Strategies in non-viral gene delivery: "to overcome serum effect"

Throughout these last years, many synthetic polymers have been developed for non-viral gene delivery systems in attempt to overcome potential shortcomings of viral vectors. However, it is still difficult to achieve highly efficient transfection under physiological condition due to severe interaction between cationic polyplexes and abundant proteins in serum. In this project, we aim to suggest different strategies to enhance transfection efficiency of non-viral polymeric vector, which was already developed in our research group, i.e. poly(N-acryloylpiperazine) (PAZ). As a first strategy, cancer-targeting delivery was attempted with a folic acid (FA)-conjugated poly(N-acryloylpiperazine) (PAZ-FA), because folic acid receptors (FRs) are over-expressed on many types of cancer cells. We hypothesized that folic acid moieties on PAZ-FA would facilitate the attachment and the internalization of polyplexes to cancer cells. As a second strategy, block copolymerization was attempted. In this approach, a diblock copolymer, i.e. poly[(N-acryloylmorpholine)-bl-(N-acryloylpiperazine)] (PAM-PAZ) was synthesized. We hypothesized that a neutral and hydrophilic poly(N-acryloylmorpholine) (PAM) block would reduce electrostatic interaction with proteins in serum which deteriorates transfection efficiency. Synthesized polymers were thoroughly characterized and details are described herein. The physical properties of polyplexes were examined such as size, surface charge by zetapotential and gel retardation assay, protection capability from enzyme, etc. To carry out transfection experiments, two types of cells, i.e. human embryonic kidney (293T) cells and human cervical epitheloid carcinoma (HeLa) cells were employed with green fluorescent protein (GFP)-expressing plasmid DNA. Transfection results were analyzed by fluorescence-activated cell sorting (FACS). As a positive control, linear poly(ethylene imine) (LPEI) was employed in the experiments. In serum-free condition, there was no substantial difference in size and/or zeta potential among PAZ-polyplexes, PAZ-FA polyplexes, PAM-PAZ polyplexes, and LPEI-polyplexes. This reveals that all PAZ derivatives are similarly capable to condense DNA into nano-sized particulate polyplexes as LPEI. PAZ and PAZ-FA showed comparable or even better transfection efficiency than LPEI, whereas PAM-PAZ showed lower transfection efficiency than the other two PAZ derivatives and LPEI. In the presence of 10% of fetal bovine serum (FBS), we observed that, the size of polyplexes of PAZ-derivatives spontaneously increased up to a few microns in an hour or two. This size increment was more severe compared to LPEI-polyplexes, although the surface charge of all polyplexes remained the same. As for transfection, with 10% of FBS in cell media, all PAZ-derivatives showed lower transfection efficiency than LPEI. To get a better insight of serum effect, we prepared a fluorescent-labeled PAZ and examined how serum affects the internalization of polyplexes. In result, we observed that serum prevents large extent of attachment on the extracellular membranes, but there was no difference in the actual amount of internalized polyplexes either with or without serum. Thus, we believe that serum does not influence on the internalization of polyplexes, but other critical pathway may involve an intracellular compartment.

Polymeric carriers for therapeutic agents

Yun Suk Jo

This thesis deals with development of polymeric carriers for many different therapeutic agents such as immune-suppressant drug (everolimus), nitric oxide (NO), and polynucleotide. Polymeric carriers developed in this project were all synthesized by reversible additionfragmentation transfer (RAFT) polymerization, which is one of the living radical polymerization techniques. Synthesis, characterization, and results from in vitro/ex vivo experiments are described. New polymeric amphiphiles were obtained from the polymers synthesized by RAFT polymerization. Monomeric building blocks were derived from N-acryloyl derivatives of the cyclic secondary amines; morpholine, piperidine and azocane. Both homopolymerization and block-copolymerization of N-acryloylmorpholine (AM), N-acryloylpiperidine (AP), and N-acryloylazocane (AH) were carried out. From diblock copolymers in these compositions, block copolymeric amphiphiles were aggregated in nano-size. Details of aggregation behaviour were studied. Everolimus, a hydrophobic immune-suppressant drug was loaded in the micelles aggregated from poly[(N-acryloylmorpholine)-block-(N-acryloylazocane)] (PAM-PAH) diblock copolymer and drug-loaded micelles released the encapsulated drug over three weeks under physiological conditions. New polymeric NO donors were conceived from piperazine derivatives such as piperazine, homopiperazine, and 2,5-dimethylpiperazine. Depending on the hydrophobicity of the grafted ring of polymeric NO donors, we observed that the releasing rate of NO varied. Furthermore, we developed self-assembled micelles releasing NO with remarkably long release time ca. over 3 weeks. By encapsulating diazeniumdiolate (NONOates) in a hydrophobic micelle core, we were able to benefit from its slow hydrolysis rate, creating this unique release pattern. As NO is released, self-assembled micelles, turning into water-soluble polymer, will ultimately be dissociated and secreted out of the body. As demonstrated by ex vivo infusion experiments with rabbit carotid arteries, NO-releasing micelles (~80 nm) readily penetrate the arterial wall at which point their therapeutic aspects could be exploited, namely the delivery of NO to restenotic lesions. A new class of polycation, namely poly(N-acryloylpiperazine) (PAZ) was synthesized by RAFT polymerization and attempted as a non-viral gene delivery system. PAZ was capable to condense plasmid DNA and formed particles with less than 100 nm of diameter. PAZ-DNA polyplexes exhibited less incidence of cytotoxicity than linear PEI (LPEI)-complexed DNA, which is a commercially available synthetic gene delivery vector. In vitro transfection experiments were carried out with various cell lines such as human embryonic kidney (HEK) 293T cell line. The results were analyzed by fluorescent-activated cell sorting (FACS). In most cases, all homo/copolymers showed comparable or better transfection efficiency than LPEI when cells were transfected without serum. However, in the presence of serum, transfection efficiency of PAZ decreased. The details of the results, characterization, and discussion are described herein.

EPFL2008

Establishment of recombinant mammalian cell lines

Madiha Derouazi

Recombinant proteins are gaining in importance for therapeutic applications. The proteins are expressed in stable cell lines, within which recombinant DNA has incorporated into the host cells genome. Identification and isolation of extremely high producers from transfected cell populations is a tedious and time-consuming effort. We proposed two different approaches for the establishment of recombinant cell lines: affinity cell sorting and microinjection. Even with the help of single cell sorting and analysis systems (flow cytometry) rarely more than thousand different cell clones, stably expressing a desired gene can be evaluated. We are considering a new approach in which polyclonal cell populations will be established which co-express a protein of interest and an epitope-tagged cell surface receptor. A microbead fixed antibody specific to the epitope tag is hoped to assist in the isolation of rare, but highly productive cells from large suspensions of transfected cells. For this reason we have also developed an efficient method for the bulk transfection of CHO cells. On the side of vector-development for this approach, we have chosen to investigate the utility of a mouse serotonin 5-HT3 receptor as a cell surface marker. This receptor can be expressed at very high levels (up to 1 x 107 receptors per cell) in heterologous systems. The receptor is detectable on the cell surface by means of fluorescent ligands, which allows the determination of transfection efficiencies in suspension cultures. A Flag-tag was fused to the amino terminal end of the receptor without adverse effects on receptor function or ligand binding. Only 5 % of receptor DNA in the transfection cocktail was sufficient to detect positive cells in a population. The specific interaction of the epitope-tagged receptor with the cognate antibody fixed to microbeads was tested in comparison to non-transfected control cells using laser scanning confocal microscopy. A 80% enrichment of cells expressing GFP and the 5-HT3 receptor was achieved in 30 minutes. We have investigated microinjection as a gene transfer method for the establishment of recombinant mammalian cell lines. The conditions for the injection of plasmid DNA into either the nucleus or the cytoplasm of CHO-DG44 cells were established using pMYK-EGFP, an expression vector with the enhanced green fluorescent protein (EGFP) gene under the control of the murine CMV promoter and the puromycin resistance gene for selection. To estimate plasmid copy number during microinjection, it was first necessary to determine the injection volume by fluorescence quantification of injected FITC-dextran. For microinjection into the cytoplasm the volume ranged from 200 to 350 fl and for nuclear microinjection it was about 180 fl. We then investigated the impact of DNA concentration and injection pressure on transient EGFP transient expression following microinjection into the cytoplasm or nucleus. As a general rule, we observed that concentration of the DNA solution injected into the cells was the most important parameter regardless of the pressure or the volume of injection. Recombinant cell lines expressing intracellular EGFP were established following microinjection into the nucleus and the cytoplasm with linear and circular DNA. Selection with puromycin, clonal expansion, and cell banking were completed within three to four weeks post-microinjection. In parallel, recombinant cell lines were established after calcium-phosphate transfection with linear and circular DNA. The number of plasmid copies integrated was determined by Southern blot and the number and localization of integration sites was determined by fluorescence in-situ hybridization (FISH). Growth of the cells and stability of EGFP expression were investigated by cultivation over a two-month period. This approach presents several advantages over other DNA delivery methods such as control of the quantity and localization of DNA delivered into the cell and speed of recovery of recombinant cell lines. This approach may be relevant for the establishment of recombinant cell lines from cells that are not easily transfected by classical methods.

EPFL2005

A novel cell culture approach for tissue engineering based on surface expansion

Hicham Majd

Most tissue engineering approaches include the delivery of reparative cells to a damaged host tissue. Generally these cells are sought to be of a stem cell character since they retain a high potential for proliferation and differentiation into diverse phenotypes. To restrict cell function to the desired phenotype and to avoid anti-immune responses, autologous stem cells are frequently isolated from intact regions of the same (damaged) organ, such as keratinocytes from the basal layer of the skin and hair follicles or from the same germ layer origin such as mesenchymal stem cells (MSC) that are obtained from the bone marrow. This approach faces two major problems: 1) The number of source cells obtained from biopsies is generally too low for direct implantation, which requires cell multiplication in culture. 2) The standard culture conditions, including growth in plastic dishes, passaging, and the composition of the growth medium, never correspond to the natural microenvironment and influence cell differentiation, often resulting in non-desired phenotypes. The objective of my thesis was to provide a solution for both problems that is applicable for the average cell culture laboratory. Our technique reduces traumatic enzymatic passaging, provides constant cell densities to keep cells in the exponential growth phase and at the same time avoids contact inhibition of cell growth. This novel approach comprises the development of a new elastic culture surface, produced from a high extension silicone rubber (HESR) that we here for the first time applied for cell culture. The HESR is enlarged by a novel mechanical approach permitting to visualize and control cells under expansion. Untreated silicone, the polymer component of the HESR, does not promote cell attachment and must therefore be functionalized. In the first part of my thesis (Chapter 2) we establish a surface treatment for silicone membranes that will resist large surface expansions. We compare established coating techniques that have originally been developed to provide static silicone surfaces with cell adhesive molecules by 1) hydrophobic, 2) electrostatic and 3) covalent interactions. Cell attachment, spreading and proliferation was evaluated between these three treatments as a function of the coating in static and stretched conditions. We show that covalent immobilization of collagen type I is most suitable to stretch cells cultured on elastic silicone substrates. We applied this technique to functionalize the HESR culture surface in all later applications. The second central part of my thesis (Chapter 3) develops a new culture method consisting of a motorized device that isotropically expands the coated HESR surface to yet unmatched 1000% of its initial surface. Proof-of-principle of this method is given by culturing human MSC, which are sensitive to passaging during expansion in vitro and that are therapeutically relevant cells to repair bone, cartilage, and cardiovascular tissue. MSC were cultured three consecutive passages on the culture surface that has been expanded in steps of 5% every 6 h for 20 days, respectively. After nine weeks of expansion culture, MSC exhibited consistently high proliferation rates, fully retained their pluripotent stem cell character and were inducible to differentiate into adipogenic, chondrogenic, osteogenic, and myogenic lineages. The cell surface expansion culture surface produced ∼10-times higher cell numbers compared with nine weeks of standard culture, which required nine events of passaging to attain the same culture surface. We propose our method to produce MSC for clinical cell therapy. In the third part of my thesis (Chapter 4) we implement our novel surface expansion method to culture fibroblasts with the aim to: 1) rapidly expand primary fibroblasts, 2) achieve higher cell yields than obtained with conventional culture, and 3) obtain homogeneous cell populations with non-fibrogenic character. The expansion of primary autologous dermal fibroblasts in culture is a pivotal step to obtain sufficiently high cell numbers for application in skin esthetics and for the treatment of large burn wounds. Dynamically enlarging the culture surface by 800% over 20 d, yielded ∼1.5 higher fibroblasts numbers than the equivalent three standard passages. By further optimizing the surface expansion speed according to the cell growth rates, the same high cell numbers were produced after only 9 d of culture. The highly compliant nature of the HESR surface had the beneficial effect of completely abolishing the formation of 'fibrotic' myofibroblasts. Hence, our novel method for fibroblast culture is faster and yields higher numbers of more homogeneous cells than standard culture passaging. It delivers a fibroblast quality and quantity that is appealing for cell therapeutic purposes. The aim of the last part of my thesis (Chapter 5) was to provide highly compliant silicone elastomers, such as the HESR, with a micropattern of cell adhesive proteins. In particular we aim to produce arrays of extracellular matrix proteins with a size and morphology to promote formation of single focal adhesions on highly elastic and expandable surfaces. This will allow future studies on cell mechanosensing through focal adhesions by discriminating between effects of focal adhesion size changes and stretch. An existing method to pattern rigid surfaces with proteins, so-called microcontact printing, is not suitable to transfer proteins onto surfaces that are softer than the stamp. We here establish a new transfer technique that is based on a stencil with micron-sized openings. The new stencil is used to expose the surface of compliant silicone substrates with Young's modulus of 10-100 kPa to a protein solution of fibronectin, collagen I and protein-repellent polymers, leaving behind the desired protein pattern. Fibroblasts were successfully attached onto the resulting focal islet arrays on compliant substrates.

EPFL2008

Establishment of recombinant mammalian cell lines

Madiha Derouazi

EPFL2005

A novel cell culture approach for tissue engineering based on surface expansion

Hicham Majd

EPFL2008

Polymeric carriers for therapeutic agents

Yun Suk Jo

EPFL2008