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Nanoparticles as non-viral vectors for gene delivery
Gene therapy refers to the transfer of genetic material encoding a therapeutic gene of interest into a cell, tissue, or whole organ with consequent expression of the transgene in order to treat a disease. However, for the success of gene therapy, the development of sophisticated and efficient delivery systems capable of transfering genes is a key factor (3,13). In brief, we can describe two classes of efficient nucleic acid carriers: viral and non-viral vectors. These delivery systems should protect nucleic acids from degradation, while providing a safe intracellular delivery (14). Over a decade ago, patients with immunodeficiency-X1 were treated with gene therapy assays based on the use of cDNA in a retrovirus (15,16). After that, gene therapy based on retrovirus vector was used to treat French patients with T cell leukemia, producing aberrant transcription and expression of LMO2 (17). These facts opened an optimistic vision about gene therapy research. Nowadays, gene therapy clinical trials are present in worldwide, mainly in United States of America (62.6%), United Kingdom (10.3%) and Germany (4.1%). These therapies are usually used in the treatment of cancer (63.8%), monogenic diseases (8.9%) and infectious diseases (8.2%) (18). The most commonly vectors used in gene therapy are still virus vectors, highlighting adenovirus (22.5%) and retrovirus (18.8%), but also lipofection (5.2%) and naked pDNA (17.5%) are raising their use (18). Notwithstanding the high efficient transfection provided by viral vectors, they can invoke immune responses or proto-oncogene activations. In this context, the non-viral vectors, particularly cationic liposomes, have a promise and potential future, taking into account their reproducibility and safety of use (5). Cationic liposomes are non-viral vectors mainly composed of cationic lipids, which guarantee their positive superficial charge. Examples of synthetic cationic lipids used in cationic liposome composition are DOTAP (1,2-dioleoyl-3- trimethylammonium propane), DOTMA (2,3-bis(oleyl)oxypropyl-trimethylammonium chloride), DDAB (dimethyl dioctadecyl ammonium bromide), DC-Chol (3 β [N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol), DMRIE (N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide), DOGS (dioctadecyl amino glycyl spermine), DOSPA (2,3 dioleyloxy-N-[2(spermine carboxaminino)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate). However, the use of cationic lipids can generate cell toxicity, and so the inclusion of others lipids, e.g. egg phosphatidylcholine (EPC), in cationic liposome composition is required to decrease the cytotoxicity (19,20). On the other hand, the positive charge of cationic liposomes provides an electrostatic interaction with negatively charged nucleic acids, forming complexes with net positive charge (21). Hence, these positive charged complexes can enter easily inside cells, whose surface membrane is negatively charged (22). Once into cells, the complexes should release the nucleic acids in cytosol, in case of RNA for rapid synthesis of a target protein (23), or in cell nucleus, in case of DNA to control gene expression for a more long-term. The inclusion of helper lipids, e.g. phosphatidylethanolamines (DOPE), in cationic liposomes composition facilitates the nucleic acid to escape inside the cytosol (24). Felgner et al. (22) were pioneers in the development of cationic liposomes for gene delivery. They produced liposomes composed of 1:1 ratio of DOTMA and DOPE, which were then commercialized as Lipofectin. For example, these cationic liposomes were used in vitro to carrier efficiently hepatitis C virus proteins into a human hepatocyte cell line (HUH7) (25) and in vivo to delivery linamarase gene for the treatment of brain tumors in animals or humans (26). Recently, other commercial cationic liposomes, such as Lipofectamine, DMRIE-C, Oligofectamine, Ambion, 293fectin, Optifecta, Invivofectamine, FuGENE, TransFast, TransFection and CLONfectin, are being used in gene therapy due to their well-established protocols and to provide high efficiency of transfection in some specific cells and with some specific nucleic acids. Our research group (27) also showed the feasibility of dehydrated-rehydrated liposomes composed of EPC, DOTAP and DOPE (50/25/25% molar, respectively) carrying polynucleotides encoding HSP65, for prevention and treatment of tuberculosis. And more recently, we obtained the same cationic liposomes produced in a large scale by ethanol injection method to delivery nucleic acid into dendritic cells (DCs) as a potential tool for cancer immunotherapy (28).
Dendritic cells are professional antigen-presenting cells widely used in immunotherapeutic approaches, particularly in immunotherapies against cancer. Since loaded with tumor antigens, mature DCs can induce an immune response against cancer by recruiting patients’ immune system (29). Different strategies are currently used to load DCs with antigens, e.g. peptide pulsing, pulsing with tumor cell lysates, infection with viral vectors, direct nucleic acid loading, or ingestion/fusion with tumor cells (30). Moreover, cationic liposomes stand out in this function as gene carriers, since besides to transfect DCs, they can also activated them (31). Particularly, our research group (32,33) showed that cationic liposomes EPC/DOTAP/DOPE were uptake by DCs, while providing cells stimulation/activation. However, dendritic cells require that these cationic liposomes have a very specific properties (size < 100 nm and polydispersity <0.2) to be internalized (32), due to the use of macropinocytosis and/or phagocytosis as transfection pathways (34). Thus, the study of methodologies that enable to form lipoplexes modulated to specific cells lines in a reproducible and controllable way is very important (35). Conventional methods of obtaining lipoplexes involve just the mixing provided by hand shaking or vortexing to reach cationic liposomes complexation with nucleic acids. However, these conventional methods introduce variability in lipoplexes formation and, as a result, provide inconsistent transfection efficiencies (6). To effectively transfect cells, the physico-chemical properties of lipoplexes should be suitable to the pathway used by the cell line to internalize nanoparticles (36).
Microfluidic droplet technologies
The general concept of microfluidics is in the manipulation of small amounts of reactants inside microchannels with the capability to control and manipulate molecules in space and time (37). In microfluidics it is possible to work with small amounts of reactants, the period of reactions is short, it is possible to work with parallel operations (38), large surface to volume ratio allows fast diffusion of compounds and fast mass/heat transfer (39). These advantages can be related to the flow in microfluidic devices, which is laminar and corresponds to a low Reynolds number (37,40). Another important consequence of this regime is that the mixture between two parallel flowing streams occurs mainly by diffusion (38).
The origin in microfluidic is in the 90´s for MicroElectroMechanical Systems area (MEMS) (39). Nowadays microfluidics has been exploited in numerous areas as: Biological analyses – detection of biomolecules (41,42), manipulation and amplification (43) and separation of DNA by capillary electrophoresis (44).
Microbial growth – Screening of variables and kinetic parameters (45,46). Nanoparticles production – polymeric particles (47,48), liposomes and lipid vesicles (49–51), metallic nanoparticles (52). Gene Delivery/Transfection – electroporation (53), hydrodynamic force and optical energy (8). Different materials can be used for the construction of microchannels and, for biological application, glass and polymers are detached (54). Glass is considered to be biocompatible, impermeable to gases (54,55), has physic and chemical stability and it is hydrophilic (54). The techniques to prepare microdevices in glass are laser ablation and wet etching (54). Microdevices can also be made by polymers which are not expensive, there is the possibility to change the chemical formulation (55), are stable (37) and hydrophobic (54). The elastomer which has been used extensively for microdevices construction is poly(dimethyl syloxane) well-known as PDMS , moreover the technique employed is soft lithography (56). In addition, it is important to consider the wettability related to the microchannel material and the droplet system. Since the continuous phase wets the walls faster than the disperse phase and forms a thin film between droplet and walls (57). The droplet breakup occurs when the continuous phase wets the device walls instead the disperse phase, thus the droplet morphology is a result of the interaction between the material which the devices is formed and the continuous phase (57). Summarizing hydrophobic channels are required to prepare water in oil systems, inversely hydrophilic channels are necessary for forming oil in in water emulsions (58). Considering the advantages of using PDMS for devices construction, in the literature different strategies are reported to change the wettability of the material, through chemical modifications (58,59).
Microfluidic systems can be classified according to the interfaces created by the fluid flows in microchannel as: pinned interfaces, in which fluids flow in parallel creating vertical interfaces, and floating interfaces, in which immiscible fluids produce droplets of precise shape (60). The use of segmented flow which the reactants are separated in different picoliter/nanoliter droplets has been used in cases when miniaturized systems has to be achieved (61). The segmented flow is the principle of emulsions which are a metastable colloidal systems (62) with two immiscible liquids. In this case there is one continuous phase and one disperse phase in droplets formats (57,61). Some advantages of droplet processes can be described in comparison with parallel flows processes. In parallel flows, in which solute are all distributed over the solvent, the efficiency of chemical reactions and the detection of some molecules inside the channels can be decreased, this phenomenon is called Taylor-Aris dispersion (63,64). The use of droplets processes cut out the contact with solid wall, reducing the probability of reagents adsorption into the channels walls (64). Droplets with samples inside can be seen as micro-reactors which allows the manipulation of small volumes (14,65). In addition, in droplets microfluidics it is possible to carry out many reactions without increasing the number and the channels size (13). Furthermore, considering the relation between the surface area and volume, the reactions inside droplets are faster because the heat and mass transfer times and also diffusion distances are shorter (13,66).
Droplet-based microfluidic platforms for lipoplexes formation
The control and rapid mixing of reagents within droplet-based microfluidic systems (micromixers) can be used, for example, to make an effective complexation between nucleic acids and liposomes in order to form lipoplexes (35). Microsystems using parallel flows, the mixing is promoted mainly by the diffusion of reactants (31). In contrast to this, microdroplets devices can add a chaotic advection contribution to the system, increasing the promoted mixing. For example, serpentine channels can generate two recirculation flows within droplets due to the shear created between the droplets and channel wall (31) (Figure 2.1). For this, droplets should have a precise size to touch the inner surface channels and generate a fluid movement relative to the stationary walls of serpentine channel (10). On the whole, micromixers can be classified as passive and active. The passive micromixers require the use of different microchannel geometries and/or liquid flow rates to generate mixing (54). On the other hand, active micromixers demand an external energy to enhance the mixing, such as pneumatic or mechanical vibration (71). Active mixers require more complex fabrication processes and they are more difficult to integrate with other microfluidic components. The passive mixers usually adopt longer mixing channels without external agitation (72). Nevertheless, some parameters of the system have to be taken into account to provide an effective mixing within droplets. One of the most important parameter is capillary number (Ca), which should be low in order to form droplets in the system (73). The Ca is proportional to the average flow velocity (U) and inversely proportional to the interfacial velocity (γ/μC, where γ is the surface tension between two immiscible phases and μC is the dynamic viscosity of continuous phase). Hence, when the flow velocity is much lower, Ca is reduced and the surface tension controls the system. In high Ca, the shear force dominates (73). As consequence, the viscosity of fluids is another parameter that interferes in interfacial velocity and in droplets mixing (74). Tice et al. (74) concluded that combination of viscous and non-viscous fluids promotes a more efficiently mixing inside droplets than the use of only non-viscous fluids. Thus, the use of fluids with approximated viscosities decreases the interfacial velocity of the system, inducing a decrease in droplets mixing (74). Moreover, the channel configuration is also fundamental in a good mixing within droplets. The channel width is often used to control the local flow velocity, when the channel depth and flow rate being kept constant (75). Furthermore, microsystems should be operate in a Peclet number between 1000 times and 100,000 times greater than Reynolds for a good mixing (76). According to the Peclet number, it is possible to determine if the convection mixing (UL, where L is a characteristic length scale) or the diffusion mixing (D, where D is a characteristic diffusion coefficient) dominates in the system (77).
Besides setting operational parameters in the droplet microfluidic system, to achieve a chaotic mixing in passive micromixers, especial geometries, such as serpentine channels outlined in Figure 2.1, can be adopted (77). Chaotic advection provides an accelerated mixing within droplet-based microfluidic devices, stretching and folding the fluid into droplets as long as they pass in these channels (40). Droplets moving downstream and upstream in serpentine channels offer an alternating motion time periodically influenced by the walls, creating fluid vortexes (40). In brief, the mechanism can be explained as follows: the part of droplet in contact with the outside arc of the channel prompts greater contact between the interface of the droplet and the channel wall, leading to a longer recirculation flow; on the other side, part of droplet in contact with the inner arc of the channel prompts smaller shear, leading to a smaller recirculation flow (13). This process repeats along the channel, in such a way that recirculating flows vary alternately on each side of the drop, generating a chaos within it (13). Passive micromixers with chaotic advection have a promising future in microfluidic field, since they allow an effective mixing on a millisecond scale without requiring moving parts in devices (76). The chaotic mixing in droplet-based microfluidic devices have various applications such as: controlling chemical reactions (40), promoting protein crystallization (73) and improving biochemical analysis (78). Among the applications, we can emphasize the complex formation between nucleic acids and liposomes (Figure 2.1) as an essential step in gene delivery process.
Table of contents :
ACKNOWLEDGEMENTS
FIGURE CAPTIONS
TABLE CAPTIONS
NOMENCLATURE
SUMMARY
Chapitre I – Introduction
1. Introduction
2. Objectifs
3. Organization de la thèse
4. Références
Chapter II – Literature review
1. Introduction
2. Nanoparticles as non-viral vectors for gene delivery
3. Microfluidic droplet technologies
4. Droplet-based microfluidic platforms for lipoplexes formation
5. Droplet-based microfluidic platforms for in vitro transfection
6. References
Chapter III – Droplet Microfluidic-Assisted Synthesis of Lipoplexes for DC Transfection
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusion
5. Acknowledgements
6. References
7. Supplementary data
Chapter IV –Tracking the Heterogeneities of CHO Cells Transiently Transfected on a Chip
1. Introduction
2. Materials and methods
3. Results and Discussion
4. Conclusions
5. Acknowledgments
6. References
7. Supplementary data
Chapter V – Conclusions
Chapter VI – Perspectives
ANNEX I – Plasmid vectors
ANNEX II –Preliminary studies in droplet microfluidic system to synthesize lipoplexes
1. Study of flow rates for droplets formation
2. Cationic liposome diluent
3. References
ANNEX III – Preliminary studies in droplet microfluidic system to transfect CHO-S cells
1. Objectives
2. Materials and methods
3. Results and discussion
4. Conclusions
5. References .
