Cationic Cholesterol Derivatives Research Articles

Abstract 5164: Liposomal Delivery of an Immunostimulatory CpG Induces Robust Antitumor Immunity and Long-Term Immune Memory by Reprogramming Tumor-Associated Macrophages

Abstract Background: The efficacy of current T cell-based immunotherapy is known to be limited by various factors that induce immune suppression in the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are representative TME-comprising immune suppressive cells and influence tumor growth and progression. TAMs are considered as an attractive target for cancer immunotherapy. However, current TAMs-targeting strategies are not sufficient to induce continuous antitumor responses. Here, we suggest a new strategy that can reprogram TAMs and further bridge innate-to-adaptive immunity by utilizing immunostimulatory CpG oligodeoxynucleotides (ODNs). In this study, we investigated the new role of TLR9 agonist CpG ODN as an immunotherapeutic agent and tested whether liposomal delivery of CpG could modulate the function of TAMs and lead to remodeling of the TME. Methods: A liposome-based nanomaterial encapsulating CpG ODN was constructed. A small lipid nanoparticle (SLNP) used as the CpG delivery platform in this study was made of a cationic cholesterol derivative and two biocompatible phospholipids. The CpG-encapsulating SLNP (SLNP@CpG) was prepared by a thin-film formation and rehydration method. In vitro studies were conducted to assess whether SLNP@CpG could reprogram macrophages. To assess antitumor therapeutic efficacy of the SLNP@CpG, two subcutaneous tumor models were established in mice and tumor growth and survival rates of mice were monitored. For tumor rechallenge experiment, mice showing complete tumor regression were rechallenged with same cancer cells and their splenocytes were analyzed to confirm the development of immune memory. To elucidate the immunological mechanism of SLNP@CpG-mediated antitumor efficacy, changes of immune cell subpopulations in treated tumors were analyzed by flow cytometry. Results: SLNP@CpG enhanced macrophage-mediated phagocytosis of cancer cells and tumor antigen cross-presentation, and skewed the polarization state of macrophages in vitro. Intratumorally injected SLNP@CpG exerted its therapeutic efficacy in an established E.G7-OVA tumor via uptake by TAMs. SLNP@CpG treatment significantly suppressed the E.G7 tumor growth and also considerably prolonged the survival of mice, with 83.3% of mice becoming tumor-free. Local administration of SLNP@CpG resisted E.G7 tumor rechallenge by inducing immunological memory and long-term antitumor immunity. Local administration of SLNP@CpG even exerted its antitumor efficacy in an aggressive B16-F10 melanoma by remodeling TME towards immune stimulation and tumor elimination. Conclusion: Liposomal delivery of CpG via local treatment reprogrammed TAMs by enhancing phagocytic activity and repolarizing M2 to M1 phenotype and also reshaped immunosuppressive TME, leading to antitumor immunity and long-term memory responses. Citation Format: Yujin Kim, Sangyong Jon. Liposomal Delivery of an Immunostimulatory CpG Induces Robust Antitumor Immunity and Long-Term Immune Memory by Reprogramming Tumor-Associated Macrophages. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5164.

Effect of pre‑freezing and saccharide types in freeze‑drying of siRNA lipoplexes on gene‑silencing effects in the cells by reverse transfection.

Our previous study reported that reverse (Rev)-transfection with small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) freeze-dried in trehalose or sucrose solution resulted in high gene-silencing activity in cells. The current study investigated whether pre-freezing or saccharide types present during the freeze-drying of siRNA lipoplexes affected gene-silencing in cells after Rev-transfection. Three types of cationic cholesterol derivatives and three types of dialkyl or trialkyl cationic lipids were used for the preparation of cationic liposomes. Additionally, six types of siRNA lipoplexes were vacuum-dried in trehalose or sucrose solution without a pre-freezing process in multi-well plates. A strong gene-silencing activity after Rev-transfection was observed regardless of the cationic lipid types in the cationic liposomes. It was also investigated whether saccharide types in the freeze-drying of siRNA lipoplexes affected gene-silencing after Rev-transfection. siRNA lipoplexes freeze-dried in monosaccharides (glucose, fructose, galactose or mannose), disaccharides (maltose, lactose, lactulose or cellobiose) and trisaccharide solution (raffinose or melezitose) demonstrated high gene-silencing activity. However, following Rev-transfection with siRNA lipoplexes freeze-dried in monosaccharides or trisaccharides, certain saccharides induced cytotoxicity and/or off-target effects. The results of the current study indicated that disaccharides may be suitable for the preparation of vacuum-dried or freeze-dried siRNA lipoplexes for Rev-transfection.

Sequential and Timely Combination of a Cancer Nanovaccine with Immune Checkpoint Blockade Effectively Inhibits Tumor Growth and Relapse

AbstractWe describe a small lipid nanoparticle (SLNP)‐based nanovaccine platform and a new combination treatment regimen. Tumor antigen‐displaying, CpG adjuvant‐embedded SLNPs (OVAPEP‐SLNP@CpG) were prepared from biocompatible phospholipids and a cationic cholesterol derivative. The resulting nanovaccine showed highly potent antitumor efficacy in both prophylactic and therapeutic E.G7 tumor models. However, this vaccine induced T cell exhaustion by elevating PD‐L1 expression, leading to tumor recurrence. Thus, the nanovaccine was combined with simultaneous anti‐PD‐1 antibody treatment, but the therapeutic efficacy of this regimen was comparable to that of the nanovaccine alone. Finally, mice that showed a good therapeutic response after the first cycle of immunization with the nanovaccine underwent a second cycle together with anti‐PD‐1 therapy, resulting in suppression of tumor relapse. This suggests that the antitumor efficacy of combinations of nanovaccines with immune checkpoint blockade therapy is dependent on treatment sequence and the timing of each modality.

Sequential and Timely Combination of a Cancer Nanovaccine with Immune Checkpoint Blockade Effectively Inhibits Tumor Growth and Relapse.

We describe a small lipid nanoparticle (SLNP)-based nanovaccine platform and a new combination treatment regimen. Tumor antigen-displaying, CpG adjuvant-embedded SLNPs (OVAPEP -SLNP@CpG) were prepared from biocompatible phospholipids and a cationic cholesterol derivative. The resulting nanovaccine showed highly potent antitumor efficacy in both prophylactic and therapeutic E.G7 tumor models. However, this vaccine induced T cell exhaustion by elevating PD-L1 expression, leading to tumor recurrence. Thus, the nanovaccine was combined with simultaneous anti-PD-1 antibody treatment, but the therapeutic efficacy of this regimen was comparable to that of the nanovaccine alone. Finally, mice that showed a good therapeutic response after the first cycle of immunization with the nanovaccine underwent a second cycle together with anti-PD-1 therapy, resulting in suppression of tumor relapse. This suggests that the antitumor efficacy of combinations of nanovaccines with immune checkpoint blockade therapy is dependent on treatment sequence and the timing of each modality.

Effects of cationic lipids in cationic liposomes and disaccharides in the freeze-drying of siRNA lipoplexes on gene silencing in cells by reverse transfection

RNA interference is a promising technology to inhibit the production of target proteins, and screening with synthetic small interfering RNA (siRNA) libraries has become a crucial research tool used to study gene function in cells. Reverse (Rev) transfection with freeze-dried siRNA/cationic liposome complexes (siRNA lipoplexes) can simplify and speed up siRNA transfection without the preparation of siRNA lipoplexes just before transfection. In this study, we examined the effects of cationic lipids in cationic liposomes and disaccharides in freeze-drying of siRNA lipoplexes on gene silencing in cells by Rev-transfection. We used three types of cationic cholesterol derivatives and three types of dialkyl or trialkyl cationic lipids for the preparation of cationic liposomes, and we prepared six types of freeze-dried siRNA lipoplexes in the presence of trehalose or sucrose solution in multi-well plates. Increasing concentrations of trehalose or sucrose included during freeze-drying of siRNA lipoplexes resulted in increased gene silencing activity upon Rev-transfection. Strong gene silencing activity was observed regardless of the type of cationic lipid in cationic liposomes when siRNA lipoplexes were freeze-dried with the disaccharides at concentrations of more than 25 mM or 100 mM. In addition, siRNA lipoplexes freeze-dried with 100 mM trehalose or sucrose showed long-term (1 month) stability without apparent loss of gene silencing activity. These findings suggested that Rev-transfection with freeze-dried siRNA lipoplexes may have potential applications in the screening of gene function using siRNA libraries.

Effect of Cationic Lipid Type in Folate-PEG-Modified Cationic Liposomes on Folate Receptor-Mediated siRNA Transfection in Tumor Cells.

In this study, we examined the effect of cationic lipid type in folate (FA)-polyethylene glycol (PEG)-modified cationic liposomes on gene-silencing effects in tumor cells using cationic liposomes/siRNA complexes (siRNA lipoplexes). We used three types of cationic cholesterol derivatives, cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol), N-(2-(2-hydroxyethylamino)ethyl)cholesteryl-3-carboxamide (OH-Chol), and cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol), and we prepared three types of FA-PEG-modified siRNA lipoplexes. The modification of cationic liposomes with 1–2 mol % PEG-lipid abolished the gene-silencing effect in human nasopharyngeal tumor KB cells, which overexpress the FA receptor (FR). In contrast, FA-PEG-modification of cationic liposomes restored gene-silencing activity regardless of the cationic lipid type in cationic liposomes. However, the optimal amount of PEG-lipid and FA-PEG-lipid in cationic liposomes for selective gene silencing and cellular uptake were different among the three types of cationic liposomes. Furthermore, in vitro transfection of polo-like kinase 1 (PLK1) siRNA by FA-PEG-modified liposomes exhibited strong cytotoxicity in KB cells, compared with PEG-modified liposomes; however, in in vivo therapy, intratumoral injection of PEG-modified PLK1 siRNA lipoplexes inhibited tumor growth of KB xenografts, as well as that of FA-PEG-modified PLK1 siRNA lipoplexes. From these results, the optimal formulation of PEG- and FA-PEG-modified liposomes for FR-selective gene silencing might be different between in vitro and in vivo transfection.

Effect of cationic lipid type in cationic liposomes for siRNA delivery into the liver by sequential injection of chondroitin sulfate and cationic lipoplex

Previously, we reported that intravenous injection of chondroitin sulfate (CS), followed by intravenous injection of cationic liposome/siRNA complex (cationic lipoplex) could deliver siRNA into the liver. In this study, we examined the effects of the type of cationic lipid in the liposome on siRNA delivery into the liver. We used 6 types of cationic cholesterol derivatives and 11 types of dialkyl or trialkyl cationic lipids, and prepared 17 types of cationic liposomes to evaluate the gene-silencing effect of siRNA in mouse liver following sequential injection of CS plus cationic lipoplex. In 14 types of cationic liposomes, siRNA was accumulated in the liver when cationic lipoplexes were injected immediately after CS. However, suppression of apolipoprotein B (ApoB) mRNA expression in the liver and reduction of low-density lipoprotein (LDL) and very-low-density lipoprotein (VLDL) cholesterol level in the serum were observed only after sequential injection of CS plus ApoB siRNA lipoplex with cationic liposomes composed of N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16)/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and N,N-dimethyl-N-octadecyloctadecan-1-aminium bromide (DC-1-18)/DOPE. Based on these findings, the type of cationic lipid strongly affected the gene-silencing effect of siRNA in the liver by sequential injection of CS plus cationic lipoplex.

Effect of cationic lipid in cationic liposomes on siRNA delivery into the lung by intravenous injection of cationic lipoplex

Cationic liposomes composed of dialkyl cationic lipid such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) can efficiently deliver siRNA to the lungs following the intravenous injection of cationic liposome/siRNA complexes (lipoplexes). In this study, we examined the effect of cationic lipid of cationic liposomes on siRNA delivery to the lungs after intravenous injection. We used six kinds of cationic cholesterol derivatives and 11 kinds of dialkyl or trialkyl cationic lipids as cationic lipids, and prepared 17 kinds of cationic liposomes composed of a cationic lipid and 1,2-dioleoyl-L-α-glycero-3-phosphatidylethanolamine (DOPE) for evaluation of siRNA biodistribution and in vivo gene silencing effects. Among cationic liposomes, those composed of N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), N,N-dimethyl-N-octadecyloctadecan-1-aminium bromide (DC-1-18), 2-((1,5-bis(octadecyloxy)-1,5-dioxopentan-2-yl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium chloride (DC-3-18D), 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12), or cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol) with DOPE exhibited high accumulation of siRNA in the lung and significant suppression of Tie2 mRNA expression after the intravenous injection of cationic lipoplexes with Tie2 siRNA. Furthermore, DC-1-16/DOPE and DC-1-18/DOPE lipoplexes with protein kinase N3 (PKN3) siRNA could suppress the tumour growth when intravenously injected into mice with lung LLC metastasis. These findings indicate that the siRNA biodistribution and in vivo knockdown efficiency after the intravenous injection of cationic lipoplexes were strongly affected by the type of cationic lipid of cationic liposomes.

Cationic cholesterol derivative efficiently delivers the genes: in silico and in vitro studies.

The aims of the research work were to synthesize ethyl(cholesteryl carbamoyl)-L-arginate (ECCA), an arginine-conjugated cholesterol derivative, and to evaluate its application as a gene delivery vector. The interactions of ECCA with DNA duplex were studied using molecular dynamics (MD) simulations. It was found that the guanidine group of ECCA could interact with the phosphate group of DNA through ionic interactions as well as hydrogen bonds. The structure of DNA was stable throughout the simulation time. Liposomes were formulated using ECCA and soya phosphatidylcholine (SPC) by a thin-film hydration method. They had the particle size of ~ 150nm and the zeta potential of + 51mV. To ensure the efficient binding of DNA to the liposomes, the ratio of DNA to ECCA was optimized using gel retardation assay. Further, serum stability, haemolysis and cytotoxicity studies were carried out to determine the stability and safety of the lipoplexes. Circular dichroism spectroscopy was used to determine the interaction of DNA and cationic liposomes. Cellular uptake pathway was determined by studying the uptake of coumarin-loaded lipoplexes at 4°C and in the presence of uptake inhibitors, i.e. genistein, chlorpromazine and methyl-β-cyclodextrin. Transfection studies were carried out to evaluate the transfection efficacy of the ECCA-loaded lipoplexes. The binding of DNA and lipoplexes was found to be stable in the presence of serum, and no degradation of DNA was observed. The lipoplexes showed low haemolysis and cytotoxicity. The uptake of coumarin-loaded liposomes was decreased up to ~ 20% in the presence of clathrin- and caveola-mediated uptake inhibitors, indicating a role of both the pathways in the uptake of the inhibitors. Satisfactory transfection efficiency was obtained compared to Lipofectamine®. Thus, cationic cholesterol derivative is a useful tool for gene delivery vector.

Efficient delivery of Notch1 siRNA to SKOV3 cells by cationic cholesterol derivative-based liposome.

A novel cationic cholesterol derivative-based small interfering RNA (siRNA) interference strategy was suggested to inhibit Notch1 activation in SKOV3 cells for the gene therapy of ovarian cancer. The cationic cholesterol derivative, N-(cholesterylhemisuccinoyl-amino-3-propyl)-N, N-dimethylamine (DMAPA-chems) liposome, was incubated with siRNA at different nitrogen-to-phosphate ratios to form stabilized, near-spherical siRNA/DMAPA-chems nanoparticles with sizes of 100–200 nm and zeta potentials of 40–50 mV. The siRNA/DMAPA-chems nanoparticles protected siRNA from nuclease degradation in 25% fetal bovine serum. The nanoparticles exhibited high cell uptake and Notch1 gene knockdown efficiency in SKOV3 cells at an nitrogen-to-phosphate ratio of 100 and an siRNA concentration of 50 nM. They also inhibited the growth and promoted the apoptosis of SKOV3 cells. These results may provide the potential for using cationic cholesterol derivatives as efficient nonviral siRNA carriers for the suppression of Notch1 activation in ovarian cancer cells.

Small interfering RNA delivery into the liver by cationic cholesterol derivative-based liposomes

Purpose: Previously, we reported that the cationic liposomes composed of a cationic cholesterol derivative, cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (termed LP-C), could deliver small interfering RNAs (siRNAs) with high transfection efficiency into tumor cells. In this study, to develop a liposomal vector for siRNA delivery in vivo, we prepared the poly(ethyleneglycol) (PEG)-modified cationic liposomes (LP-C-PEG) and evaluated their transfection efficiency in vitro and in vivo.Materials and methods: We prepared LP-C-PEG/siRNA complexes (LP-C-PEG lipoplexes) formed in water or 50 mM NaCl solution, and evaluated their siRNA biodistribution and gene silencing effect in mice after intravenous injection.Results: LP-C-PEG lipoplexes strongly exhibited in vitro gene silencing effects in human breast tumor MCF-7 cells as well as LP-C lipoplexes. In particular, formation of LP-C and LP-C-PEG lipoplexes in the NaCl solution increased the cellular association. When LP-C-PEG lipoplexes with Cy5.5-labeled siRNA formed in water or NaCl solution were injected into mice, accumulation of the siRNA was observed in the liver. Furthermore, injection of LP-C-PEG lipoplexes with ApoB siRNA could suppress ApoB mRNA levels in the liver and reduce very-low-density lipoprotein/low-density lipoprotein levels in serum compared with that after Cont siRNA transfection, although the presence of NaCl solution in forming the lipoplexes did not affect gene silencing effects in vivo.Conclusions: LP-C-PEG may have potential as a gene vector for siRNA delivery to the liver.

Mono-arginine Cholesterol-based Small Lipid Nanoparticles as a Systemic siRNA Delivery Platform for Effective Cancer Therapy.

Although efforts have been made to develop a platform carrier for the delivery of RNAi therapeutics, systemic delivery of siRNA has shown only limited success in cancer therapy. Cationic lipid-based nanoparticles have been widely used for this purpose, but their toxicity and undesired liver uptake after systemic injection owing to their cationic surfaces have hampered further clinical translation. This study describes the development of neutral, small lipid nanoparticles (SLNPs) made of a nontoxic cationic cholesterol derivative, as a suitable carrier of systemic siRNA to treat cancers. The cationic cholesterol derivative, mono arginine-cholesterol (MA-Chol), was synthesized by directly attaching an arginine moiety to cholesterol via a cleavable ester bond. siRNA-loaded SLNPs (siRNA@SLNPs) were prepared using MA-Chol and a neutral helper lipid, dioleoyl phosphatidylethanolamine (DOPE), as major components and a small amount of PEGylated phospholipid mixed with siRNA. The resulting nanoparticles were less than ~50 nm in diameter with neutral zeta potential and much lower toxicity than typical cationic cholesterol (DC-Chol)-based lipid nanoparticles. SLNPs loaded with siRNA against kinesin spindle protein (siKSP@SLNPs) exhibited a high level of target gene knockdown in various cancer cell lines, as shown by measurement of KSP mRNA and cell death assays. Furthermore, systemic injection of siKSP@SLNPs into prostate tumor-bearing mice resulted in preferential accumulation of the delivered siRNA at the tumor site and significant inhibition of tumor growth, with little apparent toxicity, as shown by body weight measurements. These results suggest that these SLNPs may provide a systemic delivery platform for RNAi-based cancer therapy.

Folate-Targeted Gadolinium-Lipid-Based Nanoparticles as a Bimodal Contrast Agent for Tumor Fluorescent and Magnetic Resonance Imaging

To enhance tumor magnetic resonance imaging (MRI) signals via the selective accumulation of contrast agents, we prepared folate-modified gadolinium-lipid-based nanoparticles as MRI contrast agents. Folate-modified nanoparticles were comprised of polyethylene glycol (PEG)-lipid, gadolinium diethylenetriamine pentaacetic acid lipid, cationic cholesterol derivatives, folate-conjugated PEG-lipid, and Cy7-PEG-lipid. Folate receptor-mediated cellular nanoparticle association was examined in KB cells, which overexpress the folate receptor. The biodistribution of nanoparticles after their intravenous injection into KB tumor-bearing mice was measured. Mice were imaged through in vivo fluorescence imaging and MRI 24 h after nanoparticle injection, and the intensity enhancement of the tumor MRI signal was evaluated. Increased cellular association of folate-modified nanoparticles was inhibited by excess free folic acid, indicating that nanoparticle association was folate receptor-mediated. Irrespective of folate modification, the amount of nanoparticles in blood 24 h after injection was ca. 10% of the injected dose. Compared with non-modified nanoparticles, folate-modified nanoparticles exhibited significant accumulation in tumor tissues without altering other biodistribution, as well as enhanced tumor fluorescence and MRI signal intensity. The results support the feasibility of MRI- and in vivo fluorescence imaging-based tumor visualization using folate-modified nanoparticles and provide opportunities to develop folate targeting-based imaging applications.

Construction of supramolecular nanofibers through electrostatic interaction between perylene and cholesterol derivatives

Abstract The self-assembly of cationic perylene diimide (PDI) and anionic cholesterol derivatives (CHOL) was conveniently achieved by the electrostatic attraction and π – π stacking interactions, exhibiting the well-defined supramolecular nanofibers ranging from hundreds of nanometers to micron dimension.

Comparison of two lipid/DNA complexes of equal composition and different morphology

Two types of complexes were prepared from a cationic cholesterol derivative, dioleoylphos-phatidylcholine and DNA. Depending on the preparation procedure complexes were either dense snarls of lipid covered DNA (type A) or multilayer liposomes with DNA between layers (type B). The transfection efficiency of the snarl-shaped complexes was low but positive. The transfection efficiency of the liposome-shaped complexes was zero, while DNA release upon their interaction with anionic liposomes was 1.7 times higher. The differences in transfection efficacy and DNA release could not be ascribed to the difference in resistance of complexes to decomposition upon interaction with anionic liposomes or intracellular environment since the lipid composition of complexes is the same. Instead the complexes in which lipoplex phase is more continuous (type A) should require more anionic lipids or more time within a cell for complete decomposition. Prolonged life time should lead to the higher probability of DNA expression.

Asymmetric lipid bilayer formation stabilized by DNA at the air/water interface

The lipid bis(guanidinium)-tren-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups used for gene transfection either alone or formulated as liposomes with the zwitterionic lipid 1,2-di-[ cis-9-octadecenoyl]- sn-glycero-3-phosphoethanolamine (DOPE). Previous investigations have shown its ability to strongly interact with DNA and form asymmetric lipid bilayers at the air/water interface when mixed with DOPE. Here, with a view to further investigate its physicochemical behavior, we studied the interactions of mixtures of BGTC with another zwitterionic lipid, 1,2-Dimyristoyl- sn-Glycero-3-Phosphocholine, (DMPC), with DNA at the air/water interface by using the Langmuir monolayer technique coupled with Brewster Angle Microscopy (BAM) and Polarization Modulation Infra Red Reflexion Absorption (PMIRRAS) spectroscopy and we investigate DNA–BGTC/DMPC interactions. We demonstrate that when DNA is injected into the subphase in excess compared to the positive charges of BGTC, it adsorbs to BGTC/DMPC monolayers at 20 mN/m whatever the lipid monolayer composition (1/5, 2/3 or 3/2 BGTC/DMPC molar ratio) and forms an incomplete monolayer of either isotropic or anisotropic double strands depending on the BGTC content in the monolayer. Compression beyond the collapse of some mixed DNA–BGTC/DMPC (2/3 and 3/2 molar ratio) systems leads to the formation of DNA monolayers underneath asymmetric lipid bilayers characterized by a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DMPC.

Brewster Angle Microscopy and PMIRRAS Study of DNA Interactions with BGTC, a Cationic Lipid Used for Gene Transfer

The lipid bis(guanidinium)-tris(2-aminoethyl)amine-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups which displays high transfection efficiency in vitro and in vivo when used alone or formulated as liposomes with the neutral colipid 1,2-di-[ cis-9-octadecenoyl]- sn-glycero-3-phosphoethanolamine (DOPE). Since transfection may be related to the structural and physicochemical properties of the self-assembled supramolecular lipid-DNA complexes, we used the Langmuir monolayer technique coupled with Brewster angle microscopy (BAM) and polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) to investigate DNA-BGTC and DNA-BGTC/DOPE interactions at the air/water interface. We herein show that BGTC forms stable monolayers at the air/water interface. When DNA is injected into the subphase, it adsorbs to BGTC at 20 mN/m. Whatever the (+/-) charge ratio of the complexes used, defined as the ratio of positive charges of BGTC in the monolayer versus negative charges of DNA injected in the subphase, the DNA interacts with the cationic lipid and forms either an incomplete (no constituent in excess) or a complete (DNA in excess) monolayer of oriented double strands parallel to the lipid monolayer plan. We also show that, under a homogeneous BGTC/DOPE (3/2) monolayer at 20 mN/m, DNA adsorbs homogeneously to form an organized but incomplete layer whatever the charge ratio used (DNA in default or in excess). Compression beyond the collapse of these mixed DNA-BGTC/DOPE systems leads to the formation of dense DNA monolayers under an asymmetric lipid bilayer with a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DOPE. These results allow a better understanding of the mechanisms underlying the formation of the supramolecular BGTC-DNA complexes efficient for gene transfection.

Hydroxyethylated cationic cholesterol derivatives in liposome vectors promote gene expression in the lung

Three cationic cholesterol derivatives (CCDs), which differ in their types of amine and bear a hydroxyethyl group at the amine group, were synthesized and formulated into liposomes and nanoparticles as gene delivery vectors. In vitro transfection into A549 cells proved that liposomes formulated with CCDs and dioleoylphosphatidylethanolamine (DOPE) of 1/2 molar ratio were more effective than the corresponding nanoparticles with CCDs and Tween 80 at charge ratios (+/−) of 1/2, 3/1 and 5/1. Among the liposomal formulations, non-hydroxyethylated CCDs were more effective than hydroxyethylated ones in vitro. However, gene transfection in the lung through intratracheal injection showed opposite results to those in vitro, with liposomes containing hydroxyethylated CCDs being more potent than those containing non-hydroxyethylated CCDs. Transfection by liposomes with N, N-methyl hydroxyethyl aminopropane carbamoyl cholesterol iodide (MHAPC) showed the highest luciferase activity, resulting in 2- and 60-fold higher gene expression than jet-PEI and naked DNA, respectively . The distribution of MHAPC lipoplex after intratracheal injection was heterogeneous, and luciferase was expressed in epithelial cells lining the bronchi and bronchioles. All the lipoplexes led to higher TNF-α levels in the lung compared to the nanoplex and jet-PEI, but our findings suggested that modification of the cationic cholesterol with a hydroxyethyl group at the tertiary amine terminal, MHAPC, promoted gene expression in the lung without increasing the toxicity compared with other CCDs. This work firstly proved that liposomes containing hydroxyethylated CCDs could promote gene expression in the lung through intratracheal injection.

The role of dioleoylphosphatidylethanolamine (DOPE) in targeted gene delivery with mannosylated cationic liposomes via intravenous route

We have previously reported that mannosylated cationic liposome consisting with the mannosylated cationic cholesterol derivative Man-C4-Chol (Man) and dioleoylphosphatidylethanolamine (DOPE) (Man/DOPE) could deliver DNA to the liver by intravenous administration via mannose receptor-mediated endocytosis, however, rapid degradation in lysosomes might be a rate-limiting step in its gene transfection. In this study, we tried to evaluate the role of DOPE in in vivo gene transfer by comparing its transfection efficacy with mannosylated liposomes composed of Man and dioleoylphosphatidylcholine (DOPC) (Man/DOPC). In vitro studies showed that the cellular association of both liposome/pCMV-Luc complexes was almost the same, although Man/DOPE complex showed about 10-fold higher transfection activity than Man/DOPC complex. After intraportal administration into mice, Man/DOPE complex showed higher gene expression than Man/DOPC complex, suggesting that DOPE improves intracellular trafficking in target cells under in vivo conditions. An intravenous administration study demonstrated that Man/DOPE complex was accumulated in the liver more efficiently and achieved a higher gene expression in the liver than Man/DOPC complex. Thus, we conclude that the property of DOPE in mannosylated liposomes contributes to the efficient gene expression in the target site through enhanced distribution to the target site and intracellular sorting in the target cells under in vivo conditions.

Synthesis of Cationic Cholesterol Derivatives with Fragments of Aliphatic Nitrogen‐Containing Bases.

AbstractFor Abstract see ChemInform Abstract in Full Text.