential material to induce directionality of neuronal growth in engineering applications for sensory nerve regeneration.

Engineering functional bladder tissues.

PURPOSE: End stage bladder disease can seriously affect patient quality of life and often requires surgical reconstruction with bowel tissue, which is associated with numerous complications. Bioengineering of functional bladder tissue using tissue-engineering techniques could provide new functional tissues for reconstruction. In this review, we discuss the current state of this field and address different approaches to enable physiologic voiding in engineered bladder tissues in the near future. MATERIALS AND METHODS: In a collaborative effort, we gathered researchers from four institutions to discuss the current state of functional bladder engineering. A MEDLINE® and PubMed® search was conducted for articles related to tissue engineering of the bladder, with special focus on the cells and biomaterials employed as well as the microenvironment, vascularisation and innervation strategies used. RESULTS: Over the last decade, advances in tissue engineering technology have laid the groundwork for the development of a biological substitute for bladder tissue that can support storage of urine and restore physiologic voiding. Although many researchers have been able to demonstrate the formation of engineered tissue with a structure similar to that of native bladder tissue, restoration of physiologic voiding using these constructs has never been demonstrated. The main issues hindering the development of larger contractile tissues that allow physiologic voiding include the development of correct muscle alignment, proper innervation and vascularization. CONCLUSION: Tissue engineering of a construct that will support the contractile properties that allow physiologic voiding is a complex process. The combination of smart scaffolds with controlled topography, the ability to deliver multiple trophic factors and an optimal cell source will allow for the engineering of functional bladder tissues in the near future.

The angiogenic response to PLL-g-PEG-mediated HIF-1α plasmid DNA delivery in healthy and diabetic rats.

Impaired angiogenesis is a major clinical problem and affects wound healing especially in diabetic patients. Improving angiogenesis is a reasonable strategy to increase diabetes-impaired wound healing. Recently, our lab described a system of transient gene expression due to pegylated poly-l-lysine (PLL-g-PEG) polymer-mediated plasmid DNA delivery in vitro. Here we synthesized peptide-modified PLL-g-PEG polymers with two functionalities, characterized them in vitro and utilized them in vivo via a fibrin-based delivery matrix to induce dermal wound angiogenesis in diabetic rats. The two peptides were 1) a TG-peptide to covalently bind these nanocondensates to the fibrin matrix (TG-peptide) for a sustained release and 2) a polyR peptide to improve cellular uptake of these nanocondensates. In order to induce angiogenesis in vivo we condensed modified and non-modified polymers with plasmid DNA encoding a truncated form of the therapeutic candidate gene hypoxia-inducible transcription factor 1α (HIF-1α). HIF-1α is the primarily oxygen-dependent regulated subunit of the heterodimeric transcription factor HIF-1, which controls angiogenesis among other physiological pathways. The truncated form of HIF-1α lacks the oxygen-dependent degradation domain (ODD) and therefore escapes degradation under normoxic conditions. PLL-g-PEG polymer-mediated HIF-1α-ΔODD plasmid DNA delivery was found to lead to a transiently induced gene expression of angiogenesis-related genes Acta2 and Pecam1 as well as the HIF-1α target gene Vegf in vivo. Furthermore, HIF-1α gene delivery was shown to enhance the number endothelial cells and smooth muscle cells - precursors for mature blood vessels - during wound healing. We show that - depending on the selection of the therapeutic target gene - PLL-g-PEG nanocondensates are a promising alternative to viral DNA delivery approaches, which might pose a risk to health.

Orthogonal nanometer-micrometer roughness gradients probe morphological influences on cell behavior.

Surface gradients facilitate rapid, high-throughput, systematic investigations in cell biology, materials science, and other fields. An important surface parameter is the surface roughness on both the micrometer and nanometer scales in the lateral direction. Two approaches have been combined to create two-dimensional roughness gradients by adding a nanoparticle density gradient onto a gradient of micro-featured roughness. All fabricated gradients were extensively characterized by SEM, AFM and optical profilometry to ensure their quality and to determine the roughness parameter Ra along the gradient. Additionally, a Fourier-transform approach was applied that allows a wavelength-dependent analysis of the surface topography. Since cell-culture assays require replicate experiments, a replica technique was used to create copies of the master gradient. Creating a negative replica in an elastomeric material served as a mold for a subsequent ceramic-casting process. A positive replica was then formed from epoxy resin, which was subsequently coated with titanium and used for cell studies. Finally, these gradients were used in cell-culture assays to determine cellular response to surface roughness. The results clearly demonstrate the influence of surface roughness on the production by osteoblasts of markers for osteogenesis. It was shown that high roughness in the micrometer range, combined with an intermediate nanofeature density (30-40 features/µm2), leads to the highest degree of osteopontin production after 14 days.

Influence of the fiber diameter and surface roughness of electrospun vascular grafts on blood activation.

Electrospun grafts have been widely investigated for vascular graft replacement due to their ease and compatibility with many natural and synthetic polymers. Here, the effect of the processing parameters on the scaffold's architecture and subsequent reactions of partially heparinized blood triggered by contacting these topographies were studied. Degrapol® (DP) and poly(lactic-co-glycolic acid) (PLGA) electrospun fibrous scaffolds were characterized with regard to fiber diameter, pore area and scaffold roughness. The study showed that electrospinning parameters greatly affect fiber diameter together with pore dimension and overall scaffold roughness. Coagulation cascade activation, early platelet adhesion and activation were analyzed after 2h of exposure of blood to the biomaterials. While no differences were found between DP and PLGA with similar topographies, the blood reactions were observed to be dependent on the fiber diameter and scaffold roughness. Scaffolds composed of thin fibers (diameter <1µm) triggered very low coagulation and almost no platelets adhered. On the other hand, scaffolds with a bigger fiber diameter (2-3µm) triggered higher thrombin formation and more platelets adhered. The highest platelet adhesion and activations rates as well as coagulation cascade activation were found in blood incubated in contact with the scaffolds produced with the biggest fiber diameter (5µm). These findings indicate that electrospun grafts with small fiber diameter (<1µm) could perform better with reduced early thrombogenicity due to lower platelet adhesion and lower activation of platelets and coagulation cascade.

Response of osteoclasts to titanium surfaces with increasing surface roughness: an in vitro study.

Osteoclasts are responsible for bone resorption and implant surface roughness promotes osseointegration. However, little is known about the effect of roughness on osteoclast activity. This study aims at the characterization of osteoclastic response to surface roughness. The number of osteoclasts, the tartrate-resistant acid phosphatase and matrix metalloproteinase (MMP) activities, the cell morphology and the actin-ring formation were examined on smooth (TS), acid-etched (TA) and sandblasted acid-etched (TLA) titanium and on native bone. Cell morphology was comparable on TA, TLA and bone, actin rings being similar in size on TLA and bone, but smaller on TA and virtually absent on TS. Gelatin zymography revealed increased proMMP-9 expression on TA, TLA, and bone compared to TS. In general, osteoclasts show similar characteristics on rough titanium surfaces and on bone, but reduced activity on smooth titanium surfaces. These results offer some insight into the involvement of osteoclasts in remodeling processes around implant surfaces.

The role of plasma proteins in cell adhesion to PEG surface-density-gradient-modified titanium oxide.

Surface-density gradients of poly(ethylene glycol) (PEG) were fabricated, in order to carry out a systematic study of the influence of PEG chain density on protein adsorption and cell-adhesion behavior, as well as the correlation between them. Gradients with a linear change in coverage of the polycationic polymer Poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) were prepared on titanium dioxide surfaces by a controlled dipping process and characterized by variable-angle spectroscopic ellipsometry and fluorescence microscopy. The adsorption behavior of single proteins (fibrinogen and albumin) generally correlated with semiempirical geometric models, illustrating the effect of the PEG-chain surface distribution on the inhibition of protein adsorption. Distinct differences could be observed between individual adsorbing proteins, attributable to their mode of surface attachment. The single and competitive adsorption of protein solutions containing albumin and fibrinogen was then investigated by fluorescence microscopy, indicating a larger amount of fibrinogen adsorption compared with albumin adsorption (in minutes to hours) along the entire PLL-g-PEG gradient samples. To further elucidate the underlying mechanism of cell adhesion and spreading as a function of PEG coverage and the potential involvement of integrins, cell-adhesion assays were carried out with human foreskin fibroblasts (hFF). The use of surface-gradient samples demonstrated the importance for protein adsorption of PEG conformation, the amount of exposed titanium dioxide surface area (and its distribution), and the structure and chemistry of the proteins involved. Correspondingly the influence of these factors on cell adhesion could be directly observed, and insights gained into the roles of both nonspecific binding and specific integrin binding in cell adhesion.

Formation and characterization of DNA-polymer-condensates based on poly(2-methyl-2-oxazoline) grafted poly(L-lysine) for non-viral delivery of therapeutic DNA.

Successful gene delivery systems deliver DNA in a controlled manner combined with minimal toxicity and high transfection efficiency. Here we investigated 15 different copolymers of poly(l-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA) of variable grafting densities and PMOXA molecular weights for their potential to complex and deliver plasmid DNA. PLL(20)g(7)PMOXA(4) formed at N/P charge ratio of 3.125 was found to transfect 9 ± 1.6% of COS-7 cells without impairment of cell viability. Furthermore these PLL-g-PMOXA-DNA condensates were internalized 2 h after transfection and localized in the perinuclear region after 6 h. The condensates displayed a hydrodynamic diameter of ∼100 nm and were found to be stable in serum and after 70 °C heat treatment, moreover the condensates protected DNA against DNase-I digestion. The findings suggest that DNA-PMOXA-g-PLL condensate formation for efficient DNA-delivery strongly depends on PMOXA grafting density and molecular weight showing an optimum at low grafting density between 7 and 14% and medium N/P charge ratio (3.125-6.25). Thus, PLL(20)g(7)PMOXA(4) copolymers might be promising as alternative to PLL-g-PEG-DNA condensates for delivery of therapeutic DNA.