Delivery Of Lipoplexes Research Articles

Lipoplexes' Structure, Preparation, and Role in Managing Different Diseases.

Lipid-based vectors are becoming promising alternatives to traditional therapies over the last 2 decades specially for managing life-threatening diseases like cancer. Cationic lipids are the most prevalent non-viral vectors utilized in gene delivery.The increasing number of clinical trials about lipoplex-based gene therapy demonstrates their potential as well-established technology that can provide robust gene transfection. In this regard, this review will summarize this important point.These vectors however have a modest transfection efficiency. This limitation can be partly addressed by using functional lipids that provide a plethora of options for investigating nucleic acid-lipid interactions as well as in vitro and in vivo nucleic acid delivery for biomedical applications. Despite their lower gene transfer efficiency, lipid-based vectors such as lipoplexes have several advantages over viral ones: they are less toxic and immunogenic, can be targeted, and are simple to produce on a large scale. Researchers are actively investigating the parameters that are essential for an effective lipoplex delivery method. These include factors that influence the structure, stability, internalization, and transfection of the lipoplex.Thorough understanding of the design principles will enable synthesis of customized lipoplex formulations for life-saving therapy.

Poly(vinyl pyrrolidone) derivatives as PEG alternatives for stealth, non-toxic and less immunogenic siRNA-containing lipoplex delivery

The recent approval of Onpattro® and COVID-19 vaccines has highlighted the value of lipid nanoparticles (LNPs) for the delivery of genetic material. If it is known that PEGylation is crucial to confer stealth properties to LNPs, it is also known that PEGylation is responsible for the decrease of the cellular uptake and endosomal escape and for the production of anti-PEG antibodies inducing accelerated blood clearance (ABC) and hypersensitivity reactions. Today, the development of PEG alternatives is crucial. Poly(N-vinyl pyrrolidone) (PNVP) has shown promising results for liposome decoration but has never been tested for the delivery of nucleic acids. Our aim is to develop a series of amphiphilic PNVP compounds to replace lipids-PEG for the post-insertion of lipoplexes dedicated to siRNA delivery. PNVP compounds with different degrees of polymerization and hydrophobic segments, such as octadecyl, dioctadecyl and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), were generated. Based on the physicochemical properties and the efficiency to reduce protein corona formation, we showed that the DSPE segment is essential for the integration into the lipoplexes. Lipoplexes post-grafted with 15% DSPE-PNVP30 resulted in gene silencing efficiency close to that of lipoplexes grafted with 15% DSPE-PEG. Finally, an in vivo study in mice confirmed the stealth properties of DSPE-PNVP30 lipoplexes as well as a lower immune response ABC effect compared to DSPE-PEG lipoplexes. Furthermore, we showed a lower immune response after the second injection with DSPE-PNVP30 lipoplexes compared to DSPE-PEG lipoplexes. All these observations suggest that DSPE-PNVP30 appears to be a promising alternative to PEG, with no toxicity, good stealth properties and lower immunological response.

Targeted Knockout of the Vegfa Gene in the Retina by Subretinal Injection of RNP Complexes Containing Cas9 Protein and Modified sgRNAs

The therapeutic effect of retinal gene therapy using CRISPR/Cas9-mediated genome editing and knockout applications is dependent on efficient and safe delivery of gene-modifying tool kits. Recently, transient administration of single guide RNAs (sgRNAs) and SpCas9 proteins delivered as ribonucleoproteins (RNPs) has provided potent gene knockout in vitro. To improve efficacy of CRISPR-based gene therapy, we delivered RNPs containing SpCas9 protein complexed to chemically modified sgRNAs (msgRNAs). In K562 cells, msgRNAs significantly increased the insertion/deletion (indel) frequency (25%) compared with unmodified counterparts leading to robust knockout of the VEGFA gene encoding vascular endothelial growth factor A (96% indels). Likewise, in HEK293 cells, lipoplexes containing varying amounts of RNP and EGFP mRNA showed efficient VEGFA knockout (43% indels) and strong EGFP expression, indicative of efficacious functional knockout using small amounts of RNP. In mice, subretinal injections of equivalent lipoplexes yielded 6% indels in Vegfa of isolated EGFP-positive RPE cells. However, signs of toxicity following delivery of lipoplexes containing high amounts of RNP were observed. Although the mechanism resulting in the varying efficacy remains to be elucidated, our data suggest that a single subretinal injection of RNPs carrying msgRNAs and SpCas9 induces targeted retinal indel formation, thus providing a clinically relevant strategy relying on nonviral delivery of short-lived nuclease activity.

Improvement of DNA Vector Delivery of DOTAP Lipoplexes by Short-Chain Aminolipids.

Cellular delivery of DNA vectors for the expression of therapeutic proteins is a promising approach to treat monogenic disorders or cancer. Significant efforts in a preclinical and clinical setting have been made to develop potent nonviral gene delivery systems based on lipoplexes composed of permanently cationic lipids. However, transfection efficiency and tolerability of such systems are in most cases not satisfactory. Here, we present a one-pot combinatorial method based on double-reductive amination for the synthesis of short-chain aminolipids. These lipids can be used to maximize the DNA vector delivery when combined with the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). We incorporated various aminolipids into such lipoplexes to complex minicircle DNA and screened these systems in a human liver-derived cell line (HuH7) for gene expression and cytotoxicity. The lead aminolipid AL-A12 showed twofold enhanced gene delivery and reduced toxicity compared to the native DOTAP:cholesterol lipoplexes. Moreover, AL-A12-containing lipoplexes enabled enhanced transgene expression in vivo in the zebrafish embryo model.

Targeted Delivery of siRNA Lipoplexes to Cancer Cells Using Macrophage Transient Horizontal Gene Transfer.

Delivery of nucleic acids into solid tumor environments remains a pressing challenge. This study examines the ability of macrophages to horizontally transfer small interfering RNA (siRNA) lipoplexes to cancer cells. Macrophages are a natural candidate for a drug carrier because of their ability to accumulate at high densities into many cancer types, including, breast, prostate, brain, and colon cancer. Here, it is demonstrated that macrophages can horizontally transfer siRNA to cancer cells during in vitro coculture. The amount of transfer can be dosed depending on the amount of siRNA loaded and total number of macrophages delivered. Macrophages loaded with calcium integrin binding protein‐1 (CIB1)‐siRNA result in decreased tumorsphere growth and decreased mRNA expression of CIB1 and KI67 in MDA‐MB‐468 human breast cancer cells. Adoptive transfer of macrophages transfected with CIB1‐siRNA localizes to the orthotopic MDA‐MB‐468 tumor. Furthermore, it is reported that macrophage activation can modulate this transfer process as well as intracellular trafficking protein Rab27a. As macrophages are heavily involved in tumor progression, understanding how to use macrophages for drug delivery can substantially benefit the treatment of tumors.

A microparticle approach for non-viral gene delivery within 3D human mesenchymal stromal cell aggregates

Three-dimensional (3D) multicellular aggregates, in comparison to two-dimensional monolayer culture, can provide tissue culture models that better recapitulate the abundant cell-cell and cell-matrix interactions found in vivo. In addition, aggregates are potentially useful building blocks for tissue engineering. However, control over the interior aggregate microenvironment is challenging due to inherent barriers for diffusion of biological mediators (e.g. growth factors) throughout the multicellular aggregates. Previous studies have shown that incorporation of biomaterials into multicellular aggregates can support cell survival and control differentiation of stem cell aggregates by delivering morphogens from within the 3D construct. In this study, we developed a highly efficient microparticle-based gene delivery approach to uniformly transfect human mesenchymal stromal cells (hMSC) within multicellular aggregates and cell sheets. We hypothesized that release of plasmid DNA (pDNA) lipoplexes from mineral-coated microparticles (MCMs) within 3D hMSC constructs would improve transfection in comparison to standard free pDNA lipoplex delivery in the media. Our approach increased transfection efficiency 5-fold over delivery of free pDNA lipoplexes in the media and resulted in homogenous distribution of transfected cells throughout the 3D constructs. Additionally, we found that MCMs improved hMSC transfection by specifically increasing macropinocytosis-mediated uptake of pDNA. Finally, we showed up to a three-fold increase of bone morphogenetic protein-2 (BMP-2) expression and enhanced calcium deposition within 3D hMSC constructs following MCM-mediated delivery of a BMP-2 encoding plasmid and culture in osteogenic medium. The technology described here provides a valuable tool for achieving efficient and homogenous transfection of 3D cell constructs and is therefore of particular value in tissue engineering and regenerative medicine applications. Statement of SignificanceThis original research describes a materials-based approach, whereby use of mineral-coated microparticles improves the efficiency of non-viral gene delivery in three-dimensional human mesenchymal stromal cell constructs. Specifically, it demonstrates the use of mineral-coated microparticles to enable highly efficient transfection of human mesenchymal stromal cells in large, 3D culture formats. The manuscript also identifies specific endocytosis pathways that interact with the mineral coating to afford the improved transfection efficiency, as well as demonstrates the utility of this approach toward improving differentiation of large cell constructs. We feel that this manuscript will impact the current understanding and near-term development of materials for non-viral gene delivery in broad tissue engineering and biofabrication applications, and therefore be of interest to a diverse biomaterials audience.

Targeted Delivery of siRNA Lipoplexes to Cancer Cells Using Macrophage Transient Horizontal Gene Transfer

Targeted Delivery of siRNA Lipoplexes to Cancer Cells Using Macrophage Transient Horizontal Gene Transfer

Injectable Biodegradable Chitosan-Alginate 3D Porous Gel Scaffold for mRNA Vaccine Delivery.

mRNA vaccines have proven to be more stable, effective, and specific than protein/peptide-based vaccines in stimulating both humoral and cellular immune response. However, mRNA's fast degradation rate and low-transfection efficiency in vivo impede its potential in vaccination. Recent research in gene delivery has focused on nonviral vaccine carriers and either implantable or injectable delivery systems to improve transgene expression in vivo. Here, an injectable chitosan-alginate gel scaffold for the local delivery of mRNA vaccines is reported. Gel scaffold biodegradation rates and biocompatibility are quantified. Scaffold-mediated mRNA in vivo transgene expression as well as ovalbumin antigen specific cellular and humoral immune responses are evaluated in vivo. Luciferase reporter protein expression resulting from mRNA lipoplex-loaded gel scaffolds is five times higher than systemic injection. Compared to systemic injections of naked mRNA or mRNA:lipoplexes, elevated levels of T cell proliferation and IFN-γ secretion are seen with in vivo scaffold-mediated mRNA lipoplex delivery. Furthermore, a humoral response (ovalbumin antigen specific IgG levels) is observed as early as week 1 for scaffold-mediated mRNA lipoplex delivery, while protein-based immunization did not elicit IgG production until 2 weeks post-injection. Results suggest that injectable scaffold mRNA vaccine delivery maybe a viable alternative to traditional nucleic acid immunization methods.

Colloidally Stable Small Unilamellar Stearyl Amine Lipoplexes for Effective BMP-9 Gene Delivery to Stem Cells for Osteogenic Differentiation.

The biocompatibility of cationic liposomes has led to their clinical translation in gene delivery and their application apart from cancer to cardiovascular diseases, osteoporosis, metabolic diseases, and more. We have prepared PEGylated stearyl amine (pegSA) lipoplexes meticulously considering the physicochemical properties and formulation parameters to prepare single unilamellar vesicles (SUV) of < 100nm size which retain their SUV nature upon complexation with pDNA rather than the conventional lipoplexes which show multilamellar nature. The developed PEGylated SA lipoplexes (pegSA lipoplexes) showed a lower N/P ratio (1.5) for BMP-9 gene complexation while maintaining the SUV character with a unique shape (square and triangular lipoplexes). Colloidal and pDNA complexation stability in the presence of electrolytes and serum indicates the suitability for intravenous administration for delivery of lipoplexes to bone marrow mesenchymal stem cells through sinusoidal vessels in bone marrow. Moreover, lower charge density of lipoplexes and low oxidative stress led to lower toxicity of lipoplexes to the C2C12 cells, NIH 3T3 cells, and erythrocytes. Transfection studies showed efficient gene delivery to C2C12 cells inducing osteogenic differentiation through BMP-9 expression as shown by enhanced calcium deposition in vitro, proving the potential of lipoplexes for bone regeneration. In vivo acute toxicity studies further demonstrated safety of the developed lipoplexes. Developed pegSA lipoplexes show potential for further in vivo preclinical evaluation to establish the proof of concept.

Exploring membrane permeability of Tomatidine to enhance lipid mediated nucleic acid transfections

Intracellular delivery of nucleic acids is one of the critical steps in the transfections. Prior findings demonstrated various strategies including membrane fusion, endosomal escape for the efficient cytoplasmic delivery. In our continuing efforts to improve the nucleic acids transfections, we harnessed cell permeable properties of Tomatidine (T), a steroidal alkaloid abundantly found in green tomatoes for maximizing intracellular delivery of lipoplexes. We doped Tomatidine into liposomes of cationic lipid with amide linker (A) from our lipid library. Six liposomal formulations (AT) of Lipid A (1 mM) with varying concentrations of Tomatidine (0–1 mM) were prepared and evaluated for their transfection efficacies. Owing to its signature characteristic of cell membrane permeability, Tomatidine modulated endocytosis process, enhanced the intracellular delivery of the lipoplexes, and in turn increased the transfection efficacy of cationic liposomes. Our findings provide ‘proof of concept’ for enhancing transfections in gene delivery applications with Tomatidine in cationic liposomal formulations. These findings can be further applied in lipid mediated gene therapy and drug delivery applications.

Cationic Lipid-Based Nucleic Acid Vectors.

The delivery of nucleic acids into cells remains an important laboratory cell culture technique and potential clinical therapy, based upon the initial cellular uptake, then translation into protein (in the case of DNA), or gene deletion by RNA interference (RNAi). Although viral delivery vectors are more efficient, the high production costs, limited cargo capacity, and the potential for clinical adverse events make nonviral strategies attractive. Cationic lipids are the most widely applied and studied nonviral vectors; however, much remains to be solved to overcome limitations of these systems. Advances in the field of cationic lipid-based nucleic acid (lipoplex) delivery rely upon the development of robust and reproducible lipoplex formulations, together with the use of cell culture assays. This chapter provides detailed protocols towards the formulation, delivery, and assessment of in vitro cationic lipid-based delivery of DNA.

Characterization of exogenous DNA mobility in live cells through fluctuation correlation spectroscopy.

The spatial-temporal dynamics of delivered DNA is a critical aspect influencing successful gene delivery. A comprehensive model of DNA lipoplex trafficking through live cells has yet to be demonstrated. Here the bioimaging approaches Raster Image Correlation Spectroscopy (RICS) and image-Means Square Displacement (iMSD) were applied to quantify DNA mechanical dynamics in live cells. DNA lipoplexes formed from DNA with a range of 21 bp to 5.5 kbp exhibited a similar range of motion within the cytoplasm of myoblast cells regardless of size. However, the rate of motion was dictated by the intracellular location, and DNA cluster size. This analysis demonstrated that the different transport mechanisms either had a size dependent mobility, including random diffusion, whereas other mechanisms were not influenced by the DNA size such as active transport. The transport mechanisms identified followed a spatial dependence comparable to viral trafficking of non-active transport mechanism upon cellular entry, active transport within the cytoplasm and further inactive transportation along the peri-nuclear region. This study provides the first real-time insight into the trafficking of DNA delivered through lipofection using image-based fluctuation correlation spectroscopy approaches. Thereby, gaining information with single particle sensitivity to develop a deeper understanding of DNA lipoplex delivery through the cell.

Efficient in vivo transfection and safety profile of a CpG-free and codon optimized luciferase plasmid using a cationic lipophosphoramidate in a multiple intravenous administration procedure

As any drug, the success of gene therapy is largely dependent on the vehicle that has to selectively and efficiently deliver therapeutic nucleic acids into targeted cells with minimal side-effects. In the case of chronic diseases that require a life-long treatment, non-viral gene delivery vehicles are less likely to induce an immune response, thereby allowing for repeated administration. Beyond the gene delivery efficiency of a given vector, the nature of nucleic acid constructs also has a central importance in gene therapy protocols.Herein, we investigated the impact of two firefly luciferase encoding plasmids on the transgene expression profile following systemic delivery of lipoplexes in mice, as well as their potential to be safely and efficiently readministered. Whereas pTG11033 plasmid is driven by a strong ubiquitous cytomegalovirus promoter, pGM144 plasmid, which has been designed to avoid inflammation and provide sustained transgene expression in lungs, is CpG-free and is under control of the human elongation factor-1 alpha promoter.Combined to the efficient cationic lipophosphoramidate BSV4, bioluminescence data showed that both plasmids were mostly expressed in the lungs of mice following a primary injection of lipoplexes. However, mice transfected with pGM144 exhibited a higher and more sustained transgene expression than those treated with pTG11033. Repeated administration studies revealed that several injections of lipoplexes could lead to similar transgene expression profiles if an interval of several weeks between subsequent injections was respected. A transient hepatotoxicity and a partial inflammatory response were caused by lipoplex injection, irrespective of the plasmid used.Altogether, these results indicate that repeated systemic administration of lipophosphoramidate-based lipoplexes in mice conducts to an effective lung transfection without serious side effects, and highlight the need to use long-lasting expressing and well tolerated plasmids in order to efficiently renew transgene expression by the successive doses.

Microwell array-mediated delivery of lipoplexes containing nucleic acids for enhanced therapeutic efficacy.

Many delivery methods have been developed to improve the therapeutic efficacy and facilitate the clinical translation of nucleic acids-based therapeutics. We present a facile microwell array to mediate the delivery of nucleic acids carried by lipoplexes, which combines the advantages of lipoplexes as an efficient carrier system, the surface mediated delivery, and the control of surface topography. This method shows much higher transfection efficiency than conventional transfection method for oligodeoxynucleotides and microRNAs, and thus significantly reduces the effective therapeutic dosages. Microwell array is also a very flexible platform. Multifunctional lipoplexes containing both nucleic acid therapeutics and imaging reagents can be easily prepared in the microwell array and efficiently delivered to cells, demonstrating its potential applications in theranostic medicine.

Delivery of Therapeutic siRNA to the Lung Endothelium via Novel Lipoplex Formulation DACC

Posttranscriptional gene silencing by RNA interference can be therapeutically exploited to inhibit pathophysiological gene expression. However, in contrast to the established effectiveness of RNAi in vitro, safe and effective delivery of siRNAs to specific organs and cell types in vivo remains the major hurdle. Here, we report the development and in vivo characterization of a novel siRNA delivery system (DACC lipoplex) suitable for modulating target gene expression specifically in the lung vasculature. Systemic administration of DACC in mice delivered siRNA cargo functionally to the lung pulmonary endothelium. A single dose of DACC lipoplexes administered by bolus injection or by infusion was sufficient to specifically silence genes expressed in pulmonary endothelial cells such as CD31, Tie-2, VE-cadherin, or BMP-R2. When tested in a mouse model for lung cancer, repeated treatment with DACC/siRNA(CD31) reduced formation of lung metastases and increased life span in a mouse model of experimental lung metastasis.

Integrating mitosis, toxicity, and transgene expression in a telecommunications packet-switched network model of lipoplex-mediated gene delivery.

Gene delivery systems transport exogenous genetic information to cells or biological systems with the potential to directly alter endogenous gene expression and behavior with applications in functional genomics, tissue engineering, medical devices, and gene therapy. Nonviral systems offer advantages over viral systems because of their low immunogenicity, inexpensive synthesis, and easy modification but suffer from lower transfection levels. The representation of gene transfer using models offers perspective and interpretation of complex cellular mechanisms,including nonviral gene delivery where exact mechanisms are unknown. Here, we introduce a novel telecommunications model of the nonviral gene delivery process in which the delivery of the gene to a cell is synonymous with delivery of a packet of information to a destination computer within a packet-switched computer network. Such a model uses nodes and layers to simplify the complexity of modeling the transfection process and to overcome several challenges of existing models. These challenges include a limited scope and limited time frame, which often does not incorporate biological effects known to affect transfection. The telecommunication model was constructed in MATLAB to model lipoplex delivery of the gene encoding the green fluorescent protein to HeLa cells. Mitosis and toxicity events were included in the model resulting in simulation outputs of nuclear internalization and transfection efficiency that correlated with experimental data. A priori predictions based on model sensitivity analysis suggest that increasing endosomal escape and decreasing lysosomal degradation, protein degradation, and GFP-induced toxicity can improve transfection efficiency by three-fold. Application of the telecommunications model to nonviral gene delivery offers insight into the development of new gene delivery systems with therapeutically relevant transfection levels.

Surface-mediated nucleic acid delivery by lipoplexes prepared in microwell arrays.

Many delivery methods have been developed to improve the therapeutic efficacy and facilitate the clinical translation of nucleic acid-based therapeutics. A facile surface-mediated nucleic acid delivery by lipoplexes is prepared in a microwell array, which combines the advantages of lipoplexes as an efficient carrier system, surface-mediated delivery, and the control of surface topography. Uniform disc-like lipoplexes containing nucleic acids are formed in the microwell array with a diameter of ∼818 nm and thickness of ∼195 nm. The microwell array-mediated delivery of lipoplexes containing FAM-oligodeoxynucleotides is ∼18.6 and ∼10.6 times more efficient than the conventional transfection method in an adherent cell line (A549 non-small cell lung cancer cells) and a suspension cell line (KG-1a acute myelogenous leukemia cells), respectively. MicroRNA-29b is then used as a model nucleic acid to investigate the therapeutic efficacy of lipoplexes delivered by the microwell array. Compared to conventional transfection methods, the effective therapeutic dosage of microRNA-29b is reduced from the microgram level to the nanogram level by lipoplexes prepared in the microwell array. The microwell array is also a very flexible platform. Both nucleic acid therapeutics and imaging reagents are incorporated in lipoplexes and successfully delivered to A549 cells, demonstrating its potential applications in theranostic medicine.

Therapeutic Delivery of MicroRNA-29b by Cationic Lipoplexes for Lung Cancer

MicroRNA-29b (miR-29b) expression has been shown to be reduced in non-small–cell lung cancer (NSCLC) tissues. Here, we have identified the oncogene cyclin-dependent protein kinase 6 (CDK6) as a direct target of miR-29b in lung cancer. We hypothesized that in vivo restoration of miR-29b and thus targeting of genes important to tumor initiation and progression may represent an option for lung cancer treatment. We developed a cationic lipoplexes (LPs)-based carrier that efficiently delivered miR-29b both in vitro and in vivo. LPs containing miR-29b (LP-miR-29b) efficiently delivered miR-29b to NSCLC A549 cells, reduced the expression of key targets CDK6, DNMT3B, and myeloid cell leukemia sequence 1 (MCL1), as well as cell growth and clonogenicity of A549 cells. In addition, the IC50 for cisplatin in the miR-29b–treated cells was effectively reduced. In a xenograft murine model, LPs efficiently accumulated at tumor sites. Systemic delivery of LP-miR-29b increased the tumor miR-29b expression by approximately fivefold, downregulated the tumor mRNA expression of CDK6, DNMT3B, and MCL1 by ~57.4, ~40.5, and ~52.4%, respectively, and significantly inhibited tumor growth by ~60% compared with LP-miR-NC (negative control). Our results demonstrate that cationic LPs represent an efficient delivery system that holds great potential in the development of miRNA-based therapeutics for lung cancer treatment.

The gene transfection properties of a lipophosphoramidate derivative with two phytanyl chains

Development of efficient and non-toxic gene delivery systems is among the most challenging requirements for successful gene therapy. Cationic lipophosphoramidates constitute a class of cationic lipids we have already shown to be efficient for in vivo gene transfer. Herein, we report the synthesis of a cationic lipophosphoramidate bearing two phytanyl chains (BSV18) as hydrophobic domain, and studied its gene transfection properties. In vitro, BSV18 exhibited a high transfection efficacy associated with a low cytotoxicity. 31P NMR studies of various cationic lipophosphoramidates in water solution suggested that the phytanyl chains may favor the formation of an inverted hexagonal phase, a supramolecular arrangement which is presumed to enhance the endosomal escape and consequently increase the transfection efficiency. In vivo, systemic delivery of BSV18-based lipoplexes allowed a high efficiency of gene transfection into the mouse lung. With a view to clinical application, we evaluated not only the efficiency of lung transfection but also the eventual in vivo side-effects. Thus, in addition to monitoring the in vivo transfection efficiency by bioluminescent imaging and identifying by immunohistochemistry the cell types transfected, we also assessed in living animals the potential liver reaction as well as the inflammatory and immune responses induced by BSV18-mediated transfection. All those adverse effects were actually highly transient. Thus, taken together, these results indicate that lipophosphoramidates equipped with two phytanyl chains may have great potential for lung gene therapy, in particular for Cystic Fibrosis.

Hydration of surfactant-modified and PEGylated cationic cholesterol-based liposomes and corresponding lipoplexes by monitoring a fluorescent probe and the dielectric relaxation time

For the optimization of plasmid DNA (pDNA)-cationic lipid complexes and lipoplex delivery, proper indexes of the physicochemical properties of lipoplexes are required. In general, the characteristics of lipoplexes are defined by particle size and zeta-potential at various mixing ratios of cationic liposomes and pDNA. In this study, we characterized the hydration level of surfactant-modified and PEGylated cationic cholesterol-based (OH-Chol) liposomes and their lipoplexes by monitoring both the fluorescent probe laurdan and the dielectric relaxation time. Fluorescence measurement using laurdan detected hydration of the headgroup of lipids in surfactant-modified liposomes and PEGylated DOTAP-liposomes, but hardly any fluorescence was detected in PEGylated OH-Chol-liposomes because the PEG layers may extend and cover the fluorescent maker. On the other hand, the measurement of dielectric relaxation time of water molecules revealed total hydration, including hydration of the PEG layer and the headgroup of cationic lipids. Furthermore, we found an inverse correlation between hydration level and cellular uptake of PEGylated lipoplexes (R=0.946). This finding indicated that the dielectric relaxation time of water molecules provides an important indicator of hydration of liposome and lipoplexes along with the fluorescence intensity of laurdan.