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Wasungu, L and Hoekstra, D. Cationic lipids, lipoplexes and intracellular delivery of genes. J Control Release 116: 255-264
Abstract
As a consequence of several setbacks encountered by viral technology in achieving efficient and safe gene therapy in clinical trials, non-viral gene delivery vectors are considered to date as a valuable alternative and to hold promise for future therapeutic applications. Nevertheless, the transfection efficiency mediated by these non-viral gene delivery vectors has to be improved, especially in vivo, to benefit fully from their advantages. Cationic lipid/nucleic acid complexes or lipoplexes have been the subject of intensive investigations in recent years to understand the parameters governing the efficiency of transfection. Specifically, the comprehension of such mechanisms, from the formation of the complexes to their intracellular delivery, will lead to the design of better adapted non-viral vectors for gene therapy applications. Here, we will discuss some recent developments in the field on the structure/function relationship of cationic lipids in the mechanism of transfection, and where appropriate, we will make a comparison with mechanisms of viral and polyplex-mediated gene delivery. Cationic lipids are often used in combination with helper lipids such as DOPE or cholesterol. The effect of DOPE on lipoplex assembly and the relevance of the structural properties of the lipoplexes in destabilizing endosomal membranes and mediating endosomal escape of DNA will be discussed.
... Lipid-based nanoparticles containing lipids that adopt inverted hexagonal phases, such as DOPE, have better delivery efficiency since they can promote the lipid transition process from lamellar phase to non-lamellar phase and destabilize the structure of the endosomal membrane, which subsequently facilitates endosomal escape, and nucleic acids can be delivered to the cytosol [150,155,156]. [3]. ...
... If ½ < p <1, lipids with cylindrical shape tend to adopt a lamellar phase. If p > 1, inverted conical-shaped lipids will adopt an inverted hexagonal phase [3,155]. Copyright 2022 American Chemical Society. ...
... If 1 2 < p < 1, lipids with cylindrical shape tend to adopt a lamellar phase. If p > 1, inverted conical-shaped lipids will adopt an inverted hexagonal phase [3,155]. Copyright 2022 American Chemical Society. ...
Lipid-based nanoparticles (LBNPs) are biocompatible and biodegradable vesicles that are considered to be one of the most efficient drug delivery platforms. Due to the prominent advantages, such as long circulation time, slow drug release, reduced toxicity, high transfection efficiency, and endosomal escape capacity, such synthetic nanoparticles have been widely used for carrying genetic therapeutics, particularly nucleic acids that can be applied in the treatment for various diseases, including congenital diseases, cancers, virus infections, and chronic inflammations. Despite great merits and multiple successful applications, many extracellular and intracellular barriers remain and greatly impair delivery efficacy and therapeutic outcomes. As such, the current state of knowledge and pitfalls regarding the gene delivery and construction of LBNPs will be initially summarized. In order to develop a new generation of LBNPs for improved delivery profiles and therapeutic effects, the modification strategies of LBNPs will be reviewed. On the basis of these developed modifications, the performance of LBNPs as therapeutic nanoplatforms have been greatly improved and extensively applied in immunotherapies, including infectious diseases and cancers. However, the therapeutic applications of LBNPs systems are still limited due to the undesirable endosomal escape, potential aggregation, and the inefficient encapsulation of therapeutics. Herein, we will review and discuss recent advances and remaining challenges in the development of LBNPs for nucleic acid-based immunotherapy.
... Herein, for the first time, we have shown that liposomes containing RGDK-lipopeptide I, endosome-disrupting histidinylated II and DOPE (co-lipid) in 0.5:0.5:1.0 mole ratio hold promise for delivering DNA vaccines into hard-to-transfect macrophage cells. As to the basis of selecting DOPE as the co-lipid in the present investigation, prior studies demonstrated higher transfection efficiencies of cationic liposomes containing DOPE as colipid due to their abilities to form highly biomembrane fusogenic non-lamellar inverted hexagonal structures [38,50]. Clearly, studies aimed at examining the transfection efficacies of cationic liposomes of RGDK-lipopeptide I containing other previously used auxiliary lipids including cholesterol, DSPC, circulation stability enhancing pegylated lipids, etc. need to be undertaken in future. ...
... Notably, liposomes containg only RGDKlipopeptide I and DOPE (in 1:1 mole ratio) were found to be somewhat less efficient (29%) in transfecting RAW 264.7 cells. In addition to DOPE, many other lipids including DOPC, DSPC, Cholesterol, etc. have been used as co-lipids in cationic lipid mediated gene delivery [23,38,50]. Recent studies have shown DOPE to be better co-lipid than DOPC, and between DSPC & DOPC, DOPC has shown more efficiency as co-lipid [20,23,56]. ...
To ensure effective immune response in genetic immunizations, DNA/mRNA vaccines need to be delivered to body’s antigen presenting cells (APCs) which is a challenging task. This is primarily due to presence of high concentrations of various degradative enzymes inside them. To this end, mannose receptor (over expressed in APCs) selective cationic liposomes have been used in the past for delivering antigen-encoded plasmid DNA to APCs. APCs also express integrin receptors on their cell surfaces. However, studies aimed at delivering DNA vaccines into APCs via integrin receptors have not yet been undertaken. Herein, we report on the use of cationic liposomes of a priorly disclosed α5β1 integrin receptor selective RGDK-lipopeptide for macrophage transfection. In this study, we have used pCMV-GFP (as model DNA vaccine) and RAW 264.7 cells (mouse macrophages cells) as model APC. We show that the liposomes of RGDK-lipopeptide containing a previously reported endosome-disrupting histidinylated lipid and DOPE (as co-lipid) in 0.5:0.5:1.0 mole ratio are the most competent in transfecting macrophage cells (44%). Findings in the fluorescence resonance energy transfer based membrane fusogencity assay revealed that the enhanced macrophage transfection efficiency of the liposomes containing RGDK-lipopeptide, endosome-disrupting histidinylated and DOPE may originate from its higher membrane fusogenicity than that for liposomes containing only RGDK-lipopeptide and DOPE. The presently described biologically safe liposomal formulations of RGDK-lipopeptide are expected to find biomedical applications in future for combating cancer and infectious diseases through genetic immunizations.
... 29,37,38 Non-linear and linear PB studies suggest a release of bound counterions as a mechanism of the entropic stabilization of DNA-membrane complexes, 39,40 which has also been supported by experiments. 34,[41][42][43] The helicity of DNA charge distribution incorporated into theoretical models shows the attractive behavior between DNA molecules. 44 With a linear Debye-Huckel treatment with helical DNA charge distribution and the assumption of low dielectric permittivity at the DNA core, Cherstvy et al. proposed a model to explain divalent cation-induced CL-DNA condensation. ...
... This result is also consistent with earlier studies. 34,[41][42][43]50 Experimental findings have indicated that a localization of Mg 2+ cations situated between DNA pairs contributes to DNA attraction in the presence of lipids. 29 To scrutinize this hypothesis, we computed the average density of Mg 2+ ions located between DNA pairs within both the DNA-in-lipid system and DNA in an aqueous solution ( Fig. 4i-j). ...
The condensation of nucleic acids by lipids is a widespread phenomenon in biology with crucial implications for drug delivery. However, due to the challenges in measuring and assessing the contribution of each component in the lipid-DNA-cation system, the intricate physical mechanisms underlying the assembly of DNAs in lipid bilayers remain insufficiently understood. Specifically, the role of divalent cations in the like-charge attraction of DNA pairs remains a topic of debate. This study utilizes all-atom molecular dynamics simulations and free energy calculations to investigate the condensation of DNA duplexes in cationic lipid bilayers. Our exhaustive exploration of the thermodynamic factors inducing DNA condensation reveals unique roles for phospholipid head groups and cations. We observed that bridging cations between the lipid head groups and DNA, drastically reduce DNA charges, while mobile magnesium cations ping-ponging between DNA double strands, create charge fluctuations. While the first factor stabilizes the DNA-lipid complex, the latter creates attractive forces to induce the condensation of DNA pairs. This novel mechanism not only sheds light on the current data regarding cationic lipid-induced DNA condensation but also provides potential design strategies for creating efficient gene delivery vectors for drug delivery.
... It has been shown that 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), a zwitterionic helper lipid, is able to form an inverted hexagonal phase at the acidic pH of endosomes, which increases the efficiency of the NA molecules' release into the cytoplasm [17,18]. It has also been shown that NA transfection efficiency depends on the molar ratio of cationic lipids to the helper lipid DOPE; in addition, such ratios vary for different types of NA (plasmid DNA, mRNA, and siRNA) [13,[19][20][21][22][23]. ...
... Cationic amphiphilic lipid 2X3 contains two hydrophobic cholesterol residues [10]. It has previously been shown that these structures can induce the formation of inverted non-lamellar phases in lipid systems [22,23]. It can be assumed that a decrease in the molar ratio of cationic lipid 2X3 in the composition of liposomes 2X3-DOPE leads to a decrease in the number of inverted structures. ...
The design of cationic liposomes for efficient mRNA delivery can significantly improve mRNA-based therapies. Lipoplexes based on polycationic lipid 1,26-bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were formulated in different molar ratios (1:1, 1:2, 1:3) to efficiently deliver model mRNAs to BHK-21 and A549. The objective of this study was to examine the effect of 2X3-DOPE composition as well as lipid-to-mRNA ratio (amino-to-phosphate group ratio, N/P) on mRNA transfection. We found that lipoplex-mediated transfection efficiency depends on both liposome composition and the N/P ratio. Lipoplexes with an N/P ratio of 10/1 showed nanometric hydrodynamic size, positive ζ potential, maximum loading, and transfection efficiency. Liposomes 2X3-DOPE (1:3) provided the superior delivery of both mRNA coding firefly luciferase and mRNA-eGFP into BHK-21 cells and A549 cells, compared with commercial Lipofectamine MessengerMax.
... Based on the chemical group, this domain can be quaternary ammonium, an amine (primary, secondary, tertiary), an amino acid or peptide, a guanidine, heterocyclic headgroups, and some specific headgroups. Quaternary ammonium is the most frequent group because of its permanent positive charge that provides high solubility in aqueous environments and strong interaction with the NAs [61,62]. ...
... that provides high solubility in aqueous environments and strong interaction with the NAs[61,62]. ...
Cancer is the second most frequent cause of death worldwide, with 28.4 million new cases expected for 2040. Despite de advances in the treatment, it remains a challenge because of the tumor heterogenicity and the increase in multidrug resistance mechanisms. Thus, gene therapy has been a potential therapeutic approach owing to its ability to introduce, silence, or change the content of the human genetic code for inhibiting tumor progression, angiogenesis, and metastasis. For the proper delivery of genes to tumor cells, it requires the use of gene vectors for protecting the therapeutic gene and transporting it into cells. Among these vectors, liposomes have been the nonviral vector most used because of their low immunogenicity and low toxicity. Furthermore, this nanosystem can have its surface modified with ligands (e.g., antibodies, peptides, aptamers, folic acid, carbohydrates, and others) that can be recognized with high specificity and affinity by receptor overexpressed in tumor cells, increasing the selective delivery of genes to tumors. In this context, the present review address and discuss the main targeting ligands used to functionalize liposomes for improving gene delivery with potential application in cancer treatment.
... Herein, for the first time, we have shown that liposomes containing RGDK-lipopeptide I, endosome-disrupting histidinylated II and DOPE (co-lipid) in 0.5:0.5:1.0 mole ratio hold promise for delivering DNA vaccines into hard-to-transfect macrophage cells. As to the basis of selecting DOPE as the co-lipid in the present investigation, prior studies demonstrated higher transfection efficiencies of cationic liposomes containing DOPE as colipid due to their abilities to form highly biomembrane fusogenic non-lamellar inverted hexagonal structures [38,50]. Clearly, studies aimed at examining the transfection efficacies of cationic liposomes of RGDK-lipopeptide I containing other previously used auxiliary lipids including cholesterol, DSPC, circulation stability enhancing pegylated lipids, etc. need to be undertaken in future. ...
... Notably, liposomes containg only RGDKlipopeptide I and DOPE (in 1:1 mole ratio) were found to be somewhat less efficient (29%) in transfecting RAW 264.7 cells. In addition to DOPE, many other lipids including DOPC, DSPC, Cholesterol, etc. have been used as co-lipids in cationic lipid mediated gene delivery [23,38,50]. Recent studies have shown DOPE to be better co-lipid than DOPC, and between DSPC & DOPC, DOPC has shown more efficiency as co-lipid [20,23,56]. ...
... Nevertheless, the internalization of SLN and the subsequent events are influenced by the SLN's components, physicochemical properties, and the cell type under investigation (Moraes-Lacerda and de Jesus 2022). These factors influence the chosen endosomal route, which, in turn, determines the fate of the particle and associated molecules, ultimately affecting transfection efficiency and cellular function (Wasungu and Hoekstra 2006, Kou et al 2013, Radaic and de Jesus 2018. ...
Solid lipid nanoparticles (SLN) are widely recognized for their biocompatibility, scalability, and long-term stability, making them versatile formulations for drug and gene delivery. Cellular interactions, governed by complex endocytic and signaling pathways, are pivotal for successfully applying SLN as a therapeutic agent. This study aims to enhance our understanding of the intricate interplay between SLN and cells by investigating the influence of specific endocytic and cell signaling pathways, with a focus on the impact of the TGF-β pathway on SLN-mediated cell transfection in both cancerous and non-cancerous prostate cells. Here, we systematically explored the intricate mechanisms governing the interactions between solid lipid nanoparticles and cells. By pharmacologically manipulating endocytic and signaling pathways, we analyzed alterations in SLNplex internalization, intracellular traffic, and cell transfection dynamics. Our findings highlight the significant role of macropinocytosis in the internalization and transfection processes of SLNplex in both cancer and non-cancer prostate cells. Moreover, we demonstrated that the TGF-β pathway is an important factor influencing endosomal release, potentially impacting gene expression and modulating cell transfection efficiency. This study provides novel insights into the dynamic mechanisms governing the interaction between cells and SLN, emphasizing the pivotal role of TGF-β signaling in SLN-mediated transfection, affecting internalization, intracellular transport, and release of the genetic cargo. These findings provide valuable insight for the optimization of SLN-based therapeutic strategies in prostate-related applications.
... Additionally, the composition of liposomes is a determinant factor in the successful delivery of loaded cargoes to the intracellular location upon entrance to target cells through endosomal escape [32]. The process of endosomal escape in liposomal preparations entails the cytosolic lipidmembrane interactions between liposomes and their encircling endosomes, which cause transient and localized disturbances within the endosomal membrane [33,34]. In this light, a myriad of modifications, such as the integration of fusogenic lipids into liposomes, hold the promise of bolstering endosomal evasion by amplifying the interaction between the endosomal and liposomal membranes [32]. ...
... These lipids are composed of a positively charged amine head group and a hydrocarbon chain or cholesterol derivative, linked together through a linker such as glycerol, making them amphiphilic in nature. The head group of the lipid, which carries a positive charge, has the ability to form an electrostatic interaction with nucleic acids that are negatively charged, thereby enabling them to entrap within lipid-based nanoparticles [119,120]. Ethyl phosphatidylcholine (ePC) ...
Lipid nanoparticles (LNPs) have gained prominence as primary carriers for delivering a diverse array of therapeutic agents. Biological products have achieved a solid presence in clinical settings, and the anticipation of creating novel variants is increasing. These products predominantly encompass therapeutic proteins, nucleic acids and messenger RNA. The advancement of efficient LNP-based delivery systems for biologics that can overcome their limitations remains a highly favorable formulation strategy. Moreover, given their small size, biocompatibility, and biodegradation, LNPs can proficiently transport therapeutic moiety into the cells without significant toxicity and adverse reactions. This is especially crucial for the existing and upcoming biopharmaceuticals since large molecules as a group present several challenges that can be overcome by LNPs. This review describes the LNP technology for the delivery of biologics and summarizes the developments in the chemistry, manufacturing, and characterization of lipids used in the development of LNPs for biologics. Finally, we present a perspective on the potential opportunities and the current challenges pertaining to LNP technology.
... The helper lipid was used to stabilize the mRNA-LNP delivery system, such as 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC) 29 or 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE). 30 These helper lipids not only stabilize the particles but also enhance mRNA delivery efficiency. 31 Ionizable lipids are of great importance for the encapsulation and delivery of mRNAs because their positively charged head groups can interact with the negatively charged phosphate backbone of the mRNA molecule. ...
The fight against COVID-19 pandemic has gained a strong consensus about the importance of developing mRNA vaccines to rapidly respond to an outbreak. Several studies have shown that mRNA vaccines formulated as mRNA-lipid nanoparticles (LNPs) for vaccination can elicit a robust and efficient immune response. In this study, we report the preparation of mRNA-bifunctional lipid nanoparticles (mRNA-BLNPs) as vaccines for targeted delivery to dendritic cells (DCs) to improve safety and enhance immune response. Using this DC-targeted delivery system, mice immunized with SARS-CoV-2 spike mRNA-BLNP vaccine elicited a stronger immune response with higher titer of neutralizing IgG antibody response than the LNP-formulated vaccine against SARS-CoV-2. In addition, the spike mRNA-BLNP vaccine with deletion of glycosites in the stem elicited a broadly protective immune response against SARS-CoV-2 and variants. These findings suggest the importance and potential of developing DC-targeted mRNA vaccines to elicit broadly protective immune responses against human viruses.
... Originally discovered in the 1960s [24], the concept of using liposomes as carriers for gene delivery originated in the 1980s [25][26][27]. Despite early challenges of low transfection efficiency, liposome formulations have been developed to deliver siRNA, plasmid DNA, and mRNA for gene silencing, replacement, and protein expression [25][26][27][28][29][30][31][32][33][34]. Surface modifications of liposomes with ligands, such as antibodies or peptides, have been a focus of research, enabling selective binding to specific receptors on target cells and improving delivery efficiency [12,13,[35][36][37][38]. ...
The CRISPR-based genome editing technology, known as clustered regularly interspaced short palindromic repeats (CRISPR), has sparked renewed interest in gene therapy. This interest is accompanied by the development of single-guide RNAs (sgRNAs), which enable the introduction of desired genetic modifications at the targeted site when used alongside the CRISPR components. However, the efficient delivery of CRISPR/Cas remains a challenge. Successful gene editing relies on the development of a delivery strategy that can effectively deliver the CRISPR cargo to the target site. To overcome this obstacle, researchers have extensively explored non-viral, viral, and physical methods for targeted delivery of CRISPR/Cas9 and a guide RNA (gRNA) into cells and tissues. Among those methods, liposomes offer a promising approach to enhance the delivery of CRISPR/Cas and gRNA. Liposomes facilitate endosomal escape and leverage various stimuli such as light, pH, ultrasound, and environmental cues to provide both spatial and temporal control of cargo release. Thus, the combination of the CRISPR-based system with liposome delivery technology enables precise and efficient genetic modifications in cells and tissues. This approach has numerous applications in basic research, biotechnology, and therapeutic interventions. For instance, it can be employed to correct genetic mutations associated with inherited diseases and other disorders or to modify immune cells to enhance their disease-fighting capabilities. In summary, liposome-based CRISPR genome editing provides a valuable tool for achieving precise and efficient genetic modifications. This review discusses future directions and opportunities to further advance this rapidly evolving field.
... It is worth noting that the structure of CLs can be intentionally modified to enhance their interaction and delivery capabilities for various types of NAs, including pDNA as well as RNA in the form of small interfering RNA (siRNA) and micro RNA (miRNA) (Fig. 1B) [53,106]. While highly effective in binding anionic NAs, CLs are frequently used in combination with ionic phospholipids or non-ionic surfactants to give supramolecular arrangements such as liposomes or niosomes (Fig. 1A), respectively, that allow for improving their delivery performances [107][108][109]. Nevertheless, despite the virtually endless list of vectors available, most gene delivery strategies devised for MSCs of different origins rely on the use of Lipofectamine ® [20,[110][111][112][113][114][115][116][117][118][119][120], the gold standard, commercially-available lipid transfection reagent consisting of a 3:1 (w/w) mixture of the cationic lipid 2,3-dioleoyloxy-N-[2(sperminecarboxiamido) ethyl[-N,N-dimethyl-1-propaniminium trifluoroacetate (DOSPA) and the zwitterionic 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE) [121]. ...
In recent decades, human mesenchymal stem cells (hMSCs) have gained momentum in the field of cell therapy for treating cartilage and bone injuries. Despite the tri-lineage multipotency, proliferative properties, and potent immunomodulatory effects of hMSCs, their clinical potential is hindered by donor variations, limiting their use in medical settings. To address this challenge, gene delivery technologies have emerged as a promising approach to modulate the phenotype and commitment of hMSCs towards specific cell lineages, thereby enhancing osteochondral repair strategies. This review provides a comprehensive overview of current non-viral gene delivery approaches used to engineer MSCs, highlighting key factors such as the choice of nucleic acid or delivery vector, transfection strategies, and experimental parameters. Additionally, it outlines various protocols and methods for qualitative and quantitative evaluation of their therapeutic potential as a delivery system in osteochondral regenerative applications. In summary, this technical review offers a practical guide for optimizing non-viral systems in osteochondral regenerative approaches.
Graphical Abstract
hMSCs constitute a key target population for gene therapy techniques. Nevertheless, there is a long way to go for their translation into clinical treatments. In this review, we remind the most relevant transfection conditions to be optimized, such as the type of nucleic acid or delivery vector, the transfection strategy, and the experimental parameters to accurately evaluate a delivery system. This survey provides a practical guide to optimizing non-viral systems for osteochondral regenerative approaches.
... Some of the commonly used polymers are poly(lacticco-glycolic acid) (PLGA), polycaprolactone (PCL), chitosan, and so on. The most commonly used lipids are lecithin, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, myristic acid, and so on [44][45][46]. Joshi et al. (2013) described the fabrication of hybrid lipopolymeric nanocarrier system consisting carbocymethyl chitosan (CMC), covalently tethered to phosphatidylethanolamine units on the surface of lipid nanovesicles, for oral delivery of paclitaxel. Paclitaxel-loaded carboxymethyl chitosan-hybrid NPs exhibited size in the range of 200-300 nm. ...
Nanocarriers have revolutionized the field of pharmacy and medicine with proven record as versatile platforms in drug delivery. However, single-component nanosystems suffer from certain limitations, namely, low drug loading, physical instability, high immunogenicity, and uneven drug release. The pragmatic approach to overcome these problems associated with single-component nanosystems is to follow a combinatorial approach of hybrid nanocarriers. These hybrid nanosytems not only offer a plethora of advantages like improved drug loading, surface functionality, better drug release profiles, and high entrapment efficiency of multiple therapeutic agents but also help in resolving the drawbacks of single-component nanosystems. Hybrid nanoparticles are structured from the combination of different nanoparticles that altogether act synergistically and, furthermore, impart new characteristics and functionality to the desired system. Hybrid nanoparticles can be categorized into organic (lipid-polymer based), inorganic (metal-based), and gene-coupled nanoparticles. Various hybrid nanoparticles exhibit potential to be employed as diagnostic agents in novel multimodal, biosensing, photoacoustic imaging, and in photodynamic therapy, gene therapy, and management of neurological disorders. This chapter is an attempt to accentuate different types of hybrid nanoparticles and compile the different research investigations pertaining to therapeutic and theranostic applications for intervention of diseases like cancer, neurological disorders, lifestyle disorders, and infectious diseases.KeywordsHybrid nanoparticlesCancerTargeted drug deliveryTheranostics
... Cationic lipids, predominantly DOTAP has been regularly used to prepare cationic liposomes; these present an additional advantage of electrolytically binding with the negatively charged cell membrane [155]. In addition, these cationic liposomes can enhance the loading efficiency of negatively charged protein antigens, and have been regularly used with negatively charged nucleic acid antigens to make lipoplexes [156]. Although cationic lipids and liposomes have significant advantages, the net high positive charge might induce hemolytic and cell lytic properties, which is recognized as a limitation [157]. ...
Bolus vaccines are often administered multiple times due to rapid clearance and reduced transportation to draining lymph nodes resulting in inadequate activation of T and B lymphocytes. In order to achieve adaptive immunity, prolonged exposure of antigens to these immune cells is crucial. Recent research has been focusing on developing long-acting biomaterial-based vaccine delivery systems, which can modulate the release of encapsulated antigens or epitopes to facilitate enhanced antigen presentation in lymph nodes and subsequently achieve robust T and B cell responses. Over the past few years, various polymers and lipids have been extensively explored to develop effective biomaterial-based vaccine strategies. The article reviews relevant polymer and lipid-based strategies used to prepare long-acting vaccine carriers and discusses their results concerning immune responses.
... Lipid-based droplets are essential carriers for mRNA encapsulation, which plays an important role in mRNA delivery. Lipoplexes are formed by cationic liposeomes and the phosphate backbone of mRNA (Elouahabi and Ruysschaert, 2005;Wasungu and Hoekstra, 2006;Guevara et al., 2020). Lipoplexes sometimes perform high instability and low transfection efficiency, and lipid nanoparticles are promising alternatives (Guevara et al., 2020;2019). ...
This review presents an integrated theoretical and computational characterization and analysis of surface pattern formation in chiral and achiral liquid crystal self-assembly and the mechanical/optical/tribological/tissue engineering surface functionalities that emerge from various wrinkling processes. Strategies to target surface patterns include linear, non-linear, multidirectional and multiscale wrinkling phenomena. The focus of the review is to show the unique surface structure-functionalities that emerge from anisotropic liquid crystal soft matter, eliminating or reducing the need of aggressive solvents, extreme pressure/temperature conditions, erosion and other surface morphing approaches. The surface pattern formation theoretical-modelling- computational results are then connected and validated with actual biological surfaces that are considered solid liquid crystal analogues, such as exocuticles of insects, fish scales, and flowers. A unique feature of the in silico surface pattern formation platform used throughout this review is the generalized liquid crystal shape equation that includes surface anchoring elasticity, membrane elasticity, and stress loads from liquid crystals orientation gradients. Clear characterization of surface shapes, curvatures, roughness, that are behind surface functionalities are introduced and applied to strengthen validation of predictions with actual nature’s surfaces. Wrinkling scaling laws, and the dependence of material properties on morphing mechanisms are elucidated. The predictions capture very well the two-scale wrinkling patterns in tulips, wrinkling gradients that display water sensor capabilities, egg carton shapes in rose petals and their potential for cell alignment, and the ability to create surface roughness with targeted kurtosis and skewness to control and optimize friction and tribological functionalities. The results are summarized in terms of surface geometry (open or closed) mechanisms and phenomena (anchoring, membrane elasticity), material properties (anchoring coefficients, membrane bending modulus, Frank elasticity), wrinkling scales and scaling laws (amplitude, wave-lengths, skewness, kurtosis) and functionalities (optical iridescence, friction, wettability, structural color, curvature-driven cell alignment and differentiation). Taken together, the range of surface geometries and surface functionalities captured by the liquid crystal biomimetic in silico platform provides a foundation for future experimental green manufacturing pathways based on anisotropic soft matter.
... If the lipoplexes go through the lysosomal pathway, plasmid DNA may get degraded while reaching the lysosomes by nucleases. Hence, for efficient transfection, plasmid DNA needs to be released to the cytosol from the early endosomes [127]. The pH of early endosomes is slightly acidic (i.e., pH 6.0-6.5) that results in the release of plasmid DNA from the endosomes. ...
... Loading anionic nucleic acids onto cationic delivery systems resulted in an enhanced binding on the cell surface and enhanced entrance of genetic material into the cell by endocytosis [1,2]. Easy manufacturing, low immune response, cost-effectiveness, and safety are some of the advantages of non-viral vectors, representing alternative drug delivery systems to viral delivery of nucleic acids [3][4][5][6][7]. ...
The case of ready to use gene delivery systems like cationic nanoemulsions is not reflecting the truth. Thus, delivery systems for applicating genes like nucleic acids have to be prepared freshly before each application. This study is focused on the preparation and characterization of cationic nanoemulsions using phytosphingosine for plasmid DNA delivery. Repurposing of cationic agents guided us to phytosphingosine, previously used for enhanced interaction with negatively charged surfaces. It was reported that phytosphingosine may act anti-apoptotic, but without using it in an appropriate delivery system like nanoemulsions. This gap attracted our interest about preparing and characterizing long-term stable cationic nanoemulsions and their cytotoxic effects on MDA-MB-231 and MCF-7 breast cancer cells using phytosphingosine. The cationic nanoemulsions 1, 2, and 3 were prepared and characterized in terms of droplet size, polydispersity index, and zeta potential, long-term stability after storage at 25 and 40 °C, complexation with pDNA, release and cytotoxicity on MDA-MB-231 and MCF-7 cells. The CNEs showed appropriate properties like a small droplet size (+30 mV). Unfortunately, each cationic nanoemulsion showed some disadvantages. Cationic nanoemulsion 1 decreased the viability of cancer cells to only 25 %. Phase separation was observed for cationic nanoemulsion 2 after storage of six months at 40 °C. And cationic nanoemulsion 3 was not able to form a complex with pDNA. However, cationic nanoemulsion 1 is more appropriate than the other cationic nanoemulsions for delivering pDNA.
... Cationic/ ionizable lipids can have various dynamic structural phases, including the micellar, lamellar, cubic and inverted hexagonal phase [119]. The type of structure can be predicted by the packing parameter (P), which represents the ratio of the area occupied by the hydrophobic tails versus the hydrophilic head [120][121][122]. When P value exceeds 1, the area occupied by the hydrocarbon lipid tails is much larger than the head group, the lipid tends to adopt the inverted hexagonal phase, which is in favor of endosomal membrane destabilization, which is crucial for LNP gene delivery [112,123]. ...
Hereditary genetic diseases, cancer, and infectious diseases are affecting global health and become major health issues, but the treatment development remains challenging. Gene therapies using DNA plasmid, RNAi, miRNA, mRNA, and gene editing hold great promise. Lipid nanoparticle (LNP) delivery technology has been a revolutionary development, which has been granted for clinical applications, including mRNA vaccines against SARS-CoV-2 infections. Due to the success of LNP systems, understanding the structure, formulation, and function relationship of the lipid components in LNP systems is crucial for design more effective LNP. Here, we highlight the key considerations for developing an LNP system. The evolution of structure and function of lipids as well as their LNP formulation from the early-stage simple formulations to multi-components LNP and multifunctional ionizable lipids have been discussed. The flexibility and platform nature of LNP enable efficient intracellular delivery of a variety of therapeutic nucleic acids and provide many novel treatment options for the diseases that are previously untreatable.
... Cationic nanostructured carriers contain cationic lipids that consist of a cationic head group, a hydrophobic tail, and a linker between these two domains [107,108]. Traditionally, monovalent cationic lipids such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) or 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) have been used to form lipoplexes and are sometimes considered fusogenic [109,110]. These molecules bind to negatively charged polyanionic nucleic acids to carry them across the cellular membrane [107,111,112]. ...
Lysosomal storage disorders are a group of progressive multisystemic hereditary diseases with a combined incidence of 1:4,800. Here we review the clinical and molecular characteristics of these diseases, with a special focus on Mucopolysaccharidoses, caused primarily by the lysosomal storage of glycosaminoglycans. Different gene editing techniques can be used to ameliorate their symptoms, using both viral and nonviral delivery methods. Whereas these are still being tested in animal models, early results of phase I/II clinical trials of gene therapy show how this technology may impact the future treatment of these diseases. Hurdles related to specific hard-to-reach organs, such as the central nervous system, heart, joints, and the eye must be tackled. Finally, the regulatory framework necessary to advance into clinical practice is also discussed.
... Lipid nanoparticle vaccine properties are affected by changes in pH levels, which facilitate mRNA encapsulation and host-cell endocytosis. 34 The mRNA nanoparticle vaccine carrier system recently gained attention following the approval of the first two lipid nanoparticle vaccine-SARS-CoV-2 vaccines. 36,37 Ongoing mRNA nanoparticle vaccine trials are listed in table 1 and clinical trial results are listed in table 3. Two lipid nanoparticle mRNA cancer vaccines encoding several neoantigens (mRNA-4157) are being assessed in combination with pembrolizumab as adjuvant therapy in patients with high-risk cutaneous melanoma following complete resection (NCT03313778 and NCT03897881). ...
Years of research exploring mRNA vaccines for cancer treatment in preclinical and clinical trials have set the stage for the rapid development of mRNA vaccines during the COVID-19 pandemic. Therapeutic cancer vaccines based on mRNA are well tolerated, and the inherent advantage in ease of production, which rivals the best available conventional vaccine manufacture methods, renders mRNA vaccines a promising option for cancer immunotherapy. Technological advances have optimised mRNA-based vaccine stability, structure, and delivery methods, and multiple clinical trials investigating mRNA vaccine therapy are now enrolling patients with various cancer diagnoses. Although therapeutic mRNA-based cancer vaccines have not yet been approved for standard treatment, encouraging results from early clinical trials with mRNA vaccines as monotherapy and in combination with checkpoint inhibitors have been obtained. This Review summarises the latest clinical advances in mRNA-based vaccines for cancer treatment and reflects on future perspectives and challenges for this new and promising treatment approach.
... Finally, the gene must penetrate the nuclear membrane into the nucleus before successfully expressing the nucleolar target protein. 21,22 The idea of gene therapy seems simple, but the easy degradation of genes in blood transport, low cell uptake rate, poor pharmacokinetic properties and other reasons limit the application of naked genes in vivo. 23 Excellent gene vectors must encapsulate genes, prolong blood circulation, protect them from degradation by circulating pathways and mediate genes entry into the nucleus for cellular transcriptional mechanisms. ...
Immunotherapy has offered an alternative therapy method for cancer patients with metastatic tumors or who are not suitable for surgical resection. Different from traditional surgery, radiotherapy and chemotherapy, immunotherapy mainly restores the activity of the body’s own immune cells silenced in the tumor microenvironment to achieve anticancer therapy. Gene therapy which corrects abnormal expression of immune cells in tumor microenvironment by delivering exogenous genes to specific immune cells, is the most widely studied immunotherapy. Although most available gene delivery vectors are still viral vectors, the further application of viral vectors is still limited by the immunogenicity and mutagenesis. Based on this, cationic polymeric gene vectors with high flexibility, high feasibility, low cost and high safety have been widely used in gene delivery. The structural variability of polymers allows specific chemical modifications to be incorporated into polymer scaffolds to improve their physicochemical properties for more stable loading of genes or more targeted delivery to specific cells. In this review, we have summarized the structural characteristics and application potential in cancer immunotherapy of these polymeric gene vectors based on poly([Formula: see text]-lysine), poly(lactic-co-glycolic acid), polyethyleneimine, poly(amidoamine) and hydrogel system.
... The role of this DOPE is attributed to its ability to promote liposome formation and its tendency to transfer liposomes from bilayer structure to hexagonal arrangement at acidic pH levels, which may contribute to intimal fusion or instability (86). Besides, DOPE can also be used as an auxiliary lipid of DOTAP-based liposomes to reduce lipid aggregation (87). ...
Vaccines can prevent many millions of illnesses against infectious diseases and save numerous lives every year. However, traditional vaccines such as inactivated viral and live attenuated vaccines cannot adapt to emerging pandemics due to their time-consuming development. With the global outbreak of the COVID-19 epidemic, the virus continues to evolve and mutate, producing mutants with enhanced transmissibility and virulence; the rapid development of vaccines against such emerging global pandemics becomes more and more critical. In recent years, mRNA vaccines have been of significant interest in combating emerging infectious diseases due to their rapid development and large-scale production advantages. However, their development still suffers from many hurdles such as their safety, cellular delivery, uptake, and response to their manufacturing, logistics, and storage. More efforts are still required to optimize the molecular designs of mRNA molecules with increased protein expression and enhanced structural stability. In addition, a variety of delivery systems are also needed to achieve effective delivery of vaccines. In this review, we highlight the advances in mRNA vaccines against various infectious diseases and discuss the molecular design principles and delivery systems of associated mRNA vaccines. The current state of the clinical application of mRNA vaccine pipelines against various infectious diseases and the challenge, safety, and protective effect of associated vaccines are also discussed.
... Since that time, a large number of commercially available cationic liposome/lipid-based systems, such as Lipofectamine and TransIT TKO, Oligofectamine, Lipofectin, DOTAP and RNAifect, have been developed [105][106][107][108][109]. A major advantage for using liposomes is their flexible physicochemical and biophysical properties, which allows manipulation for different delivery considerations [110]. For instance, adding cholesterol modifiers and/or polymers, such as 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and polyethylene glycol (PEG), to liposomes has reduced inflammatory responses and enhanced the stability of the liposomes and cellular uptake, allowing for more effective delivery and hepatic clearance in vivo [111]. Introducing helper cationic polymers in the formulation has been further shown to increase the siRNA entrapment inside the liposome core [112]. ...
Sickle cell disease (SCD) is one of the most common inherited hemoglobinopathy disorders that affects millions of people worldwide. Reactivation of HBG (HBG1, HBG2) gene expression and induction of fetal hemoglobin (HbF) is an important therapeutic strategy for ameliorating the clinical symptoms and severity of SCD. Hydroxyurea is the only US FDA-approved drug with proven efficacy to induce HbF in SCD patients, yet serious complications have been associated with its use. Over the last three decades, numerous additional pharmacological agents that reactivate HBG transcription in vitro have been investigated, but few have proceeded to FDA approval, with the exception of arginine butyrate and decitabine; however, neither drug met the requirements for routine clinical use due to difficulties with oral delivery and inability to achieve therapeutic levels. Thus, novel approaches that produce sufficient efficacy, specificity, and sustainable HbF induction with low adverse effects are desirable. More recently, microRNAs (miRNAs) have gained attention for their diagnostic and therapeutic potential to treat various diseases ranging from cancer to Alzheimer’s disease via targeting oncogenes and their gene products. Thus, it is plausible that miRNAs that target HBG regulatory genes may be useful for inducing HbF as a treatment for SCD. Our laboratory and others have documented the association of miRNAs with HBG activation or suppression via silencing transcriptional repressors and activators, respectively, of HBG expression. Herein, we review progress made in understanding molecular mechanisms of miRNA-mediated HBG regulation and discuss the extent to which molecular targets of HBG might be suitable prospects for development of SCD clinical therapy. Lastly, we discuss challenges with the application of miRNA delivery in vivo and provide potential strategies for overcoming barriers in the future.
... Cationic liposomes, also known as lipoplexes, can stably compact negatively charged nucleic acids, leading to the formation of nanomeric vesicular structure. 120 Cationic liposomes are commonly produced with a combination of a cationic lipid such as DOTAP, DOTMA, DOGS, DOSPA, and neutral lipids, such as DOPE and cholesterol. 121 These liposomes are stable enough to protect their bound nucleic acids from degradation and are competent to enter cells via endocytosis. ...
Mesenchymal stem cells (MSCs) are considered a promising regenerative therapy due to their ability to migrate toward damaged tissues. The homing ability of MSCs is unique compared with that of non-migrating cells and MSCs are considered promising therapeutic vectors for targeting major cells in many pathophysiological sites. MSCs have many advantages in the treatment of malignant diseases, particularly rheumatoid arthritis (RA). RA is a representative autoimmune disease that primarily affects joints, and secreted chemokines in the joints are well recognized by MSCs following their migration to the joints. Furthermore, MSCs can regulate the inflammatory process and repair damaged cells in the joints. However, the functionality and migration ability of MSCs injected in vivo still show insufficient. The targeting ability and migration efficiency of MSCs can be enhanced by genetic engineering or modification, eg, overexpressing chemokine receptors or migration-related genes, thus maximizing their therapeutic effect. However, there are concerns about genetic changes due to the increased probability of oncogenesis resulting from genome integration of the viral vector, and thus, clinical application is limited. Furthermore, it is suspected that administering MSCs can promote tumor growth and metastasis in xenograft and orthotopic models. For this reason, MSC mimicking nanoencapsulations are an alternative strategy that does not involve using MSCs or bioengineered MSCs. MSC mimicking nanoencapsulations consist of MSC membrane-coated nanoparticles, MSC-derived exosomes and artificial ectosomes, and MSC membrane-fused liposomes with natural or genetically engineered MSC membranes. MSC mimicking nanoencapsulations not only retain the targeting ability of MSCs but also have many advantages in terms of targeted drug delivery. Specifically, MSC mimicking nanoencapsulations are capable of encapsulating drugs with various components, including chemotherapeutic agents, nucleic acids, and proteins. Furthermore, there are fewer concerns over safety issues on MSC mimicking nanoencapsulations associated with mutagenesis even when using genetically engineered MSCs, because MSC mimicking nanoencapsulations use only the membrane fraction of MSCs. Genetic engineering is a promising route in clinical settings, where nano-encapsulated technology strategies are combined. In this review, the mechanism underlying MSC homing and the advantages of MSC mimicking nanoencapsulations are discussed. In addition, genetic engineering of MSCs and MSC mimicking nanoencapsulation is described as a promising strategy for the treatment of immune-related diseases.
... Therefore, DC-Chol was prepared in an acidic or neutral buffer solution to actively facilitate binding affinity for the negatively charged payload. Furthermore, the transfection efficiency was noticeably poor when DW was used as the liposome formation solution (Figure 2a), and this could have been caused by insufficient ionic strength to screen charges and avoid particle aggregation of liposomes before interaction with pDNA [41]. ...
Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.
... The structure of liposomes is the main characteristic necessary for their utilization as a gene or siRNA carrier. The molecular structure of liposomes or amphiphiles affects the packing ability to form self-assemblies. Akin to any amphiphiles, cationic lipids or surfactants can form various structures in an aqueous environment, such as lamellar, micellar, cubic, or inverted hexagonal structures [104]. The form of the assembly can be estimated by the critical packing parameter (CPP), which is based on the fraction of the hydrocarbon volume, multiplication of the effective area of the head group, and critical length of the lipid tail. ...
Small interfering ribonucleic acids (siRNAs) are originally recognized as an intermediate of the RNA interference (RNAi) pathway. They can inhibit or silence various cellular pathways by knocking down specific messenger RNA molecules. In cancer cells, siRNAs can suppress the expression of several multidrug-resistant genes, leading to the increased deposition of chemotherapeutic drugs at the tumor site. siRNA therapy can be used to selectively increase apoptosis of cancer cells or activate an immune response to the cancer. However, delivering siRNAs to the targeted location is the main limitation in achieving safe and effective delivery of siRNAs. This review highlights some representative examples of nonviral delivery systems, especially nanovesicles such as exosomes, liposomes, and niosomes. Nanovesicles can improve the delivery of siRNAs by increasing their intracellular delivery, and they have demonstrated excellent potential for cancer therapy. This review focuses on recent discoveries of siRNA targets for cancer therapy and the use of siRNAs to successfully silence these targets. In addition, this review summarizes the recent progress in designing nanovesicles (liposomes or niosomes) for siRNA delivery to cancer cells and the effects of a combination of anticancer drugs and siRNA therapy in cancer therapy.
Gene therapy is widely recognized as a promising method in combating diseases caused by gene abnormalities or deletions. The effects of these deletions and mutations are ameliorated through gene therapy by means of transfection vectors. These delivery vehicles are tasked with protecting the gene and transporting it to the cell nucleus when necessary. Nano-sized hydrogel particles, also known as nanogels, are crosslinked polymeric nanoparticles that are promising materials for such biomedical applications. Whereas most cationic carriers for gene delivery are nitrogen-based, we are interested in utilizing a sulfonium moiety to this end. Diversifying the available gene vectors not only satisfies scientific curiosity, it could also offer improved gene delivery efficiencies. Here we describe the synthesis of glycidyl methacrylate (GMA) nanogels as a platform for subsequent functionalization. Ring-opening reactions with diethyl sulfide were carried out to install permanent cationic sulfonium groups on the nanogels, yielding readily water-soluble nanogels with a zeta potential of ζ = +40 ± 0.5 mV at neutral pH and a mean diameter of D = 29 ± 10 nm as determined by transmission electron microscopy (TEM). The degree of functionalization with sulfonium groups was found to be tunable. These nanogels were subjected to post-synthesis modifications resulting in biocompatible sulfonium nanogels containing a thioglycerol moiety. Polyplexes were formed by successful incubation with plasmid DNA encoding for green fluorescent protein (pCMV-GFP), at various ratios. In a next step, nucleic acid delivery by sulfonium nanogels was probed for various cell lines for the first time, showing poor delivery properties.
We report the synthesis of a series of amphiphilic p‐sulfonatocalix[4]arenes with varying alkyl chain lengths (CX4‐Cn) and their application as efficient counterion activators for membrane transport of cell‐penetrating peptides (CPPs). The enhanced membrane activity is confirmed with the carboxyfluorescein (CF) assay in vesicles and by the direct cytosolic delivery of CPPs into CHO−K1, HCT 116, and KTC‐1 cells enabling excellent cellular uptake of the CPPs into two cancer cell lines. Intracellular delivery was confirmed by fluorescence microscopy after CPP entry into live cells mediated by CX4‐Cn, which was also quantified after cell lysis by fluorescence spectroscopy. The results present the first systematic exploration of structure‐activity relationships for calixarene‐based counterion activators and show that CX4‐Cn are exceptionally effective in cellular delivery of CPPs. The dodecyl derivative, CX4‐C12, serves as best activator. A first mechanistic insight is provided by efficient CPP uptake at 4 °C and in the presence of the endocytosis inhibitor dynasore, which indicates a direct translocation of the CPP‐counterion complexes into the cytosol and highlights the potential benefits of CX4‐Cn for efficient and direct translocation of CPPs and CPP‐conjugated cargo molecules into the cytosol of live cells.
Despite its high potential, non‐viral gene therapy of cancer remains challenging due to inefficient nucleic acid delivery. Ultrasound (US) with microbubbles (MB) can open biological barriers and thus improve DNA and mRNA passage. Polymeric MB are an interesting alternative to clinically used lipid‐coated MB because of their high stability, narrow size distribution, and easy functionalization. However, besides choosing the ideal MB, it remains unclear whether nanocarrier‐encapsulated mRNA should be administered separately (co‐administration) or conjugated to MB (co‐formulation). Therefore, the impact of poly(n‐butyl cyanoacrylate) MB co‐administration with mRNA‐DOTAP/DOPE lipoplexes or their co‐formulation on the transfection of cancer cells in vitro and in vivo is analyzed. Sonotransfection improved mRNA delivery into 4T1 breast cancer cells in vitro with co‐administration being more efficient than co‐formulation. In vivo, the co‐administration sonotransfection approach also resulted in higher transfection efficiency and reached deeper into the tumor tissue. On the contrary, co‐formulation mainly promoted transfection of endothelial and perivascular cells. Furthermore, the co‐formulation approach is much more dependent on the US trigger, resulting in significantly lower off‐site transfection. Thus, the findings indicate that the choice of co‐administration or co‐formulation in sonotransfection should depend on the targeted cell population, tolerable off‐site transfection, and the therapeutic purpose.
Lipid Nanoparticles (LNPs) are the most advanced non-viral platform for nucleic acid delivery, holding enormous potential applications in clinical therapy. However, some shortcomings remain, such as complex synthetic routes, low...
Gene therapy has garnered a lot of interest in the recent past for the treatment of various life-threatening genetic diseases. Gene therapy involves using genes that should be delivered to the infected cells to treat diseases. The materials used for gene delivery play a vital role in successful gene therapy. The common viruses used in gene delivery are adenovirus, adeno-associated virus, vaccinia virus, retrovirus, etc. This chapter will discuss the different viral and nonviral vectors that are used in gene delivery. Furthermore, lipid-, polymer-, and peptide-based gene delivery methods are discussed in this chapter. The physical method of gene delivery uses various techniques such as electroporation, sonoporation, needle injection, hydroboration, and magneto-fiction. In the future, standard DNA and RNA molecular techniques can be used as the principal mode of treatment in biomedical applications.
The co-assembly of lipids and other compounds has recently gained increasing interest. Here, we report the formation of stimuli-responsive lipid-DNA origami fibers through the electrostatic co-assembly of cationic lipids and 6-helix bundle (6HB) DNA origami. The photosensitive lipid degrades when exposed to UV-A light, which allows a photoinduced, controlled release of the 6HBs from the fibers. The presented complexation strategy may find uses in developing responsive nanomaterials e.g. for therapeutics.
In the realm of gene therapy, a pivotal moment arrived with Paul Berg’s groundbreaking identification of the first recombinant DNA in 1972. This achievement set the stage for future breakthroughs. Conditions once considered undefeatable, like melanoma, pancreatic cancer, and a host of other ailments, are now being addressed at their root cause—the genetic level. Presently, the gene therapy landscape stands adorned with 22 approved in vivo and ex vivo products, including IMLYGIC, LUXTURNA, Zolgensma, Spinraza, Patisiran, and many more. In this comprehensive exploration, we delve into a rich assortment of 16 drugs, from siRNA, miRNA, and CRISPR/Cas9 to DNA aptamers and TRAIL/APO2L, as well as 46 carriers, from AAV, AdV, LNPs, and exosomes to naked mRNA, sonoporation, and magnetofection. The article also discusses the advantages and disadvantages of each product and vector type, as well as the current challenges faced in the practical use of gene therapy and its future potential.
1H NMR relaxometry was applied for molecular-level structural analysis of siRNA-loaded lipid nanoparticles (LNPs) to clarify the impact of the neutral lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, on the physicochemical properties of LNP. Incorporating DSPC and cholesterol in ionizable lipid-based LNP decreased the molecular mobility of ionizable lipids. DSPC reduced the overall molecular mobility of ionizable lipids, while cholesterol specifically decreased the mobility of the hydrophobic tails of ionizable lipids, suggesting that cholesterol filled the gap between the hydrophobic tails of ionizable lipids. The decrease in molecular mobility and change in orientation of lipid mixtures contributed to the maintenance of the stacked bilayer structure of siRNA and ionizable lipids, thereby increasing the siRNA encapsulation efficiency. Furthermore, NMR relaxometry revealed that incorporating those neutral lipids enhanced PEG chain flexibility at the LNP interface. Notably, a small amount of DSPC effectively increased PEG chain flexibility, possibly contributing to the improved dispersion stability and narrower size distribution of LNPs. However, cryogenic transmission electron microscopy represented that adding excess amounts of DSPC and cholesterol into LNP resulted in the formation of deformed particles and demixing cholesterol within the LNP, respectively. The optimal lipid composition of ionizable lipid-based LNPs in terms of siRNA encapsulation efficiency and PEG chain flexibility was rationalized based on the molecular-level characterization of LNPs. Moreover, the NMR relaxation rate of tertiary amine protons of ionizable lipids, which are the interaction site with siRNA, can be a valuable indicator of the encapsulated amount of siRNA within LNPs. Thus, NMR-based analysis can be a powerful tool for efficiently designing LNP formulations and their quality control based on the molecular-level elucidation of the physicochemical properties of LNPs.
Messenger ribonucleic acid (mRNA) was found as the intermediary that transfers genetic information from DNA to ribosomes for protein synthesis in 1961. The emergency use authorization of the two covid-19 mRNA vaccines, BNT162b2 and mRNA-1273, is a significant achievement in the history of vaccine development. Because they are generated in a cell-free environment using the in vitro transcription (IVT) process, mRNA vaccines are risk-free. Moreover, chemical modifications to the mRNA molecule, such as cap structures and changed nucleosides, have proved critical in overcoming immunogenicity concerns, achieving sustained stability, and achieving effective, accurate protein production in vivo. Several vaccine delivery strategies (including protamine, lipid nanoparticles (LNPs), polymers, nanoemulsions, and cell-based administration) were also optimized to load and transport RNA into the cytosol. LNPs, which are composed of a cationic or a pH-dependent ionizable lipid layer, a polyethylene glycol (PEG) component, phospholipids, and cholesterol, are the most advanced systems for delivering mRNA vaccines. Moreover, modifications of the four components that make up the LNPs showed to increase vaccine effectiveness and reduce side effects. Furthermore, the introduction of biodegradable lipids improved LNP biocompatibility. Furthermore, mRNA-based therapies are expected to be effective treatments for a variety of refractory conditions, including infectious diseases, metabolic genetic diseases, cancer, cardiovascular and cerebrovascular diseases. Therefore, the present review aims to provide the scientific community with up-to-date information on mRNA vaccines and their delivery systems.
Approximately 2% to 3% of men and 6% to 7% of women suffer from severe depressive disorders. The existing drugs only partially relieve symptoms for roughly 40% of these patients. The majority of antidepressant drugs are based on theories that are now 50 to 60 years old, and the sector is in critical need of new drug development targets. In the recent decade, numerous genes have been connected to depression in animal models, and serious depression does run in families in humans, indicating both a genetic and environmental component. Depression has been linked to the malfunctioning of serotonin signaling genes, including p11, SERT, etc, according to earlier research. Gene therapy for depression has been found in some instances to be relatively safe, despite the fact that it may seem riskier and more invasive than medication. Hence, there is a growing field regarding the safest delivery mechanisms of these genes that treat major depressive disorders permanently. Hence, the present review summarized the delivery mechanisms of various genes responsible for depressive disorders along with their molecular mechanisms and delivery at the cellular level.
Heterogeneity and drug resistance of tumor cells are the leading causes of incurability and poor survival for patients with recurrent breast cancer. In order to accurately deliver the biological anticancer drugs to different subtypes of malignant tumor cells for omnidirectional targeted treatment of recurrent breast cancer, a distinct design is demonstrated by embedding liposome-based nanocomplexes containing pro-apoptotic peptide and survivin siRNA drugs (LPR) into Herceptin/hyaluronic acid cross-linked nanohydrogels (Herceptin-HA) to fabricate a HER2/CD44-targeted hydrogel nanobot (named as ALPR). ALPR delivered cargoes to the cells overexpressing CD44 and HER2, followed by Herceptin-HA biodegradation, subsequently, the exposed lipid component containing DOPE fused with the endosomal membrane and released peptide and siRNA into the cytoplasm. These experiments indicated that ALPR can specifically deliver Herceptin, peptide, and siRNA drugs to HER2-positive SKBR-3, triple-negative MDA-MB-231, and HER2-negative drug-resistant MCF-7 human breast cancer cells. ALPR completely inhibited the growth of heterogeneous breast tumors via multichannel synergistic effects: disrupting mitochondria, downregulating the survivin gene, and blocking HER2 receptors on the surface of HER2-positive cells. The present design overcomes the chemical drug resistance and opens a feasible route for the combinative treatment of recurrent breast cancer, even other solid tumors, utilizing different kinds of biological drugs.
Lung cancer remains a major public health problem both in terms of incidence and specific mortality despite recent developments in terms of prevention, such as smoking reduction policies and clinical management advances. Better lung cancer prognosis could be achieved by early and accurate diagnosis and improved therapeutic interventions. Nanotechnology is a dynamic and fast-developing field; various medical applications have been developed and deployed, and more exist as proofs of concepts or experimental models. We aim to summarize current knowledge relevant to the use of nanotechnology in lung cancer management. Starting from the chemical structure-based classification of nanoparticles, we identify and review various practical implementations roughly organized as diagnostic or therapeutic in scope, ranging from innovative contrast agents to targeted drug carriers. Available data are presented starting with standards of practice and moving to highly experimental methods and proofs of concept; particularities, advantages, limits and future directions are explored, focusing on the potential impact on lung cancer clinical prognosis.
The recent development of RNA‐based therapeutics in delivering nucleic acids for gene editing and regulating protein translation has led to the effective treatment of various diseases including cancer, inflammatory and genetic disorder, as well as infectious diseases. Among these, lipid nanoparticles (LNP) have emerged as a promising platform for RNA delivery and have shed light by resolving the inherent instability issues of naked RNA and thereby enhancing the therapeutic potency. These LNP consisting of ionizable lipid, helper lipid, cholesterol, and poly(ethylene glycol)‐anchored lipid can stably enclose RNA and help them release into the cells’ cytosol. Herein, the significant progress made in LNP research starting from the LNP constituents, formulation, and their diverse applications is summarized first. Moreover, the microfluidic methodologies which allow precise assembly of these newly developed constituents to achieve LNP with controllable composition and size, high encapsulation efficiency as well as scalable production are highlighted. Furthermore, a short discussion on current challenges as well as an outlook will be given on emerging approaches to resolving these issues.
Several human pathogens can cause long-lasting neurological damage. Despite the increasing clinical knowledge about these conditions, most still lack efficient therapeutic interventions. Gene therapy (GT) approaches comprise strategies to modify or adjust the expression or function of a gene, thus providing therapy for human diseases. Since recombinant nucleic acids used in GT have physicochemical limitations and can fail to reach the desired tissue, viral and non-viral vectors are applied to mediate gene delivery. Although viral vectors are associated to high levels of transfection, non-viral vectors are safer and have been further explored. Different types of nanosystems consisting of lipids, polymeric and inorganic materials are applied as non-viral vectors. In this review, we discuss potential targets for GT intervention in order to prevent neurological damage associated to infectious diseases as well as the role of nanosized non-viral vectors as agents to help the selective delivery of these gene-modifying molecules. Application of non-viral vectors for delivery of GT effectors comprise a promising alternative to treat brain inflammation induced by viral infections.
Temperature and oxidation-sensitive liposomes were prepared by stabilizing dioleoylphophatidylethanolamine (DOPE) bilayers with the ion pair of poly(ethyleneimine)/(phenylthio)acetic acid (PEI/PTA). An upper critical solution temperature (UCST) behavior was observed when PEI/PTA ion pair was suspended in an aqueous solution. It was observed that the UCST increased with increasing PTA content. The ion pair was self-assembled into nanospheres owing to its amphiphilic property which was confirmed by transmission electron microscopy. The FT-IR spectroscopic spectrum showed that the ion pair formed a salt bridge between the amino group and the carboxyl group and the PTA content in the ion pair was readily oxidized by H2O2. Further, DOPE liposomal membranes could be stabilized with PEI/PTA ion pair. Due to the amphiphilic property, the ion pair played a role as a stabilizer for the formation of DOPE liposomes. The liposome released its payload in a temperature-responsive manner, possibly because when the temperature is raised, the ion pair loses its amphiphilic property and can be detached from the liposomal membrane. The liposome was also oxidation-responsive in terms of release, possibly because the amphiphilic property of the ion pair disappears when the PTA is oxidized.
The production of efficient vectors is an essential segment for gene therapy, which is a revolutionary method for delivering therapeutic nucleic acids. Targetable gene therapy is a current challenging task...
Targeted gene delivery is a scientific approach with numerous advantages in the fields of bio- and personalized medicine. The delivery of genes and generally nucleic acids (i.e., DNA, RNA, etc.) into cellular and subcellular organelles for the treatment of several diseases seems to be the future in the design and development of medicines with limited adverse drug reactions and a high ratio of effectiveness and efficacy. The increased impact of the targeted gene delivery is in line with the increased usage of the nanosystems and their applications in the development of gene delivery systems. Lipoplexes and polyplexes are the most important nanosystems that are used for targeted gene delivery. They are composed of lipids (cationic and helper lipids) and polymers (cationic and/or stimuli-responsive polymers), respectively. These cationic materials can self-assemble into complexes in the presence of nucleic acids, which are negatively charged. They are also characterized as nonviral (synthetic) carriers of nucleic acids. This chapter aims to present the technology and the applications of lipoplexes and polyplexes in the field of targeted gene delivery. Special attention will be given to the mechanisms by that lipoplexes and polyplexes are utilized for the delivery and release of the complexed nucleic acids.
Cell Permeation and Oral Bioavailability. Peptide drug discovery has been a source of significant challenge, innovation, and achievement for several decades. This work highlights the breakthrough stage of drug discovery, providing a consolidated resource for drug hunters and academics pursuing peptide drug discovery and targeting challenging intracellular therapeutic targets. Specific articles describe hit finding, computational methods, binding affinihankty, and cellular permeability. Readers seeking to understand state-of-the-art peptide drug discovery and development, as well as those looking for areas of future growth, will find this volume useful.
Lipid‐based nanoparticles have emerged as a clinically viable platform technology to deliver nucleic acids for a wide range of healthcare applications. Within this scope, one of the most exciting areas of recent progress and future innovation potential lies in the material science of lipid‐based nanoparticles, both to refine existing nanoparticle strategies and to develop new ones. Herein, the latest efforts to develop next‐generation lipid‐based nanoparticles are covered by taking a nanoarchitectonics perspective and the design, nucleic acid encapsulation methods, scalable production, and application prospects are critically analyzed for three classes of lipid‐based nanoparticles: 1) traditional lipid nanoparticles (LNPs); 2) lipoplexes; and 3) bicelles. Particular focus is placed on rationalizing how molecular self‐assembly principles enable advanced functionalities along with comparing and contrasting the different nanoarchitectures. The current development status of each class of lipid‐based nanoparticle is also evaluated and possible future directions in terms of overcoming clinical translation challenges and realizing new application opportunities are suggested.
L’injection hydrodynamique par la veine (HLV) est une méthode physique d’administration locorégionale utile en transfert de gènes. Elle permet en effet de cibler l’ensemble de la musculature d’un membre isolé par un garrot via la délivrance d’un volume important contenant des constructions d’acides nucléiques d’intérêt complexés ou non à des vecteurs synthétiques. Cette approche a pour avantages de conserver son efficacité du petit au grand animal tout en étant cliniquement applicable. Elle se révèle donc attractive pour le traitement par thérapie génique de myopathies comme celle de Duchenne. Lorsqu’utilisée dans sa configuration originelle, l’injection HLV permet de transfecter de l’ADN nu avec une certaine efficacité. Cependant, les niveaux de transfection obtenus de la sorte sont limités et insuffisants pour conférer un réel bénéfice thérapeutique. Dans ce contexte, ce travail s’est premièrement intéressé à l’identification des étapes potentiellement limitantes pour le transfert de gènes par administration HLV. Différentes pistes d’optimisations ont été proposées et ensuite testées en utilisant la souris en tant qu’animal modèle. Ainsi, divers vecteurs synthétiques ont été utilisés, notamment des copolymères tribloc amphiphiles neutres, pouvant aider au franchissement du sarcolemme des fibres musculaires. De même, une série d’ADN plasmidiques comportant des séquences spécifiques pouvant faciliter l’étape de l’import nucléaire (appelées « DNA nuclear Targeting Sequences » ou DTS) a été mise au point et évaluée en exploitant la procédure HLV.
Neurological and neuropsychiatric disorders are the main risks for the health care system, exhibiting a huge socioeconomic load. The available range of pharmacotherapeutics mostly provides palliative consequences and fails to treat such conditions. The molecular etiology ofvarious neurological and neuropsychiatric disorders is mostly associated with a change in genetic background, which can be inherited/triggered by other environmental factors. To address such conditions, gene therapy is considered a potential approach claiming a permanent cure of the disease primarily by deletion, silencing, or edition of faulty genes and by insertion of healthier genes. In gene therapy, vectors (viral/non-vial) play an important role in delivering the desired gene to a specific region of the brain. Targeted gene therapy hasunraveled opportunitiesfor the treatment of many neurological and neuropsychiatric disorders. For improved gene delivery, the current techniques are mainly focusing on designing a precise viral vector, plasmid transfection, nanotechnology, microRNA, and in vivo clustered regulatory interspaced short palindromic repeats (CRISPR)-based therapy. These latest techniques have great benefits in treating predominant neurological and neurodevelopmental disorders, including Parkinson's disease, Alzheimer's disease, and autism spectrum disorder, as well as rarer diseases. Nevertheless, all these delivery methods have their limitations, including immunogenic reactions, off-target effects, and a deficiency of effective biomarkers to appreciate the effectiveness of therapy. In this review, we present a summary of the current methods in targeted gene delivery, followed by the limitations and future direction of gene therapy for the cure of neurological and neuropsychiatric disorders.
While delivery of chemotherapeutics to cancer cells by nanomedicines can improve therapeutic outcomes, many fail due to the low drug loading (DL), poor cellular uptake and endosomal entrapment. This study investigated the potential to overcome these limitations using pH-sensitive liposomes (PSL) empowered by the use of calcium acetate. An acidic dinitrobenzamide mustard prodrug SN25860 was used as a model drug, with non pH-sensitive liposomes (NPSL) as a reference. Calcium acetate as a remote loading agent allowed to engineer PSL- and NPSL-SN25860 with DL of > 31.1% (w/w). The IC50 of PSL-SN25860 was 21- and 141-fold lower than NPSL and free drug, respectively. At 48 h following injection of PSL-SN25860, NPSL-SN25860 and the free drug, drug concentrations in EMT6-nfsB murine breast tumors were 56.3 µg/g, 6.76 µg/g and undetectable (< 0.015 µg/g), respectively (n = 3). Meanwhile, the ex vivo tumor clonogenic assay showed 9.1%, 19.4% and 42.7% cell survival in the respective tumors. Live-cell imaging and co-localization analysis suggested endosomal escape was accomplished by destabilization of PSL followed by release of Ca²⁺ in endosomes allowing induction of a proton sponge effect. Subsequent endosomal rupture was observed approximately 30 min following endocytosis of PSL containing Ca²⁺. Additionally, calcium in liposomes promoted internalization of both PSL and NPSL. Taken together, this study demonstrated multifaceted functions of calcium acetate in promoting drug loading into liposomes, cellular uptake, and endosomal escape of PSL for efficient cytoplasmic drug delivery. The results shed light on designing nano-platforms for cytoplasmic delivery of various therapeutics.
Graphical abstract
The process by which viruses destabilize endosomal membranes in an acidification dependent manner has been mimicked with synthetic peptides that are able to disrupt liposomes, erythrocytes, or endosomes of cultured cells. Peptides containing the 20 amino-terminal amino acid sequence of influenza virus hemagglutinin as well as acidic derivatives showed erythrocyte lysis activity only when peptides were elongated by an amphipathic helix or by carboxyl-terminal dimerization. Interestingly, peptides consisting of the 23 amino-terminal amino acids of influenza virus hemagglutinin were also active in erythrocyte lysis. When peptides were in corporated into DNA complexes that utilize a receptor-mediated endocytosis pathway for uptake into cultured cells, either by ionic interaction with positively charged polylysine-DNA complexes or by a streptavidin-biotin bridge, a strong correlation between pH-specific erythrocyte disruption activity and gene transfer was observed. A high-level expression of luciferase or interleukin-2 was obtained with optimized gene transfer complexes in human melanoma cells and several cell lines.
Noninvasive optical bioluminescence imaging systems are important tools for evaluating gene expression in vivo for study of individual and temporal variation in a living animal. In this report, we demonstrate that expression of the firefly luciferase reporter gene (fl) delivered by transferrin (Tf) targeted polyethylenimine (PEI) complexes with, or without, poly(ethylene glycol) (PEG) modifications can be imaged in living A/J mice bearing N2A tumors using a cooled charged coupled device (CCD) camera. Tf-PEI-PEG, Tf-PEI, and PEI (positive control) complexes were tail-vein injected and mice were imaged at 5, 24, 48, and 72 h after complex injection. After imaging, the organs were analyzed ex vivo for firefly luciferase protein (FL) activity. The Tf and PEG modified formulations show significantly (P<0.05) higher FL activity in vivo and ex vivo at the tumor as compared to other organs, including the lungs (a site of high expression with PEI, the positive control). Furthermore, the in vivo bioluminescent signal correlated well (R(2)=0.83) with ex vivo FL activity. These data support that noninvasive imaging of fl reporter expression can be used to monitor the specificity of Tf-PEI and Tf-PEI-PEG polyplex targeting of N2A tumors in A/J mice
In 1989, Rosenberg et al. performed the first human gene therapy trial when they used a retrovirus to introduce the gene coding for resistance to neomycin into human tumor-infiltrating lymphocytes before infusing them into five patients with advanced melanoma (N Engl J Med. 1990, 323(9): 570-8). This study demonstrated the feasibility of using retroviral gene transduction in humans and set the stage for further studies. Since then, over 900 clinical trials have been completed, are ongoing or have been approved worldwide. These trials have been designed to establish feasibility and safety, to demonstrate the reality of expression of therapeutic protein(s) in vivo by the genes transferred and, in some cases, to show therapeutic benefit. In 1990, 2 trials were initiated. The number then climbed rapidly as hopes for early therapeutic success prompted an ever-increasing number of trials. The peak was reached in 1999 with 113 trials initiated. With the realisation that numerous obstacles needed to be overcome, and the occurrence of some untoward effects, the pace slowed somewhat with 53 trials approved in 2003.
Parameters that affect cellular transfection as accomplished by introducing DNA via carriers composed of cationic synthetic amphiphiles, have been investigated, with the aim to obtain insight into the mechanism of DNA translocation. Such insight may be exploited in optimizing carrier properties of synthetic amphiphiles for molecules other than nucleic acids. In the present work, the interaction of vesicles composed of the cationic amphiphile dioleyloxy-propyl-trimethylammonium chloride (DOTMA) with cultured cells was examined. The results show that optimal transfection is dependent on the concentration of lipid, which determines the efficiency of vesicle interaction with the target cell membrane, as well as the toxicity of the amphiphiles towards the cell. A low lipid/DNA ratio prevents the complex from interacting with the cell surface, whereas at a relatively high amphiphile concentration the complex becomes toxic. Translocation efficiency is independent of the initial vesicle size but is affected by the size of the DNA. An incubation time of the DNA/amphiphile complex and cells of approx. 2-4 h is required for obtaining efficient transfection. In conjunction with observations on DNA/amphiphile complex-induced hemolysis of erythrocytes, a mechanism of DNA-entry is proposed which involves translocation of the nucleic acids through pores across the membranes rather than delivery via fusion or endocytosis. Dioleoylphosphatidylethanolamine, a phospholipid frequently used in a mixture with DOTMA ('lipofectin') strongly facilitates this pore formation. Translocation of the DNA is effectively prevented when the cells are pretreated with Ca2+ or pronase. These observations suggest that Ca(2+)-sensitive cell surface proteins play a role in amphiphile-mediated DNA translocation.
Cationic lipids are widely used for gene transfer in vitro and show promise as a vector for in vivo gene therapy applications. However, there is limited understanding of the cellular and molecular mechanisms involved. We investigated the individual steps in cationic lipid-mediated gene transfer to cultured cell lines. We used DMRIE/DOPE (a 1:1 mixture of N-[1-(2,3-dimyristyloxy) propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE) and dioleoyl phosphatidylethanolamine (DOPE) as a model lipid because of its efficacy and because it is being used for clinical trials in humans. The data show that cationic lipid-mediated gene transfer is an inefficient process. Part of the inefficiency may result from the fact that the population of lipid-DNA complexes was very heterogeneous, even under conditions that have been optimized to produce the best transfection. Inefficiency was not due to inability of the complex to enter the cells because most cells took up the DNA. However, in contrast to previous speculation, the results indicate that endocytosis was the major mechanism of entry. After endocytosis, the lipid-DNA aggregated into large perinuclear complexes, which often showed a highly ordered tubular structure. Although much of the DNA remained aggregated in a vesicular compartment, there was at least a small amount of DNA in the cytoplasm of most cells. That observation plus results from direct injection of DNA and lipid-DNA into the nucleus and cytoplasm indicate that movement of DNA from the cytoplasm to the nucleus may be one of the most important limitations to successful gene transfer. Finally, before transcription can occur, the data show that lipid and DNA must dissociate. These results provide new insights into the physical limitations to cationic lipid-mediated gene transfer and suggest that attention to specific steps in the cellular process may further improve the efficiency of transfection and increase its use in a number of applications.
Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.
The process by which viruses destabilize endosomal membranes in an acidification-dependent manner has been mimicked with synthetic peptides that are able to disrupt liposomes, erythrocytes, or endosomes of cultured cells. Peptides containing the 20 amino-terminal amino acid sequence of influenza virus hemagglutinin as well as acidic derivatives showed erythrocyte lysis activity only when peptides were elongated by an amphipathic helix or by carboxyl-terminal dimerization. Interestingly, peptides consisting of the 23 amino-terminal amino acids of influenza virus hemagglutinin were also active in erythrocyte lysis. When peptides were incorporated into DNA complexes that utilize a receptor-mediated endocytosis pathway for uptake into cultured cells, either by ionic interaction with positively charged polylysine-DNA complexes or by a streptavidin-biotin bridge, a strong correlation between pH-specific erythrocyte disruption activity and gene transfer was observed. A high-level expression of luciferase or interleukin-2 was obtained with optimized gene transfer complexes in human melanoma cells and several cell lines.
To understand how DNA is released from cationic liposome/DNA complexes in cells, we investigated which biomolecules mediate release of DNA from a complex with cationic liposomes. Release from monovalent[1,2-dioleoyl-3(1)-1(trimethylammonio)propane] or multivalent (dioctadecylamidoglycylspermine) lipids was quantified by an increase of ethidium bromide (EtBr) fluorescence. Plasmid sensitivity to DNAse I degradation was examined using changes in plasmid migration on agarose gel electrophoresis. Physical separation of the DNA from the cationic lipid was confirmed and quantified on sucrose density gradients. Anionic liposomes containing compositions that mimic the cytoplasmic-facing monolayer of the plasma membrane (e.g. phosphatidylserine) rapidly released DNA from the complex. Release occurred near a 1/1 charge ratio (-/+) and was unaffected by ionic strength or ion type. Water soluble molecules with a high negative linear charge density such as dextran sulfate or heparin also released DNA. However, ionic water soluble molecules such as ATP, tRNA, DNA, poly(glutamic acid), spermidine, spermine, or histone did not, even at 100-fold charge excess (-/+). On the basis of these results, we propose that after the cationic lipid/DNA complex is internalized into cells by endocytosis it destabilizes the endosomal membrane. Destabilization induces flip-flop of anionic lipids from the cytoplasmic-facing monolayer, which laterally diffuse into the complex and form a charge neutral ion pair with the cationic lipids. This results in displacement of the DNA from the cationic lipid and release of the DNA into cytoplasm. This mechanism accounts for a variety of observations on cationic lipid/DNA complex-cell interactions.
Self-assembling polycation/DNA complexes represent a promising synthetic vector for gene delivery. However, despite considerable versatility and transfectional activity in vitro, such materials are quickly eliminated from the bloodstream following intravenous injection (plasma alpha half-life typically less than 5 min). For targeted systemic delivery a more prolonged plasma circulation of the vector is essential. Here we have examined factors contributing to rapid elimination of poly(L-lysine) (pLL)/DNA complexes from the bloodstream, and implicate the binding of proteins to the polyelectrolyte complexes as a likely cause for their blood clearance. pLL/DNA complexes reisolated from serum associate with several proteins, depending on their net charge, although the major band on SDS-PAGE co-migrates with albumin. Serum albumin binds to pLL/DNA complexes in vitro, forming a ternary pLL/DNA/albumin complex which regains some ethidium bromide fluorescence and fails to move during agarose electrophoresis. Albumin also causes increased turbidity of complexes, and reduces their zeta potential to the same level (-16 mV) as is measured in serum. We propose that rapid plasma elimination of polycation/DNA complexes results from their binding serum albumin and other proteins, perhaps due to aggregation and phagocytic capture or accumulation of the ternary complexes in fine capillary beds.
Intravenous gene delivery via cationic lipidic vectors gives systemic gene expression particularly in the lung. In order to understand the mechanism of intravenous lipofection, a systematic study was performed to investigate the interactions of lipidic vectors with mouse serum emphasizing how serum affects the biophysical and biological properties of vectors of different lipid compositions. Results from this study showed that lipidic vectors underwent dynamic changes in their characteristics after exposure to serum. Addition of lipidic vectors into serum resulted in an immediate aggregation of vectors. Prolonged incubation of lipidic vectors with serum led to vector disintegration as shown in turbidity study, sucrose-gradient centrifugation analysis and fluorescence resonance energy transfer (FRET) study. Vector disintegration was associated with DNA release and degradation as shown in EtBr intercalation assay and DNA digestion study. Serum-induced disintegration of vectors is a general phenomenon for all cationic lipidic vectors tested in this study. Yet, vectors of different lipid compositions vary greatly in the rate of disintegration. There is an inverse correlation between the disintegration rate of lipidic vectors and their in vivo transfection efficiency. Vectors with a rapid rate of disintegration such as those containing dioleoyl-phosphatidylethanolamine (DOPE) poorly stayed in the lung and were barely active in transfecting cells. In contrast, cholesterol-containing vectors that had a rapid aggregation and a slow disintegration were highly efficient in transfecting cells in vivo. The results of this study explain why cationic lipidic vectors of different lipid compositions have a dramatic difference in their in vivo transfection efficiency. These results also suggest that the study of the interactions of lipidic vectors with serum may serve as a predictive model for the in vivo efficiency of a lipidic vector. Further study of the numerous interactions of lipidic vectors with serum might lead to the development of a vector which can deliver a gene to target cells in a tissue-specific manner.
Association of a targeting ligand such as transferrin, or an endosome disrupting peptide such as GALA, with cationic liposome-DNA complexes ('lipoplexes') results in a significant enhancement of transfection of several cell types (Simões S et al, Gene Therapy 1998; 5: 955-964). Although these strategies can overcome some of the barriers to gene delivery by lipoplexes, the mechanisms by which they actually enhance tranfection is not known. In studies designed to establish the targeting specificity of transferrin, we found that apo-transferrin enhances transfection to the same extent as transferrin, indicating that internalization of the lipoplexes is mostly independent of transferrin receptors. These observations were reinforced by results obtained from competitive inhibition studies either by preincubating the cells with an excess of free ligand or with various 'receptor-blocking' lipoplexes. Transfection of cells in the presence of drugs that interfere with the endocytotic pathway provided additional insights into the mechanisms of gene delivery by transferrin- or GALA-lipoplexes. Our results indicate that transferrin-lipoplexes deliver transgenes by endocytosis primarily via a non-receptor-mediated mechanism, and that acidification of the endosomes is partially involved in this process.
The interaction of DNA with a novel cationic phospholipid transfection reagent, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC), was investigated by monitoring thermal effects, particle size, vesicle rupture, and lipid mixing. By isothermal titration calorimetry, the heat of interaction between large unilamellar EDOPC vesicles and plasmid DNA was endothermic at both physiological and low ionic strength, although the heat absorbed was slightly larger at the higher ionic strength. The energetic driving force for DNA-EDOPC association is thus an increase in entropy, presumably due to release of counterions and water. The estimated minimum entropy gain per released counterion was 1.4 cal/mole- degrees K (about 0.7 kT), consistent with previous theoretical predictions. All experimental approaches revealed significant differences in the DNA-lipid particle, depending upon whether complexes were formed by the addition of DNA to lipid or vice versa. When EDOPC vesicles were titrated with DNA at physiological ionic strength, particle size increased, vesicles ruptured, and membrane lipids became mixed as the amount of DNA was added up to a 1.6:1 (+:-) charge ratio. This charge ratio also corresponded to the calorimetric end point. In contrast, when lipid was added to DNA, vesicles remained separate and intact until a charge ratio of 1:1 (+:-) was exceeded. Under such conditions, the calorimetric end point was 3:1 (+:-). Thus it is clear that fundamental differences in DNA-cationic lipid complexes exist, depending upon their mode of formation. A model is proposed to explain the major differences between these two situations. Significant effects of ionic strength were observed; these are rationalized in terms of the model. The implications of the analysis are that considerable control can be exerted over the structure of the complex by exploiting vectorial preparation methods and manipulating ionic strength.
The mechanism whereby cationic lipids destabilize cell membranes to facilitate the intracellular delivery of macromolecules such as plasmid DNA or antisense oligonucleotides is not well understood. Here, we show that cationic lipids can destabilize lipid bilayers by promoting the formation of nonbilayer lipid structures. In particular, we show that mixtures of cationic lipids and anionic phospholipids preferentially adopt the inverted hexagonal (H(II)) phase. Further, the presence of 'helper' lipids such as dioleoylphosphatidylethanolamine or cholesterol, lipids that enhance cationic lipid-mediated transfection of cells also facilitate the formation of the H(II)phase. It is suggested that the ability of cationic lipids to promote nonbilayer structures in combination with anionic phospholipids leads to disruption of the endosomal membrane following uptake of nucleic acid-cationic lipid complexes into cells, thus facilitating cytoplasmic release of the plasmid or oligonucleotide.
Pyridinium amphiphiles, abbreviated as SAINT, are highly efficient vectors for delivery of DNA into cells. Within a group of structurally related compounds that differ in transfection capacity, we have investigated the role of the shape and structure of the pyridinium molecule on the stability of bilayers formed from a given SAINT and dioleoylphosphatidylethanolamine (DOPE) and on the polymorphism of SAINT/DOPE-DNA complexes. Using electron microscopy and small angle x-ray scattering, a relationship was established between the structure, stability, and morphology of the lipoplexes and their transfection efficiency. The structure with the lowest ratio of the cross-sectional area occupied by polar over hydrophobic domains (SAINT-2) formed the most unstable bilayers when mixed with DOPE and tended to convert into the hexagonal structure. In SAINT-2-containing lipoplexes, a hexagonal topology was apparent, provided that DOPE was present and complex assembly occurred in 150 mm NaCl. If not, a lamellar phase was obtained, as for lipoplexes prepared from geometrically more balanced SAINT structures. The hexagonal topology strongly promotes transfection efficiency, whereas a strongly reduced activity is seen for complexes displaying the lamellar topology. We conclude that in the DOPE-containing complexes the molecular shape and the nonbilayer preferences of the cationic lipid control the topology of the lipoplex and thereby the transfection efficiency.
Synthetic amphiphiles are widely used as a carrier system. However, to match transfection efficiencies as obtained for viral vectors, further insight is required into the properties of lipoplexes that dictate transfection efficiency, including the mechanism of delivery. Although endocytosis is often referred to as the pathway of lipoplex entry and transfection, its precise nature has been poorly defined. Here, we demonstrate that lipoplex-mediated transfection is inhibited by more than 80%, when plasma membrane cholesterol is depleted with methyl-beta-cyclodextrin. Cholesterol replenishment restores the transfection capacity. Investigation of the cellular distribution of lipoplexes after cholesterol depletion revealed an exclusive inhibition of internalization, whereas cell-association remained unaffected. These data strongly support the notion that complex internalization, rather than the direct translocation of plasmid across the plasma membrane, is a prerequisite for accomplishing effective lipoplex-mediated transfection. We demonstrate that internalized lipoplexes colocalize with transferrin in early endocytic compartments and that lipoplex internalization is inhibited in potassium-depleted cells and in cells overexpressing dominant negative Eps15 mutants. In conjunction with the notion that caveolae-mediated internalization can be excluded, we conclude that efficient lipoplex-mediated transfection requires complex internalization via the cholesterol-dependent clathrin-mediated pathway of endocytosis.
Bacteriorhodopsin is a proton-transporting membrane protein in Halophilic archaea, and it is considered a prototype of membrane transporters and a model for G-protein-coupled receptors. Oligomerization of the protein has been reported, but it is unknown whether this feature is correlated with, for instance, light activation. Here, we have addressed this issue by reconstituting bacteriorhodopsin into giant unilamellar vesicles. The dynamics of the fully active protein was investigated using fluorescence correlation spectroscopy and freeze fracture electron microscopy. At low protein-to-lipid ratios (<1:10 w/w), a decrease in mobility was observed upon protein photoactivation. This process occurred on a second time scale and was fully reversible, i.e. when the dark-adapted state was reestablished the lateral diffusion rate of the protein was returned to that prior to activation. A similar decrease in lateral mobility as observed upon photoactivation was obtained when bacteriorhodopsin was reconstituted at high protein-to-lipid ratios (>1:10 w/w). We interpret the shifts in mobility during light adaptation as being caused by transient photoinduced oligomerization of bacteriorhodopsin. These observations are fully supported by freeze-fracture electron microscopy, and the size of the clusters during photoactivation was estimated to consist of two or three trimers.
The plasma membrane is the interface between cells and their harsh environment. Uptake of nutrients and all communication among cells and between cells and their environment occurs through this interface. 'Endocytosis' encompasses several diverse mechanisms by which cells internalize macromolecules and particles into transport vesicles derived from the plasma membrane. It controls entry into the cell and has a crucial role in development, the immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. As the complexity of molecular interactions governing endocytosis are revealed, it has become increasingly clear that it is tightly coordinated and coupled with overall cell physiology and thus, must be viewed in a broader context than simple vesicular trafficking.
Non-phagocytic eukaryotic cells can internalize particles <1 microm in size, encompassing pathogens, liposomes for drug delivery or lipoplexes applied in gene delivery. In the present study, we have investigated the effect of particle size on the pathway of entry and subsequent intracellular fate in non-phagocytic B16 cells, using a range of fluorescent latex beads of defined sizes (50-1000 nm). Our data reveal that particles as large as 500 nm were internalized by cells via an energy-dependent process. With an increase in size (50-500 nm), cholesterol depletion increased the efficiency of inhibition of uptake. The processing of the smaller particles was significantly perturbed upon microtubule disruption, while displaying a negligible effect on that of the 500 nm beads. Inhibitor and co-localization studies revealed that the mechanism by which the beads were internalized, and their subsequent intracellular routing, was strongly dependent on particle size. Internalization of microspheres with a diameter <200 nm involved clathrin-coated pits. With increasing size, a shift to a mechanism that relied on caveolae-mediated internalization became apparent, which became the predominant pathway of entry for particles of 500 nm in size. At these conditions, delivery to the lysosomes was no longer apparent. The data indicate that the size itself of (ligand-devoid) particles can determine the pathway of entry. The clathrin-mediated pathway of endocytosis shows an upper size limit for internalization of approx. 200 nm, and kinetic parameters may determine the almost exclusive internalization of such particles along this pathway rather than via caveolae.
GALA is a 30 amino acid synthetic peptide with a glutamic acid-alanine-leucine-alanine (EALA) repeat that also contains a histidine and tryptophan residue as spectroscopic probes. It was designed to explore how viral fusion protein sequences interact with membranes. The sequence selected was long enough to span a bilayer in the alpha-helix, the glutamic acids (Glu) were selected to provide a pH-dependent negatively charged side-chain and the EALA repeat was adjusted so that the peptide would have a hydrophobic face of sufficient hydrophobicity to interact with the bilayer when the peptide was in an alpha-helix. GALA converts from a random coil to an amphipathic alpha-helix when the pH is reduced from 7.0 to 5.0. At neutral pH, GALA is water soluble while at acid pH, GALA binds to bilayer membranes. The nature of the association and the type of peptide-peptide interactions in the membrane depend upon the physico-chemical properties of the bilayer such as the acyl chain composition of the phospholipids and the presence of cholesterol. Neutral and negatively charged bilayers composed of saturated phospholipids of 14-16 acyl chain length are solubilized into peptide-lipid discs by GALA. GALA can induce fusion between small unilamellar vesicles (SUV) composed of unsaturated phospholipids. Most importantly GALA forms a transmembrane peptide pore comprised of approximately 10 GALA alpha-helical monomers that are arrayed in an alpha-helix perpendicular to the plane of the membrane. Membrane leakage from neutral or negatively charged vesicles at pH 5.0 can be adequately explained by a mathematical model assuming that GALA becomes incorporated into the vesicle bilayer and aggregates to form a transbilayer pore consisting of 10 (+/-2) peptides. The lipid compositions of model bilayer have important effects on the GALA transbilayer insertion mechanism and peptide orientation. Insertion of the pore into the membrane dramatically accelerates transmembrane phospholipid flip-flop. Cationic peptides designed based upon GALA but containing a lysine-alanine-leucine-alanine (KALA) motif can interact with nucleic acids and perturb biomembranes. The pH-controlled membrane permealization induced by GALA and related peptides serve as a paradigm for the design of environmentally responsive peptidic delivery vehicles for drugs and genes.
In a recent report, we presented data on the rich and unusual pH-dependent aggregation behavior of a sugar-based (reduced glucose) gemini surfactant (Johnsson et al. J. Am. Chem. Soc. 2003, 125, 757). In the present study, we extend the previous investigation by introducing a different sugar headgroup (reduced mannose), by varying the spacer between the two main surfactant parts, and by introducing, in one of the surfactants, an amide linkage (instead of an amine linkage) between the headgroup and the unsaturated (C18:1) hydrocarbon tails. The aggregation behavior of these four gemini surfactants has been studied and compared by means of light scattering, cryo-transmission electron microscopy, electrophoretic mobility, and fluorescence measurements. We find that all four surfactants form vesicles near neutral or high pH. However, the vesicles made from the amine-containing geminis are transformed into cylindrical or wormlike micelles at lower pH values (pH < ~5.5). The micellization is driven mainly by an increased electrostatic repulsion, caused by the protonation of the tertiary amino groups, and we find that the nature of the sugar or spacer has little influence on this process. At low pH (pH 2), solely small globular micelles are found, and the critical micelle concentration at this pH is about 0.005-0.010 mM for the different amine-containing surfactants. As was expected, the gemini surfactant with the amide instead of the amine functional groups in the headgroup does not undergo the vesicle-to-micelle transition but displays only vesicle formation within the investigated pH range. The electrophoretic mobility measurements on the vesicular samples formed from the amine-containing geminis show that the vesicles are cationic below pH ~7-7.5; however, the vesicles acquire a substantial negative charge at a higher pH. The most probable explanation for this charge reversal is a strong adsorption (or binding) of hydroxide ions onto the vesicle surface. In accordance with this hypothesis, we find that the vesicles made from the amide-containing gemini are anionic (no protonation) even at a low pH (pH <5). Using a simple Poisson-Boltzmann model, we are able to describe the obtained ζ-potential profiles reasonably well and derive a hydroxide-ion binding constant (KOH) for the respective systems. We find that the nature of the sugar does have a small influence on KOH. The colloidal stability of all four types of the gemini vesicles seems to be well-described by the classical Derjaguin-Landau-Verwey-Overbeek theory, and the vesicles aggregate/flocculate rapidly in the limit of low surface potential. However, the flocculated vesicles can be easily redispersed by, for example, raising the pH of the solution, and this flocculation/redispersal process is completely reversible.
A number of recent studies have provided new insights into the complexity of the endocytic pathways originating at the plasma membrane of mammalian cells. Many of the molecules involved in clathrin coated pit internalization are now well understood but other pathways are less well defined. Caveolae appear to represent a low capacity but highly regulated pathway in a restricted set of tissues in vivo. A third pathway, which is both clathrin- and caveolae-independent, may constitute a specialized high capacity endocytic pathway for lipids and fluid. The relationship of this pathway, if any, to macropinocytosis or to the endocytic pathways of lower eukaryotes remains an interesting open question. Our understanding of the regulatory mechanisms and molecular components involved in this pathway are at a relatively primitive stage. In this review, we will consider some of the characteristics of different endocytic pathways in high and lower eukaryotes and consider some of the common themes in endocytosis. One theme which becomes apparent from comparison of these pathways is that apparently different pathways can share common molecular machinery and that pathways considered to be distinct actually represent similar basic pathways to which additional levels of regulatory complexity have been added. (c) 2005 Elsevier B.V. All rights reserved.
The present study is aimed to characterize the interactions between plasmid DNA and cationic, large unilamellar vesicles, 110±20nm in size, composed of lipids commonly used for transfections including DOTAP/DOPE (mole ratio 1/1), DOTAP/DOPC (mole ratio 1/1), 100% DOTAP, or DC-CHOL/DOPE (mole ratio 1/1). [Abbreviations: DOTAP, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidyl-ethanolamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DC-CHOL, 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl] cholesterol]. A novel approach of combining Gouy–Chapman calculations and fluorescence measurements of the pH at the surface of lipid assemblies by the fluorophore 4-heptadecyl-7-hydroxycoumarin showed that electrostatic parameters played a key role in the instantaneous formation of the DNA–lipid complexes upon addition of different amounts of plasmid DNA to cationic liposomes in 20mM Hepes buffer (pH 7.4). Addition of large amounts of plasmid DNA leads to neutralization of 60% of the protonated DC-CHOL in DC-CHOL/DOPE (1/1) assemblies and 80% of the DOTAP in lipid assemblies. The characterization of these electrostatic parameters of the complexes suggests better and closer surrounding of plasmid DNA by lipids when DOPE is present. Time-dependent static light-scattering measurements monitored the formation of complexes and also showed that these complexes were highly unstable with respect to size at DNA/cationic lipid molar ratios between 0.2 and 0.8.
The incorporation of polymer–lipid conjugates, initially using PEG and subsequently other selected flexible, hydrophilic polymers, into lipid bilayers gives rise to sterically stabilized liposomes that exhibit reduced blood clearance and concomitant changes in tissue distribution largely because of reduced, but not eliminated, phagocytic uptake. Changes in tissue distribution includes `passive' targeting localization into sites of tumors, infection, inflammation characterized by presence of a `leaky' vasculature which represent useful applications for drug delivery. The polymer forms a surface coating which has been characterized by physical measurements and it appears to function through steric inhibition of the protein binding and cellular interactions leading to phagocytic uptake. The current understanding of the physical and biological properties are reviewed. Ongoing work in the field involves interests to increase complexity such as addition of (1) selective targeting ligands by chemical conjugation to the exterior surface of the polymer coating, (2) capabilities for intracellular release of encapsulated agents into the cytoplasm, and (3) both simultaneously.
Cationic liposome–DNA complexes (‘lipoplexes’) are used as gene delivery vehicles and may overcome some of the limitations of viral vectors for gene therapy applications. The interaction of highly positively charged lipoplexes with biological macromolecules in blood and tissues is one of the drawbacks of this system. We examined whether coating cationic liposomes with human serum albumin (HSA) could generate complexes that maintained transfection activity. The association of HSA with liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane and dioleoylphosphatidylethanolamine, and subsequent complexation with the plasmid pCMVluc greatly increased luciferase expression in epithelial and lymphocytic cell lines above that obtained with plain lipoplexes. The percentage of cells transfected also increased by an order of magnitude. The zeta potential of the ternary complexes was lower than that of the lipoplexes. Transfection activity by HSA-lipoplexes was not inhibited by up to 30% serum. The combined use of HSA and a pH-sensitive peptide resulted in significant gene expression in human primary macrophages. HSA-lipoplexes mediated significantly higher gene expression than plain lipoplexes or naked DNA in the lungs and spleen of mice. Our results indicate that negatively charged HSA-lipoplexes can facilitate efficient transfection of cultured cells, and that they may overcome some of the problems associated with the use of highly positively charged complexes for gene delivery in vivo.
1.1. The polymorphic phase behaviour of bovine brain sphingomyelin alone and in the presence of soya phosphatidylethanolamine (which prefers the hexagonal (HII) configuration in isolation) has been investigated employing 31P NMR techniques. The influence of cholesterol on the phase behaviour of these systems has also been characterized.2.2. The 31P NMR spectra obtained from dry and hydrated sphingomyelin indicate that the local motion and conformation in the phosphate group region is similar to that of glycerol-based phospholipids. In addition, in the presence of excess water the bulk of the sphingomyelin displays 31P NMR spectra consistent with the bilayer configuration. Further, a hydrocarbon phase transition is apparent as the temperature is increased through 35°C, in agreement with calorimetric results. This transition is removed by equimolar cholesterol.3.3. In the absence of cholesterol, bovine brain sphingomyelin has a similar ability to stabilize the bilayer configuration when mixed with soya phosphatidylethanolamine as does 16 : 0/16 : 0 or egg yolk phosphatidylcholine, and relatively complete bilayer stabilization occurs at 30 mol% or higher sphingomyelin contents. The mixed systems containing sphingomyelin display much less tendency to adopt non-bilayer phases when the temperature is raised to 70°C, in contrast to their phosphatidylcholine-containing counterparts.4.4. In the presence of cholesterol, 30 mol% sphingomyelin/70 mol% soya phosphatidylethanolamine dispersions display a much greater affinity for the bilayer configuration than do similar phosphatidylcholine/soya phosphatidylethanolamine systems, which tend to adopt non-bilayer configurations at low and/or equimolar cholesterol levels.
One possibility for bringing drugs to their specific targets is to use the drug-laden liposomes that have been made target-specific by the attachment of appropriate proteins. Such 'directed' proteoliposomes and most other particles are rapidly removed from the bloodstream, however, by the mononuclear phagocytes in the liver and spleen. This causes suboptimal drug accumulation at the target site. Coating the liposome surface with poly(ethylene glycol) (PEG) may prolong the circulation time of liposomes. Using plasminogen as a homing device we have shown that the PEG-modified liposomes with such a homing device coupled to the ends of the long PEG chains may combine long vesicle circulation times in the blood with high target binding capability. The PEG-coated proteoliposomes with homing devices attached at the very bilayer surface, on the contrary, are longlived but have only little or no capability to bind to their targets.
The effect of poly(ethylene glycol)--lipid (PEG--lipid) conjugates on liposomal fusion was investigated. Incorporation of PEG--lipids into large unilamellar vesicles (LUVs) composed of equimolar phosphatidylethanolamine (PE) and phosphatidylserine (PS) inhibited calcium-induced fusion. The degree of inhibition increased with increasing molar ratio of the PEG conjugate and with increasing size of the PEG moiety. Inhibition appeared to result from the steric barrier on the surface of the liposomes which opposed apposition of bilayers and interbilayer contact. In the presence of a large excess of neutral acceptor liposomes, however, fusogenic activity was restored. The rate of fusion under these conditions depended on the initial molar ratio of the PEG conjugate in the PE:PS vesicles and the length and degree of saturation of the acyl chains which composed the lipid anchor. These results are consistent with spontaneous transfer of the PEG--lipid from PE:PS LUVs to the neutral lipid sink reducing the steric barrier and allowing fusion of the PE:PS LUVs. The primary determinant of the rate of fusion was the rate of transfer of the PEG--lipid, indicating that liposomal fusion could be programmed by incorporation of appropriate PEG--lipid conjugates. Interestingly, increasing the size of the PEG group did not appear to affect the rate of fusion. The implications of these results with respect to the design of fusogenic liposomal drug delivery systems are discussed.
The influence of poly(ethylene glycol)-lipid conjugates on phospholipid polymorphism has been examined using 31P-NMR and freeze--fracture electron microscopy. An equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and cholesterol adopts the hexagonal (HII) phase when hydrated under physiological conditions but can be stabilized in a bilayer conformation when a variety of PEG-lipid conjugates are included in the lipid mixture. These PEG conjugates produced an increase in the bilayer to hexagonal (HII) phase transition temperature and a broadening of the temperature range over which both phases coexisted. Further, the fraction of phospholipid adopting the bilayer phase increased with increasing mole fraction of PEG-lipid such that at 20 mole % DOPE--PEG2000 no HII phase phospholipid was observed up to a least 60 degrees C. Increasing the size of the PEG moiety from 2000 to 5000 Da (while maintaining the PEG--lipid molar ratio constant) increased the proportion of lipid in the bilayer phase. In contrast, varying the acyl chains of the PE anchor had no effect on polymorphic behavior. PEG--lipid conjugates in which ceramide provides the hydrophobic anchor also promoted bilayer formation in DOPE:cholesterol mixtures but at somewhat higher molar ratios compared to the corresponding PEG--PE species. The slightly greater effectiveness of the PE conjugates may result from the fact that these derivatives also possess a net negative charge. Phosphorus NMR spectroscopy indicated that a proportion of the phospholipid in DOPE:cholesterol:PEG--PE mixtures experienced isotropic motional averaging with this proportion being sensitive to both temperature and PEG molecular weight. Surprisingly, little if any isotropic signal was observed when PEG--ceramide was used in place of PEG--PE. Consistent with the 31P-NMR spectra, freeze-fracture electron microscopy showed the presence of small vesicles (diameter <200 nm) and lipidic particles in DOPE:cholesterol mixtures containing PEG--PE. We conclude that the effects of PEG--lipid conjugates on DOPE:cholesterol mixtures are 2-fold. First, the complementary "inverted cone" shape of the conjugate helps to accommodate the "cone-shaped" lipids, DOPE and cholesterol, in the bilayer phase. Second, the steric hindrance caused by the PEG group inhibits close apposition of bilayers, which is a prerequisite for the bilayer to HII phase transition.
To increase cationic liposome-mediated intravenous DNA delivery extruded DOTAP:cholesterol liposomes were used to form complexes with DNA, resulting in enhanced expression of the chloramphenicol acetyltransferase gene in most tissues examined. The DNA:liposome ratio, and mild sonication, heating, and extrusion steps used for liposome preparation were crucial for improved systemic delivery. Size fractionation studies showed that maximal gene expression was produced by a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size. Cryo-electron microscopy examination demonstrates that the DNA:liposome complexes have a novel morphology, and that the DNA is condensed on the interior of invaginated liposomes between two lipid bilayers. This structure could account for the high efficiency of gene delivery in vivo and for the broad tissue distribution of the DNA:liposome complexes. Ligands can be placed on the outside of this structure to provide for targeted gene delivery.
We have developed a new type of long-circulating immunoliposome (Fab'-PEG immunoliposomes) which is efficiently extravasated into the targeted solid tumor in vivo. Small unilamellar liposomes (100-130 nm in diameter) were prepared from distearoylphosphatidylcholine (DSPC), cholesterol (CHOL) and a dipalmitoylphosphatidylethanolamine derivative of PEG with a terminal maleimidyl group (DPPE-PEG-Mal), and conjugated Fab' fragment of antibody. Inclusion of DPPE-PEG-Mal and linkage of the Fab' fragment instead of intact antibody to PEG terminals allowed the liposomes to evade RES uptake and remain in the circulation for a long time, resulting in enhanced accumulation of the liposomes in the solid tumor. Because of the ability of such Fab'-PEG immunoliposomes to target solid tumors, they appear highly attractive as carriers of not only chemotherapeutic agents, but also of macromolecular drugs.
Unlabelled:
The present study is aimed to characterize the interactions between plasmid DNA and cationic, large unilamellar vesicles, 110+/-20nm in size, composed of lipids commonly used for transfections including DOTAP/DOPE (mole ratio 1/1), DOTAP/DOPC (mole ratio 1/1), 100% DOTAP, or DC-CHOL/DOPE (mole ratio 1/1). [
Abbreviations:
DOTAP, N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DC-CHOL, 3 beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol]. A novel approach of combining Gouy-Chapman calculations and fluorescence measurements of the pH at the surface of lipid assemblies by the fluorophore 4-heptadecyl-7-hydroxycoumarin showed that electrostatic parameters played a key role in the instantaneous formation of the DNA-lipid complexes upon addition of different amounts of plasmid DNA to cationic liposomes in 20 mM Hepes buffer (pH 7.4). Addition of large amounts of plasmid DNA leads to neutralization of 60% of the protonated DC-CHOL in DC-CHOL/DOPE (1/1) assemblies and 80% of the DOTAP in lipid assemblies. The characterization of these electrostatic parameters of the complexes suggests better and closer surrounding of plasmid DNA by lipids when DOPE is present. Time-dependent static light-scattering measurements monitored the formation of complexes and also showed that these complexes were highly unstable with respect to size at DNA/cationic lipid molar ratios between 0.2 and 0.8.
A two-dimensional columnar phase in mixtures of DNA complexed with cationic liposomes has been found in the lipid composition
regime known to be significantly more efficient at transfecting mammalian cells in culture compared to the lamellar (Lα
C) structure of cationic liposome–DNA complexes. The structure, derived from synchrotron x-ray diffraction, consists of DNA
coated by cationic lipid monolayers and arranged on a two-dimensional hexagonal lattice (HII
C). Two membrane-altering pathways induce the Lα
C → HII
C transition: one where the spontaneous curvature of the lipid monolayer is driven negative, and another where the membrane
bending rigidity is lowered with a new class of helper-lipids. Optical microscopy revealed that the Lα
C complexes bind stably to anionic vesicles (models of cellular membranes), whereas the more transfectant HII
C complexes are unstable and rapidly fuse and release DNA upon adhering to anionic vesicles.
A detergent dialysis procedure is described which allows encapsulation of plasmid DNA within a lipid envelope, where the resulting particle is stabilized in aqueous media by the presence of a poly(ethyleneglycol) (PEG) coating. These 'stabilized plasmid-lipid particles' (SPLP) exhibit an average size of 70 nm in diameter, contain one plasmid per particle and fully protect the encapsulated plasmid from digestion by serum nucleases and E. coli DNase I. Encapsulation is a sensitive function of cationic lipid content, with maximum entrapment observed at dioleoyldimethylammonium chloride (DODAC) contents of 5 to 10 mol%. The formulation process results in plasmid-trapping efficiencies of up to 70% and permits inclusion of 'fusigenic' lipids such as dioleoylphosphatidylethanolamine (DOPE). The in vitro transfection capabilities of SPLP are demonstrated to be strongly dependent on the length of the acyl chain contained in the ceramide group used to anchor the PEG polymer to the surface of the SPLP. Shorter acyl chain lengths result in a PEG coating which can dissociate from the SPLP surface, transforming the SPLP from a stable particle to a transfection-competent entity. It is suggested that SPLP may have utility as systemic gene delivery systems for gene therapy protocols.
Difficulties in specific detection of transfected DNA in cells represent an important limitation in the study of the gene transfer process. We studied the cellular entry and fate of a plasmid DNA complexed with a cationic lipid, Vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyuridine (BrdU) was incorporated into plasmid DNA under conditions that minimize plasmid alteration. BrdU was localized in cells incubated with Vectamidine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated liposome structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/DNA aggregates accumulate in large vesicles in the cell cytosol. On the other hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-conjugated antibodies permitted simultaneous detection in the cells of both components of the complexes with confocal laser scanning microscopy. The DNA and lipids co-localized at the surface of and inside the cells, indicating that the complex is internalized as a whole. Our results show that the BrdU-labeled plasmid DNA detection system can be a useful tool to visualize exogenous DNA entry into cells by a combination of electron and confocal microscopy.
Recent studies have indicated that the structural features of DNA-lipid assemblies, dictated by the lipid composition and cationic lipid-to-DNA ratio, critically affect the efficiency of these complexes in acting as vehicles for cellular delivery of genetic material. Using circular dichroism we find that upon binding DNA, positively-charged liposomes induce a secondary conformational transition of the DNA molecules from the native B form to the C motif. Liposomes composed of positively-charged and neutral 'helper' lipids, found to be particularly effective as transfecting agents, induce - in addition to secondary conformational changes - DNA condensation into a left-handed cholesteric-like phase. A structural model is presented according to which two distinct, yet inter-related modes of DNA packaging coexist within such assemblies. The results underline the notion that subtle changes in the components of a supramolecular assembly may substantially modulate the interplay of interactions which dictate its structure and functional properties.
The inhibition effect of serum on the transfection efficiency of cationic liposome-DNA complexes (lipoplexes) is a major obstacle to the application of this gene delivery vector both in vitro and in vivo. The size of the lipoplexes, as they are presented to targeted cells, is found to be the major determinant of their effectiveness in transfection. The transfection efficiency and the cell association and uptake of lipoplexes with CHO cells was found to increase with increasing lipoplex size. The influence on the transfection efficiency of lipoplexes by their cationic lipid:DNA ratios, types of liposomes, incubation time in polyanion containing media, and time of serum addition, are mediated mainly through size. Lipoplexes at a 2:1 charge ratio grow in size in media containing polyanions. The size growth may be arrested by adding serum to the incubation media. By using large lipoplexes, especially those made from multilamellar vesicles, the serum inhibition effect may be overcome.
The presence of EDTA in the suspending buffer can induce the formation of multilayer structures from a mixture of the cationic lipid 3beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol and the zwitterionic 'helper' lipid 1, 2-dimyristoyl-sn-glycero-3-phosphocholine with DNA. The resulting structures consist of stacks of alternating sheets of lipid bilayer with intercalated DNA. In the absence of EDTA, only a single layer of DNA adsorbs to the lipid membrane. The buffer composition therefore influences the morphology of the lipid-aggregate/DNA assembly, which was not known to date.
The last 10 years have seen substantial progress in the development and application of nonviral vectors in gene therapy. However, many problems remain to be resolved before nonviral gene therapy can become a standard clinical practice. This review highlights the major breakthroughs in this field. The problems and future research directions are also discussed. Gene Therapy (2000) 7, 31-34.
The lamellar/nonlamellar phase preferences of lipid model membranes composed of mixtures of several cationic lipids with various zwitterionic and anionic phospholipids were examined by a combination of differential scanning calorimetry and (31)P NMR spectroscopy. All of the cationic lipids utilized in this study form only lamellar phases in isolation. Mixtures of these cationic lipids with zwitterionic strongly lamellar phase-preferring lipids such as phosphatidylcholine form only the lamellar liquid-crystalline phase even at high temperatures, as expected. Moreover, mixtures of these cationic lipids with strongly nonlamellar phase-preferring zwitterionic lipids such as phosphatidylethanolamine exhibit a markedly reduced propensity to form inverted nonlamellar phases, again as expected. However, when mixed with anionic lipids such as phosphatidylserine, phosphatidylglycerol, cardiolipin, or phosphatidic acid, a marked enhancement of nonlamellar phase-forming propensity occurs, despite the fact both components of the mixture are nominally lamellar phase-preferring. An examination of the lamellar/nonlamellar phase transition temperatures and the nature of the nonlamellar phases formed, as a function of temperature and of the composition of the mixture, indicates that the propensity to form inverted nonlamellar phases is maximal in mixtures where the mean surface charge of the membrane surface approaches neutrality and decreases markedly with increases in the density of positive or negative charge at the membrane surface. Moreover, the onset temperatures of the reversed hexagonal phase rise more steeply than do those of the inverted cubic phase as the ratio of cationic and anionic lipids is varied, suggesting that the formation of inverted hexagonal phases is more sensitive to this surface charge effect. These results indicate that surface charge per se is a significant and effective modulator of the lamellar/nonlamellar phase preferences of membrane lipids and that charged group interactions at membrane surfaces may have a major role in regulating this particular membrane property.
Cellular transfection can be accomplished by the use of synthetic amphiphiles as gene carrier system. To understand the mechanism and hence to improve the efficiency of transfection, insight into the assembly and properties of the amphiphile/gene complex is crucial. Here, we have studied the interaction between a plasmid and cationic amphiphiles, using a monolayer technique, and have examined complex assembly by atomic force microscopy. The data reveal a three-step mechanism for complex formation. In a first step, the plasmids, interacting with the monolayer, display a strong tendency of orientational ordering. Subsequently, individual plasmids enwrap themselves with amphiphile molecules in a multilamellar fashion. The size of the complex formed is determined by the supercoiled size of the plasmid, and calculations reveal that the plasmid can be surrounded by 3 to 5 bilayers of the amphiphile. The eventual size of the transfecting complex is finally governed by fusion events between individually wrapped amphiphile/DNA complexes. In bulk phase, where complex assembly is triggered by mixing amphiphilic vesicles and plasmids, a similar wrapping process is observed. However, in this case, imperfections in this process may give rise to a partial exposure of plasmids, i.e., part of the plasmid is not covered with a layer of amphiphile. We suggest that these exposed sites may act as nucleation sites for massive lipoplex clustering, which in turn may affect transfection efficiency.
The pH-dependent fusion properties of large unilamellar vesicles (LUVs) composed of binary mixtures of anionic and cationic lipids have been investigated. It is shown that stable LUVs can be prepared from the ionizable anionic lipid cholesteryl hemisuccinate (CHEMS) and the permanently charged cationic lipid N,N-dioleoyl-N, N-dimethylammonium chloride (DODAC) at neutral pH values and that these LUVs undergo fusion as the pH is reduced. The critical pH at which fusion was observed (pH(f)) was dependent on the cationic lipid-to-anionic lipid ratio. LUVs prepared from DODAC/CHEMS mixtures at molar ratios of 0 to 0.85 resulted in vesicles with pH(f) values that ranged from pH 4.0 to 6.7, respectively. This behavior is consistent with a model in which fusion occurs at pH values such that the DODAC/CHEMS LUV surface charge is zero. Related behavior was observed for LUVs composed of the ionizable cationic lipid 3alpha-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Chol) and the acidic lipid dioleoylphosphatidic acid (DOPA). Freeze-fracture and (31)P NMR evidence is presented which indicates that pH-dependent fusion results from a preference of mixtures of cationic and anionic lipid for "inverted" nonbilayer lipid phases under conditions where the surface charge is zero. It is concluded that tunable pH-sensitive LUVs composed of cationic and anionic lipids may be of utility for drug delivery applications. It is also suggested that the ability of cationic lipids to adopt inverted nonbilayer structures in combination with anionic lipids may be related to the ability of cationic lipids to facilitate the intracellular delivery of macromolecules.
Cationic liposomes belong to the family of non-viral vectors for gene delivery. Despite several drawbacks, such as low efficiency compared to viruses and inactivation by serum, cationic liposomes remain a promising tool for gene therapy. Therefore further investigation of the mechanism of transfection and improvement of formulations are warranted.
In a comparative study, we investigated the effect of serum on the ability of SAINT, a novel synthetic amphiphile, and Lipofectin to mediate transfection in vitro, employing a variety of cell lines.
In all cell types, SAINT-mediated transfection was not significantly affected by the presence of serum, in contrast to Lipofectin-mediated transfection. Intriguingly, the extent of complex association was enhanced in the presence of serum, while cell association of the Lipofectin complex was approximately two-fold higher than that of SAINT. These data imply that transfection efficiency and the amount of cell-associated complex are not related. However, when the helper lipid dioleoylphosphatidylethanolamine (DOPE) was substituted for cholesterol, SAINT-mediated transfection was reduced in the presence of serum. This indicates that lipoplex composition rather than the cationic lipid per se codetermines the effect of serum. Also, the presence of serum decreased cytotoxicity, while no correlation could be demonstrated between toxicity and transfection efficiency. The binding of serum proteins to either complex was identical, both in terms of protein identity and relative amounts.
We propose that serum, in conjunction with cell-specific factors and lipoplex composition, determines complex (in)stability, which is crucial for effective gene delivery and expression.
Novel reduced sugar gemini amphiphiles linked through their tertiary amino head groups via alkyl spacers of 4 or 6 carbons, and with varying (unsaturated) alkyl tail lengths of 12--18, have been synthesized and tested for transfection in vitro in an adherent Chinese hamster ovary cell line (CHO-K1). Transfection efficiencies peaked at 2.7 times that of the commercial standard Lipofectamine Plus/2000 for pure solutions of the compound bearing unsaturated (oleyl) alkyl tails. For those compounds bearing saturated alkyl tails, transfection efficiency peaked at a tail length of 16, at a level similar to Lipofectamine Plus/2000. All of the amphiphiles formed bilayer vesicles at physiological pH. Some of the amino groups at the surface were protonated, and vesicles therefore bore a positive charge. Increased protonation with reduced pH resulted in greatly increased monomer solubility and a morphology change from vesicle to micelle at characteristic pH values, dependent on the tail length. For the compounds promoting high transfection efficiency, this characteristic pH was within the range found in the endosomal compartment (7.4--4.0). Formation of mixed micelles between gemini surfactant and membrane phospholipids at reduced pH may therefore provide a method of endosome rupture and subsequent escape of entrapped DNA, thus discarding the need for extra fusogenic or endosomolytic agents. The positive charge on the vesicles at physiological pH drives the colloidal association with DNA. Small angle X-ray scattering measurements indicate that lamellar aggregates are formed, which have a d spacing of 48--54 A. Preliminary differential scanning calorimetric measurements suggest that reduction of pH causes a disordering of the hydrocarbon region of the DNA-surfactant complex.
Poly(ethylene glycol)-lipid (PEG-lipid) conjugates are widely used in the field of liposomal drug delivery to provide a polymer coat that can confer favorable pharmacokinetic characteristics on particles in the circulation. More recently these lipids have been employed as an essential component in the self-assembly of cationic and neutral lipids with polynucleic acids to form small, stable lipid/DNA complexes that exhibit long circulation times in vivo and accumulate at sites of disease. However, the presence of a steric barrier lipid might be expected to inhibit the transfection activity of lipid/DNA complexes by reducing particle-membrane contact. In this study we examine what effect varying the size of the hydrophobic anchor and hydrophilic head group of PEG-lipids has on both gene and antisense delivery into cells in culture. Lipid/DNA complexes were made using unilamellar vesicles composed of 5 mole% PEG-lipids in combination with equimolar dioleoylphosphatidylethanolamine and the cationic lipid dioleyldimethylammonium chloride. Using HeLa and HepG2 cells we show that under the conditions employed PEG-lipids had a minimal effect on the binding and subsequent endocytosis of lipid/DNA complexes but they severely inhibited active gene transfer and the endosomal release of antisense oligodeoxynucleotides into the cytoplasm. Decreasing the size of the hydrophobic anchor or the size of the grafted hydrophilic PEG moiety enhanced DNA transfer by the complexes.
We have investigated the mechanism of lipoplex-mediated transfection, employing a dialkyl pyridinium surfactant (SAINT-2), and using serum as a modulator of complex stability and processing. Particle size and stability determine lipoplex internalization, the kinetics of intracellular processing, and transfection efficiency. Clustered SAINT-2 lipoplexes are obtained in the absence of serum (-FBS lipoplexes), but not in its presence (+FBS lipoplexes), or when serum was present during lipoplex formation [FBS], conditions that mimic potential penetration of serum proteins. The topology of DNA in [FBS] lipoplexes shifts from a supercoiled, as in -FBS lipoplexes, to a predominantly open-circular conformation, and is more prone to digestion by DNase. Consistently, atomic force microscopy revealed complexes with tubular extensions, reflecting DNA that protrudes from the lipoplex surface. Interestingly, the internalization of [FBS] lipoplexes is approximately three-fold higher than that of -FBS and +FBS lipoplexes, yet their transfection efficiency is approximately five-fold lower. Moreover, in contrast to -FBS and +FBS complexes, [FBS] complexes were rapidly processed into the late endosomal/lysosomal degradation pathway. Intriguingly, transfection by [FBS] complexes is greatly improved by osmotic rupture of endocytic compartments. Our data imply that constraints in size and morphology govern the complex' ability to interact with and perturb cellular membranes, required for gene release. By extrapolation, we propose that serum may regulate these parameters in an amphiphile-dependent manner, by complex 'penetration' and modulation of DNA conformation.
Cationic liposomes are applied to transfer oligonucleotides (ODNs) into cells to regulate gene expression for gene therapeutic or cell biological purposes. In vivo, poly(ethylene glycol) (PEG)-lipid derivatives are employed to stabilize and prolong the circulation lifetime of nucleic acid-containing particles, and to improve targeting strategies. In this study, we have studied the effects of PEG-lipid analogues, i.e. PEG coupled to either phosphatidylethanolamine (PE) or ceramide, on cationic-lipid-DNA complex ('lipoplex') assembly and the mechanism of cationic-lipid-mediated delivery of ODNs in vitro. Inclusion of 10 mol% PEG-PE in ODN lipoplexes inhibited their internalization in Chinese hamster ovary cells by more than 70%. The intracellular fraction remained entrapped in the endosomal/lysosomal pathway, and no release of ODNs was apparent. Similar observations were made for complexes prepared from liposomes that contained PEG-ceramides. Interestingly, delivery resumed when lipoplexes had been externally coated with PEG-ceramides. In this case, the kinetics of delivery were dependent on the length of the ceramide acyl chain, consistent with a requirement for the PEG-lipid to dissociate from the complex. Moreover, although the chemical nature of the PEG-ceramides distinctly affected the net internalization of the complexes, impediment of delivery was largely related to an inhibitory effect of the PEG-lipid on the release of ODNs from the endosomal compartment. Cryo-electron microscopy and small-angle X-ray scattering revealed that the PEG-lipids stabilize the lamellar phase of the lipoplexes, while their acyl-chain-length-dependent transfer from the complex enables adaptation of the hexagonal phase. Within the endosomal compartment, this transition appears to be instrumental in causing the dissociation and cytosolic release of the ODNs for their nuclear homing.
Annette Deichmann, Martijn H Brugman, Cynthia C Bartholomae, Kerstin Schwarzwaelder, Monique MA Verstegen, Steven J Howe, Anne Arens, Marion G Ott, Dieter Hoelzer, Reinhard Seger, Manuel Grez, Salima Hacein-Bey-Abina, Marina Cavazzana-Calvo, Alain Fischer, Anna Paruzynski, Richard Gabriel, Hanno Glimm, Ulrich Abel, Claudia Cattoglio, Fulvio Mavilio, Barbara Cassani, Alessandro Aiuti, Cynthia E Dunbar, Christopher Baum, H Bobby Gaspar, Adrian J Thrasher, Christof von Kalle, Manfred Schmidt, Gerard Wagemaker. (2011) Insertion Sites in Engrafted Cells Cluster Within a Limited Repertoire of Genomic Areas After Gammaretroviral Vector Gene Therapy. Molecular Therapy 19, 2031-2039
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Stefan Stein, Marion G Ott, Stephan Schultze-Strasser, Anna Jauch, Barbara Burwinkel, Andrea Kinner, Manfred Schmidt, Alwin Krämer, Joachim Schwäble, Hanno Glimm, Ulrike Koehl, Carolin Preiss, Claudia Ball, Hans Martin, Gudrun Göhring, Kerstin Schwarzwaelder, Wolf-Karsten Hofmann, Kadin Karakaya, Sandrine Tchatchou, Rongxi Yang, Petra Reinecke, Klaus Kühlcke, Brigitte Schlegelberger, Adrian J Thrasher, Dieter Hoelzer, Reinhard Seger, Christof von Kalle, Manuel Grez. (2010) Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nature Medicine 16, 198-204
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Steven J. Howe, Marc R. Mansour, Kerstin Schwarzwaelder, Cynthia Bartholomae, Michael Hubank, Helena Kempski, Martijn H. Brugman, Karin Pike-Overzet, Stephen J. Chatters, Dick de Ridder, Kimberly C. Gilmour, Stuart Adams, Susannah I. Thornhill, Kathryn L. Parsley, Frank J.T. Staal, Rosemary E. Gale, David C. Linch, Jinhua Bayford, Lucie Brown, Michelle Quaye, Christine Kinnon, Philip Ancliff, David K. Webb, Manfred Schmidt, Christof von Kalle, H. Bobby Gaspar, Adrian J. Thrasher. (2008) Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. Journal of Clinical Investigation 118, 3143-3150
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In plasma membranes, lipids distribute asymmetrically across the bilayer, a process that requires proteins. Recent work identified novel lipid translocators in yeast, and their activity was functionally correlated to endocytosis, thus boosting investigations on identity, mechanism, and function of lipid translocases.
Endocytosis of various endogenous plasma membrane molecules, including signalling receptors, glycosphingolipids and glycosylphosphatidylinositol (GPI)-linked proteins, occurs in the absence of functional clathrin-coated pits. Most of these molecules are found in biochemically defined lipid rafts, which suggests that at least some clathrin-independent endocytosis may be raft specific or raft mediated. However, recent studies of the uptake of raft markers have revealed a diversity of internalization methods. Although lipid rafts may somehow be recognized by endocytic machinery, at this stage the data do not readily fit with the idea of a single raft-specific or raft-dependent endocytic pathway. Many studies report uptake of raft molecules by caveolar endocytosis (defined by sensitivity to cholesterol depletion and to overexpression of a specific mutant of dynamin 2). It is now apparent that this is a highly regulated process, and caveolin 1, one of the characteristic protein components of caveolae, might in fact act to slow or inhibit endocytosis. The molecular details of caveolar endocytosis have yet to be elucidated. Several sources indicate that clathrin-independent uptake to a distinct class of caveolin-1-containing endosome, termed the caveosome, allows different types of endocytic mechanisms to have different functional consequences for the cell. It is likely that there are mechanisms that allow recruitment and targeting of specific molecules to caveosomes.