Amphiphilic Lipopeptide Research Articles

Self-assembling anti-Gram-negative bacterial peptide derivatives with potent broad-spectrum antimicrobial activity

Food contamination caused by bacteria is an important challenge in the food industry. In this study, the peptide derivative C12−CL5 was produced by attaching a 12-carbon saturated fatty acid to the N-terminus of the anti-Gram-negative bacterial peptide CL5. The amphiphilic lipopeptide C12−CL5 self-assembled into core-shell nanostructures at concentrations above its critical aggregation concentration. It was found that the self-assembling C12−CL5 showed potent activity against both Gram-negative and Gram-positive bacteria, with minimum inhibitory concentrations of 4–32 μM. Moreover, C12−CL5 exhibited good thermal stability, excellent salt tolerance, as well as strong and rapid killing kinetics. Studies on the antimicrobial mechanism of C12−CL5 indicated that it could disrupt cell membranes, leading to leakage of cellular contents and ultimately to bacterial death. This study revealed that the self-assembly of antimicrobial peptides improved their antimicrobial activity and spectrum. It provides the insights and basis for the development of potent and broad-spectrum antimicrobial peptides for use, e.g., in the food industry.

Molecularly Engineered Surfactin Analogues Induce Nonapoptotic-Like Cell Death and Increased Selectivity in Multiple Breast Cancer Cell Types.

Surfactin, a negatively charged amphiphilic lipopeptide biosurfactant, is synthesized by the bacterium Bacillus subtilis. It consists of a cyclic heptapeptide and an 11-15C β-hydroxy fatty acid. To probe how the modification of the molecular skeleton of surfactin influences its selectivity and activity against breast cancer, six synthetic surfactins were generated. Modifications were accomplished by conjugating amine-functionalized molecules to the Glu and Asp carboxyl moieties of the heptapeptide. The resulting synthetic surfactins provided a diverse series of molecules with differences in charge, size, and hydrophilicity. After purification and structural analysis, insights into biological activity and specificity were generated for each compound. Dose-dependent growth inhibition was determined for four tumorigenic breast cancer cell lines in monolayer and spheroid morphologies, as well as nontumorigenic fibroblasts and sheep erythrocytes, which were utilized to determine selectivity indices. Results indicated that two compounds, which have amplified anionic charge, had increased activity on breast cancer, with reduced activity on nontumorigenic fibroblasts and erythrocytes. Cationic derivative surf-ethylenediamine has increased activity on all cell lines tested. Novel correlations between dose-response activities and physicochemical properties of all compounds determined that there is a significant correlation between the critical micelle concentration and activity against multiple cell lines.

Molecular and supramolecular engineering on lipopeptide-based hole-punching nanotoxins to trigger multimodal death of drug-resistant tumors: Apoptosis, necrosis and autophagy

Discovering bioactive and efficient new agents for broad-spectrum multidrug resistance reversal is a worldwide challenge in the biomedical field. Herein, bioactive nanomaterials assembled from specially-designed lipopeptides are developed as a novel type of hole-punching nanotoxins to trigger multimodal tumor death for multidrug resistance reversal in vitro and in vivo. The dendritic arginine-rich peptides as hydrophilic parts could incorporate with multivalent scaffolds and multiple-interaction residues to boost membrane-binding intensity, and cholesterol as hydrophobic blocks are hopeful to improve and ameliorate membrane-targeting/inserting ability against drug-resistant tumor membranes. The amphiphilic lipopeptides are designed to drive supramolecular self-assembly into nanostructures for improving hole-punching potential and stability. As envisaged, these lipopeptide-based nanotoxins could maintain high stability, obtain strong hole-punching activity and trigger multimodal tumor death with 80.3% TUNEL-positive apoptotic cells, 97.8% PARP-positive necrotic cells and 71.0% LC3-positive autophagic cells for multidrug resistance reversal in vivo. This work opens a new opportunity to develop new-style toxins for antitumor treatment, and the lipopeptide-based nanotoxins are expected to be a candidate agent for dealing with multidrug resistance.

New Developments in Pediatric Antifungal Pharmacology.

New Developments in Pediatric Antifungal Pharmacology.

Guanidinium-rich lipopeptide functionalized bacteria-absorbing sponge as an effective trap-and-kill system for the elimination of focal bacterial infection

Focal bacterial infections are often difficult to treat due to the rapid emergence of antibiotic-resistant bacteria, high risk of relapse, and severe inflammation at local lesions. To address multidrug-resistant skin and soft tissue infections, a bacteria-absorbing sponge was prepared to involve a “trap-and-kill” mechanism. The system describes a guanidinium-rich lipopeptide functionalized lyotropic liquid-crystalline hydrogel with bicontinuous cubic networks. Amphiphilic lipopeptides can be spontaneously anchored to the lipid-water interface, exposing their bacterial targeting sequences to enhance antibacterial trapping/killing activity. Computational simulations supported our structural predictions, and the sponge was confirmed to successfully remove ∼98.8% of the bacteria in the medium. Release and degradation behavior studies indicated that the bacteria-absorbing sponge could degrade, mediate enzyme-responsive lipopeptide release, or generate ∼200 nm lipopeptide nanoparticles with environmental erosion. This implies that the sponge can effectively capture and isolate high concentrations of bacteria at the infected site and then sustainably release antimicrobial lipopeptides into deep tissues for the eradication of residual bacteria. In the animal experiment, we found that the antibacterial performance of the bacterial-absorbing sponge was significant, which demonstrated not only a long-term inhibition effect to disinfect and avoid bacterial rebound, but also a unique advantage to protect tissue from bacterial attack. Statement of significanceHost defense peptides/peptidomimetics (HDPs) have shown potential for the elimination of focal bacterial infections, but the application of their topical formulations suffers from time-consuming preparation processes, indistinctive toxicity reduction effects, and inefficient bacterial capture ability. To explore new avenues for the development of easily prepared, low-toxicity and high-efficiency topical antimicrobials, a guanidinium-rich lipopeptide was encapsulated in a lyotropic liquid-crystalline hydrogel (denoted as “bacteria-absorbing sponge”) to achieve complementary superiorities. The superior characteristic of the bacteria-absorbing sponge involves a “trap-and-kill” mechanism, which undergoes not only a long-term inhibition effect to disinfect and avoid bacterial rebound, but also effective bacterial capture and isolating action to confine bacterial diffusion and protect tissues from bacterial attack.

Enhancing Antigen Presentation and Inducing Antigen-Specific Immune Tolerance with Amphiphilic Peptides.

Ag-specific immunotherapy to restore immune tolerance to self-antigens, without global immune suppression, is a long-standing goal in the treatment of autoimmune disorders such as type 1 diabetes (T1D). However, vaccination with autoantigens such as insulin or glutamic acid decarboxylase have largely failed in human T1D trials. Induction and maintenance of peripheral tolerance by vaccination requires efficient autoantigen presentation by APCs. In this study, we show that a lipophilic modification at the N-terminal end of CD4+ epitopes (lipo-peptides) dramatically improves peptide Ag presentation. We designed amphiphilic lipo-peptides to efficiently target APCs in the lymph nodes by binding and trafficking with endogenous albumin. Additionally, we show that lipophilic modification anchors the peptide into the membranes of APCs, enabling a bivalent cell-surface Ag presentation. The s.c. injected lipo-peptide accumulates in the APCs in the lymph node, enhances the potency and duration of peptide Ag presentation by APCs, and induces Ag-specific immune tolerance that controls both T cell- and B cell-mediated immunity. Immunization with an amphiphilic insulin B chain 9-23 peptide, an immunodominant CD4+ T cell epitope in NOD mice, significantly suppresses the activation of T cells, increases inhibitory cytokine production, induces regulatory T cells, and delays the onset and lowers the incidence of T1D. Importantly, treatment with a lipophilic β-cell peptide mixture delays progression to end-stage diabetes in acutely diabetic NOD mice, whereas the same doses of standard soluble peptides were not effective. Amphiphilic modification effectively enhances Ag presentation for peptide-based immune regulation of autoimmune diseases.

Virus-inspired nanoparticles as versatile antibacterial carriers for antibiotic delivery against Gram-negative and Gram-positive bacteria

Infectious diseases become one of the leading causes of human death. Traditional treatment based on classical antibiotics could not provide enough antibacterial activity to combat bacterial infections due to low bioavailability, even leading to antibiotic resistance. In recent years, biomimetic delivery systems have been developed to improve drug therapy for various diseases, such as malignant tumor and cardiovascular disease. In this work, we designed virus-inspired nanodrugs (VNDs) through co-assembly of amphiphilic lipopeptide dendrons and poly(lactic-co-glycolic acid) polymers for high-efficiency antibiotic delivery. These VNDs had well-defined and stable nanostructures for tetracycline encapsulation and delivery. The VNDs were capable of promoting antibiotic internalization and enhancing their antibacterial effects against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Additionally, no obvious cytotoxicity of VNDs was observed to human cell lines. This work successfully demonstrated the virus-mimetic nanoparticles served as promising and applicable antibiotic delivery platform for antibacterial treatment.

Iron doped carbon dots based nanohybrids as a tetramodal imaging agent for gene delivery promotion and photothermal-chemodynamic cancer synergistic theranostics

Nonviral gene delivery vectors for cancer therapy still confronts great challenges as their low transfection efficiency, vague tracing and poor therapeutic effects. Recently, organic–inorganic nanohybrids show complementary and synergistic effects in nano-scaled delivery system, inspiring their applications in gene delivery exploration. A liposomal carbon dots nanohybrids system (PEG-RLS/Fe@CDs) have been fabricated, which was composed of iron-doped carbon dots (Fe@CDs) derived from iron (II) phthalocyanine and amphiphilic lipopeptide assemblies (DSPE-mPEG2000/RLS). With its excellent photothermal conversion capability (η = 63.4%), gene transfection efficiency was increased by 3.5-fold in 4T1 tumor cell and 2-fold in animal model. Introduction of Fe@CDs in the nanohybrids could also serve as a tetramodal contrast agent for fluorescence/photoacoustic/photothermal/magnetic resonance imaging, showing up to 3 orders of magnitude enhancement in photoacoustic signal, as well as 16 °C temperature increasement after 5 min irradiation inside tumor tissue. Furthermore, both in vitro and in vivo studies confirmed the bioactivity of PEG-RLS/Fe@CDs as nanozyme for photothermal and chemodynamic synergistic therapy, leading to a noticeable inhibited tumor growth with 80% survival for at least 50 days in a breast tumor model. These revealed the PEG-RLS/Fe@CDs is a promising candidate platform for improving gene therapy by transfection enhancement, real-time tracing and tumor synergistic destruction.

Interactions of Linear Analogues of Battacin with Negatively Charged Lipid Membranes

The increasing resistance of bacteria to available antibiotics has stimulated the search for new antimicrobial compounds with less specific mechanisms of action. These include the ability to disrupt the structure of the cell membrane, which in turn leads to its damage. In this context, amphiphilic lipopeptides belong to the class of the compounds which may fulfill this requirement. In this paper, we describe two linear analogues of battacin with modified acyl chains to tune the balance between the hydrophilic and hydrophobic portion of lipopeptides. We demonstrate that both compounds display antimicrobial activity with the lowest values of minimum inhibitory concentrations found for Gram-positive pathogens. Therefore, their mechanism of action was evaluated on a molecular level using model lipid films mimicking the membrane of Gram-positive bacteria. The surface pressure measurements revealed that both lipopeptides show ability to bind and incorporate into the lipid monolayers, resulting in decreased ordering of lipids and membrane fluidization. Atomic force microscopy (AFM) imaging demonstrated that the exposure of the model bilayers to lipopeptides leads to a transition from the ordered gel phase to disordered liquid crystalline phase. This observation was confirmed by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) results, which revealed that lipopeptide action causes a substantial increase in the average tilt angle of lipid acyl chains with respect to the surface normal to compensate for lipopeptide insertion into the membrane. Moreover, the peptide moieties in both molecules do not adopt any well-defined secondary structure upon binding with the lipid membrane. It was also observed that a small difference in the structure of a lipophilic chain, altering the balance between hydrophobic and hydrophilic portion of the molecules, results in different insertion depth of the active compounds.

Extending the Half-Life of a Protein in Vivo by Enzymatic Labeling with Amphiphilic Lipopeptides.

Synthesis of lipid-protein conjugates is one of the significant techniques in drug delivery systems of proteins; however, the intact conjugation of a lipid and protein is yet challenging due to the hydrophobicity of lipid molecules. In order to facilitate easy handling of the lipid moiety in conjugation, we have focused on a microbial transglutaminase (MTG) that can ligate specific lysine (K) and glutamine (Q) residues in lipopeptides and a protein of interest. As MTG substrates, monolipid- and dilipid-fused amphiphilic short lipopeptide substrates (lipid-G3S-RHK or lipid2-KG3S-RHK) were designed. These amphiphilic lipopeptides and a model protein (enhanced green fluorescent protein, EGFP) fused with LLQG (LQ-EGFP) were both water-soluble, and thus lipid-protein conjugates were efficiently obtained through the MTG reaction with a >80% conversion rate of LQ-EGFP even using cholesterol-G3S-RHK. In vitro cell adhesion and in vivo half-life stability of the successfully obtained lipid-protein conjugates were evaluated, showing that the monocholesterol-G3S-RHK modification of a protein gave the highest cell adhesion efficiency and longest half-life time by formation of a stable albumin/lipid-protein complex.

Multi-functional lipopeptide micelles as a vehicle for curcumin delivery

Curcumin (CUR) is a kind of natural polyphenol with low aqueous solubility, poor bioavailability and favorable antitumor activity. In order to enhance anti-tumor activity and efficient delivery of CUR, an amphiphilic lipopeptide (C18H5R7RGDS, LP) containing H5R7RGDS heads and stearic acid (C18) tails was prepared by solid-phase peptide synthesis. Spherical LP micelles of ∼50 nm were self-assembled in PBS (pH 7.0) with good dispersion and significantly improved the aqueous solubility of CUR by 5400 times more than that of free CUR. The sequence of H5 in LP endowed the CUR-loaded LP micelles with good pH-responsive drug release behavior. In the case of pH 5.0, the electrostatic repulsion interaction among the ionized H5 fraction destroyed the structure of micelles, leading to significantly accelerated CUR release behavior. Hemolysis assay and proliferation inhibition test of normal cells confirmed apparently the excellent biocompatibility of LP. CUR-loaded LP micelles showed much higher cell growth inhibition on HepG2 cells and lower cytotoxicity on L02 cells than free CUR. With integrin-targeting sequence (RGD) and cell penetrating peptide (R8), LP micelles can more specifically and efficiently deliver CUR into integrin-overexpressed HepG2 cells than C18KR8 (LP-R1) and C18KRGDS (LP-R2) micelles. Combined with the enhanced drug solubility, distinctive pH-sensitive property and good tumor targeting, such LP micelles may have the clinical potential for tumor-targeting delivery.

Preparation and evaluation of amphipathic lipopeptide-loaded PLGA microspheres as sustained-release system for AIDS prevention.

At present, AIDS drugs are typical inhibitors that cannot achieve permanent effects. Therefore, the research of blocking HIV infection is essential. Especially for people in the high‐risk environment, long‐term prevention is important, because HIV can easily infect cells once the drug is interrupted. However, there is still no long‐acting AIDS prevention drug approved. Hence, the purpose of this study is to prepare a fusion inhibitor loaded poly(d, l‐lactic‐co‐glycolic acid) (PLGA) microspheres as a sustained‐release system for long‐term AIDS prevention. As the HIV membrane fusion inhibitor (LP‐98) used in this research is amphiphilic lipopeptide, W1/O/W2 double‐emulsion method was chosen, and premix membrane emulsification technique was used for controlling the uniformity of particle size. Several process parameters that can impact drug loading efficiency were summarized: the concentration of LP‐98 and PLGA, and the preparation condition of primary emulsion. Finally, the microspheres with high loading efficiency (>8%) and encapsulation efficiency (>90%) were successfully prepared under optimum conditions. Pharmacokinetic studies showed that LP‐98‐loaded microspheres were capable to continuously release for 24 days in rats. This research can promote the application of sustained‐release microspheres in AIDS prevention, and the embedding technique used in this study can also provide references for the loading of other amphipathic drugs.

Self-Assembly, Nematic Phase Formation, and Organocatalytic Behavior of a Proline-Functionalized Lipopeptide.

The self-assembly of the amphiphilic lipopeptide PAEPKI-C16 (P = proline, A = alanine, E = glutamic acid, K = lysine, I = isoleucine, and C16 = hexadecyl) was investigated using a combination of microscopy, spectroscopy, and scattering methods and compared to that of C16-IKPEAP with the same (reversed) peptide sequence and the alkyl chain positioned at the N-terminus and lacking a free N-terminal proline residue. The catalytic activity of these peptides was then compared using a model aldol reaction system. For PAEPKI-C16, the cryo-TEM images showed the formation of micrometer-length fibers, which by small-angle X-ray scattering (SAXS) were found to have radii of 2.5–2.6 nm. Spectroscopic analysis shows that these fibers are built from β-sheets. This behavior is in complete contrast to that of C16-IKPEAP, which forms spherical micelles with peptides in a disordered conformation [HutchinsonJ. Phys. Chem. B2019, 123, 613]. In PAEPKI-C16, spontaneous alignment of fibers was observed upon increasing pH, which was accompanied by observed birefringence and anisotropy of SAXS patterns. This shows the ability to form a nematic phase, and unprecedented nematic hydrogel formation was also observed for these lipopeptides at sufficiently high concentrations. SAXS shows retention of an ultrafine (1.7 nm core radius) fibrillar network within the hydrogel. PAEPKI-C16 with free N-terminal proline shows enhanced anti:syn diastereoselectivity and better conversion compared to C16-IKPEAP. The cytotoxicity of PAEPKI-C16 was also lower than that of C16-IKPEAP for both fibroblast and cancer cell lines. These results highlight the sensitivity of lipopeptide properties to the presence of a free proline residue. The spontaneous nematic phase formation by PAEPKI-C16 points to the high anisotropy of its ultrafine fibrillar structure, and the formation of such a phase at low concentrations in aqueous solution may be valuable for future applications.

Integration of Indocyanine Green Analogs as Near-Infrared Fluorescent Carrier for Precise Imaging-Guided Gene Delivery.

Codelivery of diagnostic probes and therapeutic molecules often suffers from intrinsic complexity and premature leakage from or degradation of the nanocarrier. Inspired by the "Y" shape of indocyanine green (ICG), the dye is integrated in an amphiphilic lipopeptide (RNF). The hydrophilic segment is composed of arginine-rich dendritic peptides, while cyanine dyes are modified with two long carbon chains and employed as the hydrophobic moiety. They are linked through a disulfide linkage to improve the responsivity in the tumor microenvironment. After formulation with other lipopeptides at an optimized ratio, the theranostic system (RNS-2) forms lipid-based nanoparticles with slight positive zeta potential enabling efficient condensation of DNA. The RNS-2 displays glutathione responded gene release, activatable fluorescence recovery, and up to sevenfold higher in vitro transfection than Lipofectamine 2000. Compared with a Cy3 and Cy5 labeled fluorescence resonance energy transfer indicator for gene release, the "turn-on" indocyanine green analogs exhibit longer emission wavelength and better positive correlation with the dynamic processes of gene delivery. More importantly, the RNS-2 system enables efficient near infrared imaging guided gene transfer in tumor-bearing mice and thus provides more precise and accurate information on location of the cargo gene and synthesized carriers.

Polymorphism of asymmetric catalysts based on amphiphilic lipopeptides in solution

The self-assembly of model [P]RWG lipopeptides (P: l-proline, R: l-arginine, W: l-tryptophan, G: l-glycine), containing one or two aliphatic octadecyl (C18) chains in water and cyclohexanone/water solutions was examined. The self-assembly of mixtures of these RWG and PRWG lipopeptides was also investigated. These materials presented a similar critical aggregation concentration of ∼4.0 × 10-4 wt% and were characterized by unordered secondary structures with some β-sheet content. TEM and cryo-TEM revealed the presence of mainly nanotape structures with micelles observed for systems rich in PRWG(C18H37). Analysis of detailed SAXS form factor measurements revealed the presence of bilayers 3-4 nm thick while the PRWG(C18H37) micelles have a core radius of approximately 3 nm, and a shell thickness of 2 nm. For the cyclohexanone/water systems polymorphs containing cluster aggregates (with radius of 0.25 nm to 0.50 nm) and some elongated structures (with radius of 5.7 nm to 26.1 nm) were seen. Longer structures were formed with the increase of the proline-containing lipopeptide content. The catalytic activity of these peptides was assessed using a model nitro-aldol reaction. The concentration of water in the reaction system influenced the conversion, higher content promoted better efficiency for the water systems, but the opposite was observed for the cyclohexanone/water samples.

Investigation of the Assembly Behavior of an Amphiphilic Lipopeptide at the Liquid Crystal-Aqueous Interface.

In this article, we designed an amphiphilic lipopeptide molecule, 5(6)-carboxyfluorescein-KKKKKKSKTK-Cys(C12H25)-OMe (FAM-lipopeptide-C12), and studied its assembly behavior at the 4-cyano-4'-pentylbiphenyl (5CB)-aqueous interface. The ordering transitions of liquid crystals (LCs) revealed that FAM-lipopeptide-C12 can assemble at the LC-aqueous interface (both planar and curved interfaces). The assembly can be destroyed by adding trypsin, which catalyzes the hydrolysis of lipopeptides. Fluorescence measurements further confirmed the assembly and deassembly behavior of FAM-lipopeptide-C12 at the LC-aqueous interface. Overall, our work provides a general method for the construction of a biointerface by directly assembling amphiphilic lipopeptides at the LC-aqueous interface, which can potentially be used in selectively detecting the activity of specific enzymes and other biomolecular interactions.

Amphiphilic lipopeptide significantly enhances uptake of charge-neutral splice switching morpholino oligonucleotide in spinal muscular atrophy patient-derived fibroblasts

Splice-switching antisense oligonucleotides (SSOs) are emerging therapeutics with two SSOs recently approved by the FDA for Duchenne muscular dystrophy and spinal muscular atrophy. SSOs are administered without any delivery vector and require large doses to achieve the therapeutic benefit, primarily due to their poor cellular uptake. Although cell-penetrating peptides (CPP) have shown great potential in delivering SSOs into cells, their capacity as delivery vector is limited. Here we have studied the effect of lipid conjugation on the cell permeability of a known CPP (ApoE). Myristic acid was coupled at the N-terminus of ApoE to a C-terminal cysteine residue. The myristoylated ApoE (Myr-ApoE) was conjugated to a maleimide functionalised phosphorodiamidate morpholino oligonucleotide (PMO). The Myr-ApoE-PMO conjugate showed no cytoxicity and had significantly higher efficiency in cell permeability with 30% higher splice-switching activity compared to ApoE-PMO. The self-assembly properties of this amphiphilic lipopeptide-PMO conjugate was assessed. Transmission electron microscopy showed formation of nanoparticles with amphiphile behaviour and spherical structure. The self-assembly of Myr-ApoE-PMO into nanoparticles enabled it to better bind to cell membranes and to be more efficiently taken up by fibroblast cells. These results showed that modification of physico-chemical properties of peptides to produce peptide amphiphiles enhances cellular uptake and can be used as an efficient delivery vector for therapeutic SSOs.

Amphiphilic Lipopeptide-Mediated Transport of Insulin and Cell Membrane Penetration Mechanism.

Arginine octamer (R8) and its derivatives were developed in this study for the enhanced mucosal permeation of insulin. R8 was substituted with different aminos, then modified with stearic acid (SA). We found that the SAR6EW-insulin complex had stronger intermolecular interactions and higher complex stability. The amphiphilic lipopeptide (SAR6EW) was significantly more efficient for the permeation of insulin than R8 and R6EW both in vitro and in vivo. Interestingly, different cellular internalization mechanisms were observed for the complexes. When the effectiveness of the complexes in delivering insulin in vivo was examined, it was found that the SAR6EW-insulin complex provided a significant and sustained (six hours) reduction in the blood glucose levels of diabetic rats. The improved absorption could be the comprehensive result of stronger intermolecular interactions, better enzymatic stability, altered internalization pathways, and increased transportation efficacy. In addition, no sign of toxicity was observed after consecutive administrations of SAR6EW. These results demonstrate that SAR6EW is a promising epithelium permeation enhancer for insulin and suggest that the chemical modification of cell-penetrating peptides is a feasible strategy to enhance their potential.

New amphiphilic N-phosphoryl oligopeptides designed for gene delivery

Gene therapy is a potent tool for the treatment of cancer and other gene defect diseases, which involves using DNA that encodes a functional, therapeutic gene to replace a mutated gene. However, the DNA transfection efficiency is restricted by its negative charges and low susceptibility to endonucleases which prevent them penetrating tissue and cellular membranes. Both viral and non-viral vectors have been used for gene delivery, but the former are limited by their immunogenicity, while the latter are less efficient than their viral counterpart. Cationic amphiphilic lipopeptides whose structures can be easily modified and transformed have been used as non-viral vectors in gene delivery system due to their low cytotoxicity and high transfection efficiency. In this study, a series of cationic amphiphilic N-phosphoryl oligopeptides with varied lengths of hydrophobic tails and oligopeptide headgroups (C12-K6, C14-K6, C16-K6, Chol-K6 and C12-H6) were synthesized and used as gene delivery vectors. The affinities, abilities to condense pDNA and transfection efficiencies of the K6-lipopeptides were better than those of the H6-lipopeptides. In addition, the hydrophobic chains of the lipopeptides also affected their transfection efficiencies. The K6-lipopeptide with a hydrophobic chain of twelve carbons (C12-K6) showed the highest transfection efficiency in all these synthetic lipopeptides. At an optimal P/N ratio of 20, C12-K6 showed comparable pDNA transfection efficiency to PEI-25k, a well-defined gene delivery vector, but the cytotoxicity of C12-K6 was much lower. With acceptable gene transfection efficiency and low cytotoxicity, this cationic amphiphilic lipopeptide will have promising applications in gene therapy.

Silencing of miR-21 by locked nucleic acid–lipid nanocapsule complexes sensitize human glioblastoma cells to radiation-induced cell death

The recent discovery of microRNA (miRNA) as major post-transcriptional repressors prompt the interest of developing novel approaches to target miRNA pathways to improve therapy. In this context, although the most significant barrier to their widespread clinical use remains delivery, nuclease-resistant locked nucleic acid (LNA) that bind specifically and irreversibly to miRNA represent interesting weapons. Thus, by focusing on oncongenic miR-21 miRNA, which participate to cancer cell resistance to apoptotic signals, the aim of the present study was to investigate the possibility of silencing miRNA by LNA conjugated to lipid nanocapsules (LNCs) as miRNA-targeted nanomedicines in U87MG glioblastoma (GBM) cells. After synthesis of an amphiphilic lipopeptide affine for nucleic acids, a post-insertion procedure during the LNC phase inversion formulation process allowed to construct peptide-conjugated LNCs. Peptide-conjugated LNCs were then incubated with LNAs to allow the formation of complexes characterized in gel retardation assays and by their physicochemical properties. U87MG cell treatment by LNA-LNC complexes resulted in a marked reduction of miR-21 expression as assessed by RTqPCR. In addition, exposure of U87MG cells to LNA-LNC complexes followed by external beam radiation demonstrated a significant improvement of cell sensitivity to treatment and emphasizes the interest to investigate further this miRNA-targeted strategy.