Downloaded via BETHEL UNIV on March 24, 2019 at 00:36:01 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Chapter 2
Aerosol Delivery to the Airways Using Cationic Lipid Nanocomplexes in a Perspective of Cystic Fibrosis Treatment Mathieu Berchel,1 Paul-Alain Jaffrès,1 Tony Le Gall,2 and Tristan Montier*,2 1CEMCA
UMR CNRS 6521, Université de Brest, IBSAM, 6 Avenue Victor Le Gorgeu, F-29238 Brest, France 2UMR INSERM 1078, Université de Brest, Faculté de médecine et des sciences de la santé, CHRU de Brest, IBSAM, 22 rue Camille Desmoulins, F29609 Brest, France *E-mail:
[email protected].
Diseases affecting the respiratory tract are numerous and many are disabling or deadly. Nucleic acids are emerging as a new tool and a new therapeutic class of compounds that could be used to address some lung diseases. However, considering the different anatomical barriers and the physicochemical behavior of nucleic acids in the blood stream, lungs are not easy to treat following a general administration. In this context, aerosol delivery is largely considered as an adapted and noninvasive approach avoiding the hepatic metabolism. This local delivery leads to a lung concentration and limits eventual side effects associated with a systemic injection. However, aerosol administration of active molecules is a real challenge due to the physical, chemical, and biological obstacles, such as respiratory movement, surfactant, or bacterial strains. Nanocomplexes have the potential to increase bioavailability and favor intracellular penetration of specific drugs into the pulmonary tissue. In this chapter, we will present our strategy to develop efficient gene carriers that, from a synthesis point of view, can be prepared on a large scale. We will discuss their use in preparing various formulations and their progressive
© 2019 American Chemical Society Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
adaptation to the airway constraints. In particular, we will focus our attention on cationic amphiphilic complexes used for aerosol gene delivery and we will present promising formulations in preclinical studies in a cystic fibrosis context.
Anatomy of Lungs and Airways The upper airways (oronasal cavities, pharynx, and larynx) and lower airways (trachea, bronchi, and segmental bronchi) form the respiratory tract. The upper airways ensure the filtration, the heating, and the humidification of the air we breathe, but the key role of the respiratory tract is to perform efficient gas exchanges between the air and the blood, which is ensured at the pulmonary alveoli stage. The lower airways are structured like a tree. The trachea, corresponding to the trunk (with a diameter of 13 to 15 mm and 100 to 150 mm lengthways), divides into two main branches (right bronchus: diameter of ~ 15 mm and ~ 2.5 cm in length and left bronchus: diameter of ~ 11 mm and ~ 5 cm in length), which divide into segmental bronchi. At the extremity, the bronchioles (diameter of less than 1 mm) end with the pulmonary alveoli where the gas diffusion is performed. Most of the time, the decrease in respiratory function is associated with repeated aggression on the respiratory tract. For example, smokers or Chronic Obstructive Pulmonary Disease patients inhaled toxicants that gradually destroyed the pulmonary parenchyma, leading to a reduction in gas exchange and giving rise to an increase in the O² partial pressure. In Cystic Fibrosis (CF) patients, repeated cycles of infection and inflammation induce a progressive fibrosis of the pulmonary parenchyma and a decrease in respiratory function measured trough the Forced Expiratory Volume in 1 seconde (FEV1). The epithelial structure of the airways varies depending on the section. The bronchi correspond to a pseudo-stratified epithelium. In the bronchioles, the epithelium is cylindrical and then evolves into a cuboidal form. The tracheobronchial epithelium (trachea and bronchi) comprise ciliated cells allowing mucociliary clearance, globlet cells, and basal cells. The ciliated cells allow the elimination of pollutants trapped on the surface’s liquid, which covers the epithelium. This Airway Surface Liquid is composed of a periciliary layer and a mucus layer. The periciliary layer has a lower viscosity than the mucus layer (1). On the other hand, the mucus is composed of salts, proteins (glycoproteins, mucins, mucoproteins), and water (2). It is secreted from different kind of cells (globlet cells in the trachea and clara cells in the bronchioles). The level of hydration in the surface liquid is a function of the ionic transports (chloride ions and sodium ions in part). Some diseases, like CF, induce a misregulation or misexpression of the channels involved in ion transport, resulting in dehydration of the surface liquid and a defect in mucociliary clearance (3) progressively obstructing the luminary of the bronchi. As an additional consequence, this hyperviscous mucus constitutes a favorable environment for the development of microbial infections. 36 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
To treat epithelial cells, several obstacles have to be overcome during their transit from the upper airways. In addition to the movements induced by respiration, some extracellular barriers are facing into the lungs and are a deadly trap for inhalation treatments (4). The mucus is a natural barrier and its role is to purify breathing air by trapping inhaled particles. In a healthy person, the thickness of the mucus is around 5–10 μm and is renewed every 10 to 20 min(5). Depending on its rheology and composition, mucus also represents a sticky barrier against inhaled therapies (6). The mucosal structure allows the passage of particles around 100 to 200 nm in size (7). In some obstructive diseases, the mucus is highly viscous and the mesh is tighter (8). In addition to its high mucin concentration, mucus contains many other anionic molecules such as cellular dust, DNA fragments, etc. Due to their charge, they can also complex inhaled drugs and limit their activity (9). For nonviral gene therapy, this facilitates the concentration of negative charges that will contribute to destabilization of the nucleic acids/vector complexes. Additionally, there is a second surface liquid called a pulmonary surfactant that is present on the inner surface of the pulmonary alveoli, limiting the energetic expense and facilitating respiratory movements. This surfactant can also capture active principles. It is produced by the pneumocytes type II. It contributes to the reduction of the air/liquid surface tension on the alveoli facilitating respiration. It also acts as a gatekeeper in immune defense. It comprises 90% lipids (mainly dipalmitoylphosphatidylcholine) and 10% proteins (10). In newborns, mostly premature infants, a deficiency in pulmonary surfactant leads to respiratory failures. In adults, frequent alterations of the pulmonary surfactant are reported. They can occur as a result of drowning as well as acute respiratory distress syndrome. There are different exogenous surfactants that can be delivered endotracheally (11). Then, a natural defense system based on mucociliary clearance participates in the elimination of inhaled toxicants (12). As mentioned previously, during inhalation the particles are trapped in the mucus. The cilia present on the surface of the respiratory epithelium beat simultaneously at a frequency of 1000 to 1500 beats per minute and drive the mucus up to the trachea. The rate of upward movement of the mucus is between 5 and 20 mm/min. Once in the trachea, the mucus is eliminated by the digestive tract or by expectoration. As a consequence, mucociliary clearance is a route for rapid elimination of inhaled drugs. It is therefore necessary that these therapeutic drugs do not remain trapped in the mucus to avoid their quick elimination. Additionally, a pulmonary microbiota exists in the lower respiratory tract which had long been considered sterile, even in healthy individuals (13), and is likely present from an early age. It varies from one individual to another, depending on age and health status. Some pathologies, like Chronic Obstructive Pulmonary Disease, CF, asthma, or cancer, lead to an imbalance of this flora, favoring the progressive development of specific pathogens (14), and some bacteria responsible for lung infections produce enzymes capable of degrading drugs such as antibiotics. Bacterial strains can grow planktonically or as biofilms. The processing from planktonic bacteria to biofilm is conducive to a tolerance of treatments (15). Where 37 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
there exists a biofilm, the penetration of the active drug is deeply reduced due to the composition of the matrix formed, mainly of polysaccharides, proteins, nucleic acids, and lipids (16). In a biofilm, the proximity of the bacterial strains favors the dissemination of resistance by horizontal transmission of the genes. Finally, within a biofilm, part of the bacteria is dormant. This state of low active metabolism limits the efficacy of some antibiotics (17).
Aerosol Delivery: Rationale and Challenges Rationale Preclinical evaluations of nonviral vectors can be conducted using systemic administration methods with intravenous (iv) injection. In mice, tail vein injection is indeed an easy, commonly used delivery method. It has been employed on many occasions, facilitating high transgene expression in the lungs, not only when using luciferase-encoding plasmid DNA (pDNA) (18, 19) but also a therapeutic Cystic Fibrosis Transmembrane conductance Regulator CFTR-encoding pDNA (with very high levels of CFTR mRNA in lung homogenates and CFTR-positive alveolar pneumocytes; unpublished results). However, although iv injection is a practical way for identifying efficient in vivo gene carriers for transfection of the gas-exchange airways, its clinical relevance in CF appears unrealistic since the mechanism of transfection relies on a transient embolism at the pulmonary microvasculature level. Since the main cause of death in CF now results from progressive lung destruction, due to thickening of the airway mucus leading to repeated infections and chronic inflammation (20), the most important target is the epithelium of the conducting airways. Accordingly, airway administration appears to be the most relevant delivery route because it is the most direct access and it is also the less invasive in the CF delicate respiratory clinical context. Over the past decade, a clinically relevant strategy regarding lung gene therapy has been developed by the U.K. CF gene therapy consortium, especially by the Oxford Gene Medicine group (Drs. Deborah Gill and Steve Hyde) with whom we have developed a fruitful collaboration. This provided encouraging results (21) and allowed for the election of the GL67A formulation for clinical CF gene therapy studies with a multidose clinical trial that was completed in 2015 (22). Aerosol Delivery: Principle The aerosol delivery procedure involves administrating a formulation as an aerosol to the airways. Typically, this formulation consists of a pDNA mixed with a synthetic carrier and other components (see Evaluation Following Aerosol Delivery). To convert a liquid formulation into an aerosol, a strong air current is applied over its surface to snatch very small droplets. These droplets then form an aerosol that can be inhaled by the subjects, settling homogenously over the entire airway epithelium. Compared to the iv injection procedure, the aerosol delivery allows a more direct interaction between the DNA complexes and the airway epithelium (23, 24). This bestows an aqueous solution on the epithelium without flooding the airways. Moreover, this prevents the complexes from being 38 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
misdirected into the esophagus because no deglutition reflex is induced, hence primarily focusing the administration to the airways. Another advantage is that aerosol delivery results in very little trauma for the subject; as less stress is induced, results are more reliable. Regarding preclinical studies, this procedure can be used with minor modifications to treat animals within experimental conditions in full accordance with the “3R rules” in animal experimentation. From an experimental point of view, some specific materials are required, as depicted in Figure 1. The aerosol obtained is driven into an exposure box where it accumulates in the form of a mist, which can be breathed by animals housed within the box. Only a small part of the solution stored in the nebulizer reservoir actually reaches the animals’ lungs. Most of the aerosol collects on the wall of the box or over the fur of the mice. However, no alternative solution is currently available, and this protocol remains the best preclinical method to evaluate original synthetic formulations.
Figure 1. Aerosol experimental setup. A nebulizer (PARI LC plus, PulmoMed; inset) stores the formulation to be aerosolized. It is connected to a compressed air outlet and to a transparent box in which a group of mice can be housed simultaneously.
Typically, 20 to 25 mice are simultaneously treated, to create several groups that are analyzed at different points of time post-aerosol and to get statistically significant results (as some variability inevitably occurs between animals). Very large quantities of cationic lipids and DNA are needed to conduct a single aerosol experiment. This amount is close to what is needed to treat a human subject; 39 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
for instance, approximately 130 mg of monovalent cationic lipid are required to complex, at charge ratio (cationic lipid/DNA) 2/1, the 25 mg of pDNA needed. This huge quantity of materials is contained in 10 mL of solution. Of note, the nebulizer has a death space (up to 2 mL), meaning that only 80% of the initial formulation is effectively aerosolized. Thus, such experiments are quite expensive, implying that high quantities of both DNA and cationic lipids are available.
Evolution of Cationic Lipids To assess the transfection efficacy of new cationic amphiphiles and to deal with the large quantities of cationic lipids that are needed for aerosolization, the cationic lipids must be either commercially available or readily prepared in quite large quantities. To this end, we have developed bio-inspired cationic amphiphiles that have the capacity to compact pDNA and to transfect eukaryotic cells (25). As shown in Figure 2, our cationic amphiphiles are composed of a phosphorus functional group that links together two lipid chains and one cationic polar head group. Interestingly, the synthesis procedure that requires only a few synthesis steps (3 to 5) can be achieved on a multigram scale and the synthesis schemes are very versatile. It can be emphasized that some of these cationic amphiphiles are similar to natural phospholipids, exemplified by compounds 1–5 (Figure 2), that possess a polar head group which is closely related to the naturally occurring phosphocholine moiety. The main difference between these series of cationic lipids and natural phospholipids arises from the absence of glycerol units in their structures. This difference makes their synthesis easier because the incorporation of a glycerol unit into the structure of amphiphiles usually requires additional protection/deprotection steps (26). This versatile mode of synthesis leads us to introduce structural modulation with the aim of identifying the most efficient molecular structures to carry nucleic acids. For the transfection efficacies evaluation, in vitro assays were set up on different cell lines, including lung epithelium cell lines (e.g., 16HBE), since it is the cheaper way to discriminate the transfection potential of new cationic amphiphiles that could subsequently be used for in vivo transfection assays. Our modulation of the structure of cationic amphiphiles focused on the phosphorus function (phosphonate 1 (27), phosphoramide 2 (28, 29), thiophosphoramides 3 (30, 31), and phosphate 4–5 (32, 33)). These modifications were motivated by the goal to identify cationic amphiphiles that feature a compromise in terms of molecular stability. Indeed, the cationic amphiphiles must be sufficiently robust to face all possible degradation before cell internalization. However, after entry into the cell, the degradation of the cationic amphiphiles is highly desired to favor the plasmid release but also to avoid or to reduce toxicity. Accordingly, the initial work with phosphonate function was followed by the incorporation of a phosphoramide or a phosphate group, which are likely more adapted structures, to avoid accumulation of such types of molecules inside the cell after transfection.
40 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Figure 2. Molecular structure of synthetic phospholipids featuring a phosphonate (1), a phosphoramidate (2), a thio-phosphoramidate (3), an alkyl phosphate (4), or an aryl phosphate (5). Our investigation has focused on the modification of the polar head groups and on the structure of the hydrophobic domain. These two structural features are crucial to the transfection efficacies and, as reported later in this chapter, some modifications induced additional properties, such as bactericidal action or induced major supramolecular structure changes. The cationic polar head group of cationic amphiphiles is one of the key structural features since it plays an important role when the cationic amphiphiles interact with the negatively charged phosphate groups of nucleic acids (e.g., pDNA and mRNA). This ionic interaction, which aids the compaction process of nucleic acid with cationic amphiphiles, is a dynamic and reversible phenomenon influenced by the ionic species present in the surrounding medium and governed by enthalpic and entropic factors (34, 35). The strength of this interaction is also governed by the nature of the cation involved. In this regard, we suggested that the size and the possible cationic charge delocalization should deeply influence the strength of this ionic interaction (27). This hypothesis constitutes a possible point of structural modulation that is worthy to be explored for two 41 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
reasons: (1) A compromise in ionic strength interactions must lead to a desired compromise in stability for lipoplexes. Indeed, if stability of the supramolecular assemblies (lipoplexes) is needed outside of the cells, nucleic acid release after endocytosis is required to avoid degradation of genetic materials in lysosomes. To this end, a compromise of ionic strength interaction should contribute toward developing efficient cationic amphiphiles for gene delivery that, in a perspective of administration by aerosolization, must also support shear forces involved during aerosolization. (2) The interaction of cationic amphiphiles with anionic amphiphiles naturally present in any cells can induce clustering of lipid membrane and, therefore, membrane destabilization that can induce cytotoxicity. Once more, the modulation of these ionic interactions is likely a parameter that could induce positive effects with respect to cell cytotoxicity. The synthesis scheme used to prepare lipophosphoramides is permitted to easily introduce different types of polar head groups. As summarized in Figure 3, the two- to three-step procedure starts with an Atherton-Todd reaction (36) followed either by an alkylation of the amine (ammonium) or by a reaction with trimethylphosphine, trimethylarsine, or N-methylimidazole to produce the ammonium 2 (37), the phosphonium 6 (38), the arsonium 7 (KLN47) (18), or the imidazolium 8 (39), respectively.
Figure 3. Synthesis route for the preparation of lipophosphoramides with different cationic polar head groups.
The lipophosphoramide-arsonium 7 was identified as an efficient cationic amphiphile for gene delivery in vitro (40) and in vivo (18) that, interestingly, features a reduced cytotoxicity when compared to its ammonium equivalent 2 (27). This cationic lipid 7 (also named KLN47) is now a gold standard for our laboratory since it is efficient on a large variety of cell lines and is also efficient in vivo. As reported previously, the different transfection efficacies and cytotoxicity between cationic amphiphiles possessing a trimethylarsonium or a trimethylammonium polar head group are likely ascribed to the larger size of the trimethylarsonium fragment that likely reduce the strength of the ionic 42 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
interactions (27). The incorporation of an N-methylimidazolium polar head group that featured a delocalized positive charge also produced efficient cationic amphiphiles. For instance, compound 8 was associated with cationic polymers to design lipopolyplexes that were employed for tendon healing (41) and for the development of a cancer vaccine based on the delivery of mRNA or pDNA in dendritic cells (42, 43). Recent preclinical assays demonstrate the pertinence of the strategy and the efficacy of lipopolyplexes for this application (44). For this application, we also developed a simple synthesis protocol based on the reactivity of imine with lipophosphite to design cationic amphiphiles (45). This simple synthesis protocol offers a variety of cationic amphiphiles. Some of them were efficient for pDNA delivery in dendritic cells. The hydrophobic part of cationic amphiphiles is the second essential structural element that influences the transfection efficacies. The supramolecular hydrophobic interactions involved between the lipid chains of cationic amphiphiles induced a segregation between the hydrophobic parts and the hydrophilic parts of cationic amphiphiles that are directly involved in the compaction of nucleic acids. Consequently, the auto-assembly of cationic amphiphilic compounds, governed by hydrophobic interaction, plays a crucial role in compaction of nucleic acids. Compaction of pDNA by cationic amphiphiles must be viewed as a dynamic and a reversible supramolecular process. In this regard, the structure of the lipid domain has a strong impact on this dynamic process and, consequently, on the transfection efficacies. Indeed, robust supramolecular packing of hydrophobic moieties, which is observed with long saturated alkyl chains (e.g., stearic lipid chains C18:0), reduces the dynamic of the supramolecular interaction. This feature is desired when the lipoplexes are localized outside of the cells because it strongly protects the nucleic acid up to its target cell. However, after cell internalization that usually occurs by endocytosis pathways (46), the existence of a robust supramolecular packing is counterproductive because the nucleic acids can get stuck in the endosome due to a limited lipid mixing between the cationic amphiphiles of the lipoplexes and the amphiphilic compounds of the endosomal membrane. To illustrate this structural feature, we have shown that the cationic lipophosphoramidate 9 (Figure 4) possessing two stearyl lipid chains was almost inefficient to deliver pDNA (in vitro assays) whereas its bis-oleyl analogue 2 was efficient (47). It can therefore be concluded that the incorporation of Z-unsaturation within the lipid chain that creates a kick in the hydrophobic domain reduced the van der Waals interactions in the supramolecular packing and thus induced higher fusion properties as observed by Förster resonance energy transfer experiments (48). This thinking relative to the incorporation of a structural parameter in the hydrophobic domain that induced disorder in the supramolecular packing can be extended by hypothesizing that the length of the lipid chain, the presence of ramified lipid chains, or the presence of two different lipid chains are structural elements that will induce disorder within the hydrophobic domain and therefore will limit the interaction forces and the supramolecular cohesion. Consequently, such types of molecular modulations must have an impact on the transfection efficacies. The versatile synthesis scheme of phosphoramidates leads us to explore all of these possibilities. First, the introduction of polyunsaturated lipid 43 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
chains (linoleyl lipid chains C18:2) lead us to prepare the lipophosphoramide 10 (Figure 4) (19). This compound proved to be more fluid than its oleyl equivalent 7. Surprisingly, compound 10 was less efficient in vitro but more efficient in vivo (iv injection). If the incorporation of two unsaturations within the lipid chains (linoleyl chains) produced efficient cationic amphiphiles for the delivery of pDNA, the extension of this concept by the incorporation of additional unstaurations would render the compounds more sensitive to degradation, likely by oxidation, thus limiting this strategy. Another possibility to introduce disorder within the hydrophobic domain is to incorporate branched side chains. The presence of ramifications will reduce the van der Waals interactions between the lipid chains that will thus be less packed together. Consequently, the required energy for the destructuration of the lipoplexes after cell internalization will be lower. In this direction, we incorporated phytanyl chains within the molecular structure of lipophosphoramidate to prepare the cationic amphiphile 11 (49). Phytanyl chains possess four methyl groups regularly distributed on a C16 linear lipid chain. We found that cationic amphiphile 11 was much more efficient to deliver pDNA in vivo (iv injection). However, the difficulties associated with preparing liposomal solutions with compound 11 invited us to further explore its supramolecular assemblies. Indeed, the presence of branched side chains on the lipid chains should increase the volume of the hydrophobic domain that, according to the Israelachvili theory (50), can impact the supramolecular assembly. Since all of our cationic amphiphiles possess a phosphorus atom that is an active NMR nucleus, we recorded 31P NMR of hydrated lipids (static Hahn echo sequence) to determine the lamellar versus hexagonal packing. We found that the bis-oleyl cationic amphiphile 7 self-organized in a lamellar supramolecular structure whereas the bisphythanyl-based amphiphile 11 self-organized in a hexagonal packing. The higher in vivo transfection efficacy of compound 11 can be ascribed to its different supramolecular packing (hexagonal phases) that, as previously reported in the literature, should favor fusion via lipid mixing (51). The incorporation of branched side chains in the hydrophobic domain of cationic amphiphiles can be achieved by selecting naturally occurring ramified lipids like phytanyl chains. We also explored the possibility of preparing synthetically ramified lipid chains following a versatile synthesis scheme in order to be able to control and to modulate the nature (e.g. length) of the branched side chains. For this purpose, we looked for click reactions that can be applied to modify the structure of existing lipid chains. The simplest strategy was to make use of the C=C double bond, already present on unsaturated lipid chains, as an anchoring point. In this respect, the thiol-ene click reaction was very attractive for the following reasons: (1) The application of a thiol-ene reaction on oleyl chains introduces a thioether function. Since the sulfur atom is weakly electronegative, the resulting thioether function features a low polarity, which is compatible with our idea to introduce branched side chains within the hydrophobic domain and to keep this part still hydrophobic after modification. (2) Thiol-ene reaction is a well-documented reaction (52) that is a metal-free click reaction. Consequently, the final product will not be polluted by traces of metallic species. We found that the thiol-ene reaction was successful with different types of alkyl thiol. This reaction can be achieved at the early stage of the synthesis 44 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
of cationic amphiphiles, but we found that its implementation in the last step of the synthesis was the best option (53). That means that any type of cationic amphiphile possessing two oleyl chains can be transformed into an amphiphilic compound possessing ramified lipid chains by applying a thio-ene reaction. We applied this protocol to modify the structure of the lipophosphoramide 2 that produced, for instance, the ramified cationic amphiphiles 12 and 13 (Figure 5). In a series of four ramified lipophosphoramidates obtained by thiol-ene reaction, the supramolecular packing was studied by 31P NMR and 2H NMR. We found that whatever the structure of the thiol involved in the thiol-ene reaction all the compounds exhibited, when hydrated, an inverted hexagonal packing (HII) (53). This thiol-ene procedure was also applied to another class of cationic amphiphile (1,2-di-O-octadecenyl-3-trimethylammonium propane) and we found (by 2H NMR) that this amphiphile adopted an inverted hexagonal supramolecular packing when placed in water (53). Altogether, it can be concluded that the modification of oleyl chains by thiol-ene click reaction is a simple method to produce cationic amphiphiles that will adopt inverted hexagonal packing in water. These amphiphilic compounds were also tested as carriers of pDNA in vitro. It was found that compounds 12 and 13 were more efficient for transfection than the bis-oleyl analogue 2 when used at a charge ratio of 4.
Figure 4. Molecular structure of phosphoramide possessing different lipid domains and either a trimethylammonium or a trimethylarsonium polar head group. 45 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
Figure 5. Chemical structure of lipophosphoramidates obtained by thiol-ene reaction with dodecane-thiol (12) and cyclohexyl-thiol (13). Another possibility for introducing some disorder within the hydrophobic domain of cationic amphiphiles consists of including two different lipid chains in their structure. This strategy was, for instance, illustrated by Nantz et al. who included in the structure of a cationic lipid two linear lipid chains of different length (C12:0 and C14:0) (54). This compound, used as a gene carrier for in vivo experiments, proved to be more efficient than its analogue possessing two C14 lipid chains. Aiming to further explore this possibility, we developed a synthesis method with only a few steps to prepare cationic amphiphiles possessing two different lipid chains that could be saturated, unsaturated, or branched. The first approach applied the methodology reported in Figure 6, requiring the preparation of lipophosphite with two different lipid chains. This type of precursor is possible to prepare (55), but a simpler procedure was concomitantly developed. This procedure starts with POCl3 as a precursor and, after optimization that imposes a careful control of the temperature and a follow-up by 31P NMR, the successive addition of two different lipids chains followed by the addition of the functional amine that will subsequently form the cationic polar head group. Thus, we obtained a series of cationic amphiphiles that can be prepared at a multigram scale (Figure 6) (55). Having in hand cationic amphiphiles with two different lipid chains noted R1 and R2, and also the cationic amphiphiles possessing two identical lipid chains (R1 or R2), we wondered about the best strategy to follow to produce the most efficient formulation for pDNA delivery since two approaches were possible: (1) the association of two different lipid chains at a molecular scale (cationic lipids with two different lipid chains) versus (2) the association of different alkyl chains at a supramolecular scale by mixing two cationic amphiphiles possessing two identical lipid chains noted either R1 or R2. The systematic evaluation of the symmetric cationic amphiphiles (two identical lipid chains), the dissymmetric amphiphiles (two different lipid chains), or the mixing of two symmetric amphiphiles for in vitro transfection led to the conclusion that mixing at a molecular scale (cationic lipid with two different lipid 46 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
chains) was more efficient than mixing at a supramolecular level (mixing of two symmetric amphiphiles). The second conclusion indicated that the nature of the lipid chains included within the structure of the cationic lipid deeply influenced the transfection efficacies. The less efficient vectors possessed two different saturated lipid chains (e.g., C18:0 and C14:0). On the other hand, the best combination involved associating either a C12:0 lipid chain or an oleyl lipid chain with one phytanyl chain within the molecular structure of cationic lipid to produce compounds 14 and 15 (Figure 6). This result also confirmed the great efficacy of the cationic amphiphile possessing two phytanyl chains for gene delivery.
Figure 6. (A) Synthesis of cationic amphiphiles possessing two different lipid chains from POCl3; (B) molecular structure of the most efficient dissymmetric compounds as gene carriers.
All of these molecular variations that are possible to render thanks to the flexible synthesis scheme invited us to consider these compounds for other properties. It was indeed reported that some cationic amphiphilic compounds could exhibit bactericidal action (56, 57). This identification of bactericidal action was also motivated by our will to identify cationic amphiphiles that would be efficient to deliver nucleic acid and that would possess an additional bactericidal action. Such types of cationic amphiphiles would be adapted for the transfection of mammal cells in the context of bacterium infection. This situation is actually 47 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
occurring in CF patients who have to face opportunist bacterial infections. A systematic screening of the bactericidal action of our cationic amphiphiles (38) lead to the conclusion that: (1) The nature of the cationic polar head group is determinant to produce the most bactericidal action. In this regard, the best bactericidal action was obtained with the cationic amphiphiles possessing a trimethylarsonium polar head group. (2) The lipid chains were needed to observe a bactericidal action and the structure of the lipid chains influenced the bactericidal action. The best lipid chains identified so far were linear saturated myristic chains (C14:0). (3) The nature of the phosphorus function also influenced the bactericidal action with the best function being the phosphoramidate group. Taking into account the different structural modulation, the best bactericidal agents were compounds 7, 10, and 16 (Figure 7).
Figure 7. Molecular structure of the cationic amphiphiles exhibiting the highest bactericidal action.
Interestingly, these bactericidal agents that have the presence of a trimethylarsonium polar head group in common have the capacity to compact pDNA and to transfect mammal cells. Concomitantly, they exhibit strong bactericidal action against Gram-positive bacterial strains, including multiresistant strains. As previously shown, this type of cationic amphiphile can contribute to protect pDNA from degradation by bacteria that would be present close to the target cells. This dual property (transfection and bactericidal action) is, therefore, of valuable interest in the context of CF gene therapy. The synthetic phospholipids reported above were synthesized by making use of a versatile molecular platform where the phosphorus function plays a central role since it links the lipid domain to the cationic polar head group. The versatility of the synthesis scheme permitted us to achieve many modulations of the molecular structure that led us to identify modifications that deeply impact 48 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
the supramolecular behavior (branched lipid chains), that impact the transfection efficacy and reduced toxicity (the nature of the cationic group), and that offer an efficient methodology to associate two different lipid chains within the molecular structure of cationic amphiphiles. Additionally, some of these cationic vectors present a bactericidal action, which may be an added value for CF gene therapy. Moreover, the synthesis of all the cationic amphiphiles reported above can be achieved at a multigram scale. We therefore have serious candidate molecules for the development of formulations that would be used for the transfection of lung via aerosolization.
Evaluation Following Aerosol Delivery Among synthetic delivery systems, GL67A is still the gold standard for aerosol delivery to the lungs. Although it has demonstrated encouraging results in preclinical and clinical studies (58, 59), its efficiency remains far below that of viral vectors, with a gap of at least one or two logs (60). Thus, important improvements in gene transfer efficacy still need to be done. GL67A formulation (Figure 8) corresponds to a combination of (1) a cationic lipid (GL67 itself, which is a cholesteryl-spermine), (2) a zwitterionic colipid (DOPE, which provides fusogenicity), and (3) a PEGylated lipid (DMPE-PEG5000, used as stabilizer) all mixed in a well-defined proportion (1/2/0.05 molar ratio, respectively). It is typically used to complex 25 mg of pDNA to form lipoplexes at charge ratio ~ 2 before delivery using the aerosol delivery procedure (Figure 1). As noticed before, this protocol is highly clinically relevant as it is very similar to the aerosol administration performed by the U.K. CF gene therapy consortium in its last clinical multidose gene therapy trial in CF patients (22).
Figure 8. GL67A components; it includes GL67 (17), DOPE (18), and DMPE-PEG5K (19). 49 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
The composition of GL67A can be used as a guide to conceive original formulations based on one’s own vectors. Some of the abovementioned cationic lipids have thus been used in place of GL67 within “GL67A-derived formulation.” As regards the formulation, we have done important work dedicated to its preparation and characterization. This is indeed crucial for the rationalization of the transfection results to improve physicochemical properties and reach pharmacological standards, including homogeneity with low polydispersity. First, we found that cationic lipids, such as 7 (KLN47), can be formulated as liposomal solutions up to very high concentrations (at least up to 30 mM). However, before mixing with DNA, the addition of a PEGylated lipid was required to avoid flocculation (due to an aggregation of DNA complexes). Accordingly, as for GL67A, all formulations need to include at least three to four components, that is, the cationic lipid (e.g., 7) with an optional colipid (such as 18), a PEGylated lipid (e.g., 19), and pDNA. We determined the minimal amount of the steric stabilizer DMPE-PEG5K needed to obtain concentrated and colloidally stable (homogeneous) DNA/lipid formulations; this consisted of working out these formulations at a reduced (1/100) scale (i.e., 250 µg DNA being complexed in 100 µL). Our work has thus been subdivided into two parts. (1) The identification of concentrated lipoplex formulations based on different types of vectors previously mentioned, especially 7/PEGylated lipid/pDNA or 7/PEGylated lipid/helper lipid/pDNA, which exhibit well-defined physicochemical criteria (size and zeta potential) and colloidal stability. Other derived formulations have also been developed based on 10 (BSV4) or 11 (BSV18). On the other hand, for the carriers yielding interesting results in a first aerosol assay, we tried to make rational changes to their formulation by modifying parameters such as the lipid/colipid molar ratio, the type of colipid, the ratio of the mass of steric stabilizer to the mass of DNA, and the charge ratio of the DNA complexes finally formed when mixing the formulation with the pDNA. (2) For the formulations selected, we prepared a large amount of liposomal solution (30 mM in 5 mL) for evaluation of their transfection activity. In these experiments, formulations were mixed with the same volume (in 5 mL) of an optimized (CpG-free) luciferase-encoding pDNA (pGM144) (61). Physicochemical experiments were again performed to determine the feasibility of the mixtures in terms of stability. Next, formulations were assessed to determine whether they displayed some properties required for aerosolization, that is, resistance to the physical forces of aerosolization and thus protection of the integrity of the pDNA in the complexes and conservation of the transfection activity under in vitro experimental conditions. Finally, gene delivery efficiency was assessed in animals at different time points following nebulization via highly sensitive luminescence measurements from living animals and then from lung homogenates (after sacrificing the animals). We first noticed that all of the lipid formulations, when mixed with pDNA at high concentrations (2.5 mg/mL), allowed one to obtain apparently homogenous and colloidally-stable DNA lipoplex solutions. This validated our strategy for working out concentrated lipid formulations adequate for aerosol delivery at a small (1/100) scale, in particular as regards the amount of steric stabilizer DMPE-PEG5K used, which was always high enough to allow the formation of 50 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
colloidally stable DNA complexes that are relatively homogenous (with a low polydispersity index). Next, using the clinical nebulizer chosen (PARI LC Plus), an aerosol obtained from all of the complexes formed could be breathed by the mice hosted inside the housing box. In particular, we did not face any major aerosolization problems, such as excessive bubbling or foaming that would have made delivery impossible. Nebulization lasted between 30 to 45 minutes, which is a typical duration according to U.K. expertise. In every instance, the aerosolized formulation was well-tolerated by the mice, no clinical adverse effect (such as stress, prostration, etc.) being observed at any time during the experiment. Further, the lungs collected at different time points (from 24 hours after aerosol until 28 days later) always exhibited a normal aspect. The absence of an obvious toxic effect was further confirmed when measuring liver enzyme activities in sera sampled from the mice just after their sacrifice. Almost all of the aerosols performed allowed transfection of the lungs in mice in vivo; in many cases, the average luciferase levels were indeed measured above the background determined using naive, nontreated mice. This was a noticeable result since, in the experience of the U.K. CF gene therapy consortium, among the many formulations that have been evaluated over several years in the past, most proved to be completely inefficient (only ~ 10% of the formulations tested demonstrated some efficiency). Despite the fact that the U.K.-elected GL67A formulation showed the highest efficiency, positive results (p < 0.05) obtained with our formulations were nevertheless remarkable, as our formulations had not been worked out at length in contrast to the GL67A formulation. Incorporation of a colipid at an adequate molar ratio versus the cationic lipid appears to play an important role. It is noteworthy here that GL67A corresponds to a combination of GL67 with DOPE at 1/2 lipid/colipid molar ratio. GL67 is also a cholesteryl-based polycationic lipid whereas ours incorporates aliphatic chains linked to a monocationic head group. These differences may deserve to be compared more in depth in the future. Among the formulations we have worked out, a combination of 8 (KLN25)/MM27 1/1 yielded the best transfection efficiencies. It is noteworthy here that 8 and MM27 are two lipids incorporating aliphatic (oleyl) chains—that is, they do not contain a cholesterol moiety—and 8 incorporates a monovalent permanent cationic head group. Also, the combination of these two lipophosphoramides (which present high structural similarities because they differ only by the fact that the 8 head group is based on an imidazolium group whereas the MM27 colipid harbors an imidazole group) was previously found to be quite efficient for gene transfection in general (39). This may be due to the protonability of the imidazole at acidic endosomal pH with a proton sponge effect and an increased fusogenicity. However, at that stage of our evaluations, GL67A was still not challenged by the cationic lipid systems we have developed. Here, it must be noted that only a small set of the compounds previously mentioned have been evaluated under aerosol experimental conditions, for the obvious reasons related to the technical constraints inherent to the method. As stated above, our cationic lipid derivatives may display additional activities due to their amphiphilic cationic properties, as demonstrated under other experimental conditions (38, 40). It is noteworthy that, under the same experimental conditions, GL67A does not have such activities (unpublished 51 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
results). Thus, surpassing GL67A may require improvements of other properties. As a reminder here, CF is characterized by recurrent episodes of infection and inflammation due to chronic opportunistic pathogens having the capacity to colonize CF lungs. The results obtained from several CF clinical trials using nonviral vectors demonstrated the safety and feasibility of repeated nebulization. However, they also emphasized that higher expression of the therapeutic CFTR gene is still needed to obtain relevant clinical benefits, such as a reduction in bacterial infections. This may be achieved notably through further development and optimization of synthetic delivery systems. Given the dual properties—antibacterial and gene delivery—of the amphiphiles from our laboratories, we assume that they could demonstrate an advantage to transfect the epithelial cells in the complex environment of the CF lungs. We have reported arsonium-containing lipophosphoramides as polyfunctional nanocarriers capable of simultaneous antibacterial action against Gram-positive bacteria and gene transfer into eukaryotic cells (38). In a more recent work, we have shown that such amphiphiles can also be combined with an N-heterocyclic carbene-silver complex within multimodular gene delivery systems, accumulating the respective bioproperties of each component. Indeed, we have reported that an equimolar combination of two such compounds (1) was simultaneously active against Gram-positive and Gram-negative bacteria that frequently contaminates the lungs of CF patients, (2) retained its activity during several passages whereas conventional antibiotics did not, (3) was quite well-tolerated by human bronchial epithelial cells, and (4) showed both antibacterial and transfection activities even when delivered via an aerosol (62). This supports the potential benefits of a system displaying gene delivery activity and antibacterial properties at the same time. It could indeed provide a better protection to the DNA, leading to a more efficient transfection of bronchial epithelial cells; besides, it could participate in the eradication of undesirable bacteria, thus reducing the stress of epithelial cells and allowing a better expression of the medicine gene. Finally, the expression of a functional CFTR chloride channel at the apical membrane of lung epithelial cells might also restore the innate antibacterial defense, thus creating a virtuous therapeutic circle (63).
Conclusion Encouraging results strongly invite one to perform further preclinical evaluations of nonviral vectors as possible GL67 alternatives for aerosol gene delivery into the lungs. Obviously, selected formulations will have to be carefully worked out and extensively characterized in order to be optimal for aerosol delivery. To reach this purpose, chemists/physicochemists and biologists must continue working in very close collaboration, in order to (1) conceive new gene carriers that can be easily prepared in large quantities, (2) work out highly concentrated formulations incorporating such original vectors suitable for aerosol delivery, and (3) evaluate their in vivo lung transfection efficiency following aerosol administration in mice. First, in vivo aerosol experiments mainly involved normal mice for obvious availability reasons; the CpG-free luciferase-expressing 52 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
pGM144 plasmid, which has been optimized for in vivo transgene expression (61), should be replaced in a second step with the CpG-free CFTR-expressing pGM169 pDNA using molecular biology methods specifically detecting CFTR expression from the transferred plasmid. Finally, the best formulations may also be evaluated in CF-/- animal models, using the therapeutic pGM169 plasmid. All of this work may allow an evolution toward a multimodular “virus like” gene delivery system dedicated to the treatment of lung pathologies such as CF.
References 1.
2. 3. 4.
5.
6. 7.
8.
9.
10.
11. 12. 13.
Button, B.; Cai, L. H.; Ehre, C.; Kesimer, M.; Hill, D. B.; Sheehan, J. K.; Boucher, R. C.; Rubistein, M. A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia. Science 2012, 337, 937–941. Lai, S. K.; Wang, Y. Y.; Wirtz, D.; Hanes, J. Micro- and macrorheology of mucus. Adv. Drug Delivery Rev. 2009, 61, 86–100. Munkholm, M.; Mortensen, J. Mucociliary clearance: pathophysiological aspects. Clin. Physiol. Funct. Imaging 2014, 34, 171–177. d’Angelo, I.; Conte, C.; La Rotonda, M. I.; Miro, A.; Quaglia, F.; Ungaro, F. Improving the efficacy of inhaled drugs in cystic fibrosis: challenges and emerging drug delivery strategies. Adv. Drug Delivery Rev. 2014, 75, 92–111. Lai, S. K.; Wang, Y. Y.; Hanes, J. Mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues. Adv. Drug Delivery Rev. 2009, 61, 158–171. Duncan, G. A.; Jung, J.; Hanes, J.; Suk, J. S. The Mucus Barrier to Inhaled Gene Therapy. Mol. Ther. J. Am. Soc. Gene Ther. 2016, 24, 2043–2053. Schuster, B. S.; Suk, J. S.; Woodworth, G. F.; Hanes, J. Nanoparticle diffusion in respiratory mucus from humans without lung disease. Biomaterials 2013, 34, 3439–3946. Kim, N.; Duncan, G. A.; Hanes, J.; Suk, J. S. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. J. Controlled Release 2016, 28, 465–488. Alipour, M.; Suntres, Z. E.; Halwani, M.; Azghani, A. O.; Omri, A. Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis. PloS One 2009, 4, e5724. Hidalgo, A.; Cruz, A.; Pérez-Gil, J. Pulmonary surfactant and nanocarriers: Toxicity versus combined nanomedical applications. Biochim. Biophys. Acta 2017, 1859, 1740–1748. Speer, C. P.; Sweet, D. G.; Halliday, H. L. Surfactant therapy: past, present and future. Early Hum. Dev. 2013, 89, S22–S24. Bustamante-Marin, X. M.; Ostrowski, L. E. Cilia and Mucociliary Clearance. Cold Spring Harb. Perspect. Biol. 2017, 9, a028241. Faner, R.; Sibila, O.; Agustí, A.; Bernasconi, E.; Chalmers, J. D.; Huffnagle, G. B.; Manichanh, C.; Molyneaux, P. L.; Paredes, R.; Pérez Brocal, V.; Ponomarenko, J.; Sethi, S.; Dorca, J.; Monsó, E. The microbiome 53
Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
14. 15. 16.
17.
18.
19.
20. 21.
22.
in respiratory medicine: current challenges and future perspectives. Eur. Respir. J. 2017, 49, 1602086. Marsland, B. J.; Gollwitzer, E. S. Host-microorganism interactions in lung diseases. Nat. Rev. Immunol. 2014, 14, 827–835. Rukavina, Z.; Vanić, Ž. Current Trends in Development of Liposomes for Targeting Bacterial Biofilms. Pharmaceutics 2016, 8, 18. Ciofu, O.; Tolker-Nielsen, T.; Jensen, P. Ø.; Wang, H.; Høiby, N. Antimicrobial resistance, respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients. Adv. Drug Delivery. Rev. 2015, 85, 7–23. Walters, M. C.; Roe, F.; Bugnicourt, A.; Franklin, M. J.; Stewart, P. S. Contributions of antibiotic penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin. Antimicrob. Agents Chemother. 2003, 47, 317–323. Picquet, E.; Le Ny, K.; Delépine, P.; Montier, T.; Yaouanc, J. J.; Cartier, D.; des Abbayes, H.; Férec, C.; Clément, J. C. Cationic lipophosphoramidates and lipophosphoguanidines are very efficient for in vivo DNA delivery. Bioconjugate Chem. 2005, 16, 1051–1053. Lindberg, M. F.; Le Gall, T.; Carmoy, N.; Berchel, M.; Hyde, S. C.; Gill, D. R.; Jaffrès, P. A.; Lehn, P.; Montier, T. Efficient in vivo transfection and safety profile of a CpG-free and codon optimized luciferase plasmid using a cationic lipophosphoramidate in a multiple intravenous administration procedure. Biomaterials 2015, 59, 1–11. Ratjen, F.; Döring, G. Cystic Fibrosis. Lancet 2003, 361, 681–689. Davies, L. A.; Hyde, S. C.; Nunez-Alonso, G.; Bazzani, R. P.; HardingSmith, R.; Pringle, I. A.; Lawton, A. E.; Abdullah, S.; Roberts, T. C.; McCormick, D.; Sumner-Jones, S. G.; Gill, D. R. The Use of CpG-free Plasmids to Mediate Persistent Gene Expression Following Repeated Aerosol Delivery of pDNA/PEI Complexes. Biomaterials 2012, 33, 5618–5627. Alton, E. W. F. W.; Armstrong, D. K.; Ashby, D.; Bayfield, K. J.; Bilton, D.; Bloomfield, E. V.; Boyd, A. C.; Brand, J.; Buchan, R.; Calcedo, R.; Carvelli, P.; Chan, M.; Cheng, S. H.; Collie, D. D. S.; Cunningham, S.; Davidson, H. E.; Davies, G.; Davies, J. C.; Davies, L. A.; Dewar, M. H.; Doherty, A.; Donovan, J.; Dwyer, N. S.; Elgmati, H. I.; Featherstone, R. F.; Gavino, J.; Gea-Sorli, S.; Geddes, D. M.; Gibson, J. S. R.; Gill, D. R.; Greening, A. P.; Griesenbach, U.; Hansell, D. M.; Harman, K.; Higgins, T. E.; Hodges, S. L.; Hyde, S. C.; Hyndman, L.; Innes, J. A.; Jacob, J.; Jones, N.; Keogh, B. F.; Limberis, M. P.; Lloyd-Evans, P.; Maclean, A. W.; Manvell, M. C.; McCormick, D.; McGovern, M.; McLachlan, G.; Meng, C.; Montero, M. A.; Milligan, H.; Moyce, L. J.; Murray, G. D.; Nicholson, A. G.; Osadolor, T.; Parra-Leiton, J.; Porteous, D. J.; Pringle, I. A.; Punch, E. K.; Pytel, K. M.; Quittner, A. L.; Rivellini, G.; Saunders, C. J.; Scheule, R. K.; Sheard, S.; Simmonds, N. J.; Smith, K.; Smith, S. N.; Soussi, N.; Soussi, S.; Spearing, E. J.; Stevenson, B. J.; Sumner-Jones, S. G.; Turkkila, M.; Ureta, R. P.; Waller, M. D.; Wasowicz, M. Y.; Wilson, J. M.; Wolstenholme-Hogg, P. UK Cystic Fibrosis Gene Therapy Consortium 54
Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
23. 24.
25.
26.
27.
28.
29.
30.
31.
32.
Repeated Nebulisation of Non-Viral CFTR Gene Therapy in Patients with Cystic Fibrosis: a Randomised, Double-Blind, Placebo-Controlled, Phase 2b Trial. Lancet Respir. Med. 2015, 3, 684–691. Stribling, R.; Brunette, E.; Liggitt, D.; Gaensler, K.; Debs, R. Aerosol Gene Delivery In Vivo. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 11277–11281. McLachlan, G.; Davidson, H.; Holder, E.; Davies, L. A.; Pringle, I. A.; Sumner-Jones, S. G.; Baker, A.; Tennant, P.; Gordon, C.; Vrettou, C.; Blundell, R.; Hyndman, L.; Stevenson, B.; Wilson, A.; Doherty, A.; Shaw, D. J.; Coles, R. L.; Painter, H.; Cheng, S. H.; Scheule, R. K.; Davies, J. C.; Innes, J. A.; Hyde, S. C.; Griesenbach, U.; Alton, E. W. F. W.; Boyd, A. C.; Porteous, D. J.; Gill, D. R.; Collie, D. D. S. Pre-Clinical Evaluation of Three Non-Viral Gene Transfer Agents for Cystic Fibrosis After Aerosol Delivery to the Ovine Lung. Gene Ther. 2011, 18, 996–1005. Berchel, M.; Le Gall, T.; Couthon-Gourvès, H.; Haelters, J. P.; Montier, T.; Midoux, P.; Lehn, P.; Jaffrès, P. A. Lipophosphonate/lipophosphoramidates: A family of synthetic vectors efficient for gene delivery. Biochimie 2012, 94, 33–41. Bennett, M. J.; Malone, R. W.; Nantz, M. H. A flexible approach to synthetic lipid ammonium-salts for polynucleotide transfection. Tetrahedron Lett. 1995, 36, 2207–2210. Guénin, E.; Hervé, A. C.; Floch, V.; Loisel, S.; Yaouanc, J. J.; Clément, J. C.; Férec, C.; des Abbayes, H. Cationic Phosphonolipids Containing Quaternary Phosphonium and Arsonium Groups for DNA Transfection with Good Efficiency and Low Cellular Toxicity. Angew. Chem., Int. Ed. 2000, 39, 629–631. Lamarche, F.; Mével, M.; Montier, T.; Burel-Deschamps, L.; Giamarchi, P.; Tripier, R.; Le Gall, T.; Cartier, D.; Lehn, P.; Clément, J. C.; Jaffrès, P. A. Lipophosphoramidates as lipidic part of lipospermines for gene delivery. Bioconjugate Chem. 2007, 18, 1575–1582. Mével, M.; Montier, T.; Lamarche, F.; Delépine, P.; Le Gall, T.; Yaouanc, J. J.; Jaffrès, P. A.; Cartier, D.; Lehn, P.; Clément, J. C. Dicationic lipophosphoramidates as DNA carriers. Bioconjugate Chem. 2007, 18, 1604–1611. Fraix, A.; Montier, T.; Carmoy, N.; Loizeau, D.; Burel-Deschamps, L.; Le Gall, T.; Giamarchi, P.; Couthon-Gourvès, H.; Haelters, J. P.; Lehn, P.; Jaffrès, P. A. Cationic lipo-thiophosphoramidates for gene delivery: synthesis, physico-chemical characterization and gene transfection activity--comparison with lipo-phosphoramidates. Org. Biomol. Chem. 2011, 9, 2422–2432. Fraix, A.; Montier, T.; Le Gall, T.; Sevrain, C. M.; Carmoy, N.; Lindberg, M. F.; Lehn, P.; Jaffrès, P. A. Lipothiophosphoramidates for gene delivery: critical role of the cationic polar headgroup. Org. Biomol. Chem. 2012, 10, 2051–2058. Le Corre, S. S.; Berchel, M.; Le Gall, T.; Haelters, J. P.; Lehn, P.; Montier, T.; Jaffrès, P. A. Cationic Trialkylphosphates: Synthesis and Transfection Efficacies Compared to Phosphoramidate Analogues. Eur. J. Org. Chem. 2014, 2014, 8041–8048. 55
Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
33. Le Corre, S. S.; Belmadi, N.; Berchel, M.; Le Gall, T.; Haelters, J. P.; Lehn, P.; Montier, T.; Jaffrès, P. A. Cationic dialkylarylphosphates: a new family of bio-inspired cationic lipids for gene delivery. Org. Biomol. Chem. 2015, 13, 1122–1132. 34. Pozharski, E.; MacDonald, R. C. Lipoplex thermodynamics: determination of DNA-cationic lipoid interaction energies. Biophys. J. 2003, 85, 3969–3978. 35. Patel, M. M.; Anchordoquy, T. J. Contribution of hydrophobicity to thermodynamics of ligand-DNA binding and DNA collapse. Biophys. J. 2005, 88, 2089–2103. 36. Le Corre, S. S.; Berchel, M.; Couthon-Gourvès, H.; Haelters, J. P.; Jaffrès, P. A. Atherton-Todd reaction: mechanism, scope and applications. Beilstein J. Org. Chem. 2014, 10, 1166–1196. 37. Berchel, M.; Le Gall, T.; Haelters, J. P.; Lehn, P.; Montier, T.; Jaffrès, P. A. Cationic lipophosphoramidates containing a hydroxylated polar headgroup for improving gene delivery. Mol. Pharm. 2015, 12, 1902–1910. 38. Le Gall, T.; Berchel, M.; Le Hir, S.; Fraix, A.; Salaün, J. Y.; Férec, C.; Lehn, P.; Jaffrès, P. A.; Montier, T. Arsonium-containing lipophosphoramides, poly-functional nano-carriers for simultaneous antibacterial action and eukaryotic cell transfection. Adv. Healthcare Mater. 2013, 2, 1513–1524. 39. Mével, M.; Breuzard, G.; Yaouanc, J. J.; Clément, J. C.; Lehn, P.; Pichon, C.; Jaffrès, P. A.; Midoux, P. Synthesis and transfection activity of new cationic phosphoramidate lipids: high efficiency of an imidazolium derivative. ChemBioChem 2008, 9, 1462–1471. 40. Laurent, V.; Fraix, A.; Montier, T.; Cammas-Marion, S.; Ribault, C.; Benvegnu, T.; Jaffrès, P. A.; Loyer, P. Highly efficient gene transfer into hepatocyte-like HepaRG cells: new means for drug metabolism and toxicity studies. Biotechnology J. 2010, 5, 314–320. 41. Delalande, A.; Gosselin, M. P.; Suwalski, A.; Guilmain, W.; Leduc, C.; Berchel, M.; Jaffrès, P. A.; Baril, P.; Midoux, P.; Pichon, C. Enhanced Achilles tendon healing by fibromodulin gene transfer. Nanomedicine: NBM 2015, 11, 1735–1744. 42. Perche, F.; Gosset, D.; Mével, M.; Miramon, M. L.; Yaouanc, J. J.; Pichon, C.; Benvegnu, T.; Jaffrès, P. A.; Midoux, P. Selective gene delivery in dendritic cells with mannosylated and histidylated lipopolyplexes. J. Drug Targeting 2011, 19, 315–325. 43. Perche, F.; Benvegnu, T.; Berchel, M.; Lebegue, L.; Pichon, C.; Jaffrès, P. A.; Midoux, P. Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA. Nanomedicine: NBM 2011, 7, 445–453. 44. Le Moignic, A.; Malard, V.; Benvegnu, T.; Lemiègre, L.; Berchel, M.; Jaffrès, P. A.; Baillou, C.; Delost, M.; Macedo, R.; Rochefort, J.; Lescaille, G.; Pichon, C.; Lemoine, F. M.; Midoux, P.; Mateo, V. Preclinical evaluation of mRNA trimannosylated lipopolyplexes as therapeutic cancer vaccines targeting dendritic cells. J. Controlled Release 2018, 278, 110–121. 56 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
45. Berchel, M.; Akhter, S.; Berthe, W.; Gonçalves, C.; Dubuisson, M.; Pichon, C.; Jaffrès, P. A.; Midoux, P. Synthesis of alpha-aminolipophosphonates as cationic lipids or co-lipids for DNA transfection in dendritic cells. J. Mater. Chem. B 2017, 5, 6869–6881. 46. Billiet, L.; Gomez, JP.; Berchel, M.; Jaffrès, PA.; Le Gall, T.; Montier, T.; Bertrand, E.; Cheradame, H.; Guégan, P.; Mével, M.; Pitard, B.; Benvegnu, T.; Lehn, P.; Pichon, C.; Midoux, P. Gene transfer by chemical vectors, and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line. Biomaterials 2012, 33, 2980–2990. 47. Le Corre, S. S.; Berchel, M.; Belmadi, N.; Denis, C.; Haelters, J. P.; Le Gall, T.; Lehn, P.; Montier, T.; Jaffrès, P. A. Cationic lipophosphoramidates with two different lipid chains: synthesis and evaluation as gene carriers. Org. Biomol. Chem. 2014, 12, 1463–1474. 48. Loizeau, D.; Le Gall, T.; Mahfoudhi, S.; Berchel, M.; Maroto, A.; Yaouanc, J. J.; Jaffrès, P. A.; Lehn, P.; Deschamps, L.; Montier, T.; Giamarchi, P. Physicochemical properties of cationic lipophosphoramidates with an arsonium head group and various lipid chains: A structure-activity approach. Biophys. Chem. 2013, 171, 46–53. 49. Lindberg, M. F.; Carmoy, N.; Le Gall, T.; Fraix, A.; Berchel, M.; Lorilleux, C.; Couthon-Gourvès, H.; Bellaud, P.; Fautrel, A.; Jaffrès, P. A.; Lehn, P.; Montier, T. The gene transfection properties of a lipophosphoramidate derivative with two phytanyl chains. Biomaterials. 2012, 33, 6240–6253. 50. Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Theory of self-assembly of hydrocarbon amphiphiles into micelles and bilayers. J. Chem. Soc., Faraday Trans 1976, 72, 1525e68. 51. Koynova, R.; Wang, L.; Macdonald, R. C. Cationic phospholipids forming cubic phases: lipoplex structure and transfection efficiency. Mol. Pharm. 2008, 5, 739–744. 52. Hoyle, C. E.; Bowman, C. N. Thiol-ene click chemistry. Angew. Chem. Int. Ed. Engl. 2010, 49, 1540–1573. 53. Afonso, D.; Le Gall, T.; Couthon-Gourvès, H.; Grélard, A.; Prakash, S.; Berchel, M.; Kervarec, N.; Dufourc, E. J.; Montier, T.; Jaffrès, P. A. Triggering bilayer to inverted-hexagonal nanostructure formation by thiol-ene click chemistry on cationic lipids: consequences on gene transfection. Soft Matter 2016, 12, 4516–4520. 54. Nantz, M. H.; Dicus, C. W.; Hilliard, B.; Yellayi, S.; Zou, S.; Hecker, J. G. The benefit of hydrophobic domain asymmetry on the efficacy of transfection as measured by in vivo imaging. Mol. Pharm. 2010, 7, 786–794. 55. Berchel, M.; Haddad, J.; Le Corre, S.; Haelters, J. P.; Jaffrès, P. A. Synthesis of lipid-based unsymmetrical O,O-dialkylphosphites. Tetrahedron Lett. 2015, 56, 2345–2348. 56. Baussanne, I.; Bussiere, A.; Halder, S.; Ganem-Elbaz, C.; Ouberai, M.; Riou, M.; Paris, J.; Ennifar, M. E.; Mingeot-Leclercq, M. P.; Decout, J. L. Synthesis and antimicrobial evaluation of amphiphilic neamine derivatives. J. Med. Chem. 2010, 53, 119–127. 57 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
57. Ioannou, C. J.; Hanlon, G. W.; Denver, S. P. Action of disinfectant quaternary ammonium compounds against Staphylococcus aureus. Antimicrob. Agents Chem. Ther. 2007, 51, 296–306. 58. Lee, E. R.; Marshall, J.; Siegel, C. S.; Jiang, C.; Yew, N. S.; Nichols, M. R.; Nietupski, J. B.; Ziegler, R. J.; Lane, M. B.; Wang, K. X.; Wan, N. C.; Scheule, R. K.; Harris, D. J.; Smith, A. E.; Cheng, S. H. Detailed Analysis of Structures and Formulations of Cationic Lipids for Efficient Gene Transfer to the Lung. Hum. Gene Ther. 1996, 7, 1701–1717. 59. McLachlan, G.; Davidson, H.; Holder, E.; Davies, L. A.; Pringle, I. A.; Sumner-Jones, S. G.; Baker, A.; Tennant, P.; Gordon, C.; Vrettou, C.; Blundell, R.; Hyndman, L.; Stevenson, B.; Wilson, A.; Doherty, A.; Shaw, D. J.; Coles, R. L.; Painter, H.; Cheng, S. H.; Scheule, R. K.; Davies, J. C.; Innes, J. A.; Hyde, S. C.; Griesenbach, U.; Alton, E. W. F. W.; Boyd, A. C.; Porteous, D. J.; Gill, D. R.; Collie, D. D. S. Pre-Clinical Evaluation of Three Non-Viral Gene Transfer Agents for Cystic Fibrosis After Aerosol Delivery to the Ovine Lung. Gene Ther 2011, 18, 996–1005. 60. Mitomo, K.; Griesenbach, U.; Inoue, M.; Somerton, L.; Meng, C.; Akiba, E.; Tabata, T.; Ueda, Y.; Frankel, G. M.; Farley, R.; Singh, C.; Chan, M.; Munkonge, F.; Brum, A.; Xenariou, S.; Escudero-Garcia, S.; Hasegawa, M.; Alton, E. W. F. W. Toward Gene Therapy for Cystic Fibrosis Using a Lentivirus Pseudotyped with Sendai Virus Envelopes. Mol. Ther. 2010, 18, 1173–1182. 61. Hyde, S. C.; Pringle, I. A.; Abdullah, S.; Lawton, A. E.; Davies, L. A.; Varathalingam, A.; Nunez-Alonso, G.; Green, A. M.; Bazzani, R. P.; Sumner-Jones, S. G.; Chan, M.; Li, H.; Yew, N. S.; Cheng, S. H.; Boyd, A. C.; Davies, J. C.; Griesenbach, U.; Porteous, D. J.; Sheppard, D. N.; Munkonge, F. M.; Alton, E. W.; Gill, D. R. CpG-Free Plasmids Confer Reduced Inflammation and Sustained Pulmonary Gene Expression. Nat. Biotechnol. 2008, 26, 549–51. 62. Mottais, A.; Berchel, M.; Sibiril, Y.; Laurent, V.; Gill, D.; Hyde, S.; Jaffrès, P. A.; Montier, T.*; Le Gall T.* Antibacterial effect and DNA delivery using a combination of an arsonium-containing lipophosphoramide with an N-heterocyclic carbene-silver complex - Potential benefits for cystic fibrosis lung gene therapy. Inter. J. Pharm. 2018, 536, 29–41. 63. Hisert, K. B.; Heltshe, S. L.; Pope, C.; Jorth, P.; Wu, X.; Edwards, R. M.; Radey, M.; Accurso, F. J.; Wolter, D. J.; Cooke, G.; Adam, R. J.; Carter, S.; Grogan, B.; Launspach, J. L.; Donnelly, S. C.; Gallagher, C. G.; Bruce, J. E.; Stoltz, D. A.; Welsh, M. J.; Hoffman, L. R.; McKone, E. F.; Singh, P. K. Restoring Cystic Fibrosis Transmembrane Conductance Regulator Function Reduces Airway Bacteria and Inflammation in People with Cystic Fibrosis and Chronic Lung Infections. Am. J. Respir. Crit. Care Med. 2017, 195, 1617–1628.
58 Sakurai and Ilies; Targeted Nanosystems for Therapeutic Applications: New Concepts, Dynamic Properties, Efficiency, and Toxicity ACS Symposium Series; American Chemical Society: Washington, DC, 2019.
