Membrane-destabilizing Activity Research Articles

Characterization of TelE, a T7SS LXG Effector Exhibiting a Conserved C-Terminal Glycine Zipper Motif Required for Toxicity.

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic bacterial pathogen strongly associated with colorectal cancer. Here, through comparative genomics analysis, we demonstrated that the genetic locus encoding the type VIIb secretion system (T7SSb) machinery is uniquely present in SGG in two different arrangements. SGG UCN34 carrying the most prevalent T7SSb genetic arrangement was chosen as the reference strain. To identify the effectors secreted by this secretion system, we inactivated the essC gene encoding the motor of this machinery. A comparison of the proteins secreted by UCN34 wild type and its isogenic ΔessC mutant revealed six T7SSb effector proteins, including the expected WXG effector EsxA and three LXG-containing proteins. In this work, we characterized an LXG-family toxin named herein TelE promoting the loss of membrane integrity. Seven homologs of TelE harboring a conserved glycine zipper motif at the C terminus were identified in different SGG isolates. Scanning mutagenesis of this motif showed that the glycine residue at position 470 was crucial for TelE membrane destabilization activity. TelE activity was antagonized by a small protein TipE belonging to the DUF5085 family. Overall, we report herein a unique SGG T7SSb effector exhibiting a toxic activity against nonimmune bacteria. IMPORTANCE In this study, 38 clinical isolates of Streptococcus gallolyticus subsp. gallolyticus (SGG) were sequenced and a genetic locus encoding the type VIIb secretion system (T7SSb) was found conserved and absent from 16 genomes of the closely related S. gallolyticus subsp. pasteurianus (SGP). The T7SSb is a bona fide pathogenicity island. Here, we report that the model organism SGG strain UCN34 secretes six T7SSb effectors. One of the six effectors named TelE displayed a strong toxicity when overexpressed in Escherichia coli. Our results indicate that TelE is probably a pore-forming toxin whose activity can be antagonized by a specific immunity protein named TipE. Overall, we report a unique toxin-immunity protein pair and our data expand the range of effectors secreted through T7SSb.

2E,4E-Decadienoic Acid, a Novel Anti-Oomycete Agent from Coculture of Bacillus subtilis and Trichoderma asperellum.

ABSTRACTPhytophthora nicotianae is an oomycete pathogen of global significance threatening many important crops. It is mainly controlled by chemosynthetic fungicides, which endangers ecosystem and human health; thus, there is an urgent need to explore alternatives for these fungicides. In this study, a new anti-oomycete aliphatic compound, 2E,4E-decadienoic acid (DDA), was obtained through coculture of Bacillus subtilis Tpb55 and Trichoderma asperellum HG1. Both in vitro and in vivo tests showed that DDA had a strong inhibitory effect against P. nicotianae. In addition, rhizosphere microbiome analysis showed that DDA reduced the relative abundance of Oomycota in rhizosphere soil. Transcriptome sequencing (RNA-Seq) analysis revealed that treatment of P. nicotianae with DDA resulted in significant downregulation of antioxidant activity and energy metabolism, including antioxidant enzymes and ATP generation, and upregulation of membrane-destabilizing activity, such as phospholipid synthesis and degradation. The metabolomic analysis results implied that the pathways influenced by DDA were mainly related to carbohydrate metabolism, energy metabolism, and the cell membrane. The biophysical tests further indicated that DDA produced oxidative stress on P. nicotianae, inhibited antioxidant enzyme and ATPase activity, and increased cell membrane permeability. Overall, DDA exerts inhibitory activity by acting on multiple targets in P. nicotianae, especially on the cell membrane and mitochondria, and can therefore serve as a novel environment-friendly agent for controlling crop oomycete disease.IMPORTANCE P. nicotianae is an oomycete pathogen that is destructive to crops. Although some oomycete inhibitors have been used during crop production, most are harmful to the ecology and lead to pathogen resistance. Alternatively, medium-chain fatty acids have been reported to exhibit antimicrobial activity in the medical field in previous studies; however, their potential as biocontrol agents has rarely been evaluated. Our in vivo and in vitro analyses revealed that the medium-chain fatty acid 2E,4E-decadienoic acid (DDA) displayed specific inhibitory activity against oomycetes. Further analysis indicated that DDA may acted on multiple targets in P. nicotianae, especially on the cell membrane and mitochondria. Our findings highlight the potential of DDA in controlling oomycete diseases. In conclusion, these results provide insights regarding the future use of green and environment-friendly anti-oomycete natural products for the prevention and control of crop oomycete diseases.

Lipid Flip-Flop-Inducing Antimicrobial Phytochemicals from Gymnema sylvestre are Bacterial Membrane Permeability Enhancers.

An amphiphilic phytochemical fraction isolated from methanol extract of Gymnema sylvestre leaf powder contained six terpenoids, two flavonoids, and one alkaloid that induced rapid flip-flop of fluorescent phospholipid analog in the phosphatidyl choline bilayer. Lipid-flipping activity of the methanol-extracted fraction of G. sylvestre (MEFGS) was dose-dependent and time-dependent with a rate constant k = (12.09 ± 0.94) mg–1 min–1 that was saturable at (40 ± 1) % flipping of the fluorescent lipid analogue. Interactions of MEFGS phytochemicals with large unilamelar vesicles led to time-dependent change in their rounded morphology into irregular shapes, indicating their membrane-destabilizing activity. MEFGS exhibited antibacterial activity on Escherichia coli (MTCC-118), Staphylococcus aureus (MTCC-212), and Pseudomonas aeruginosa (MTCC-1035) with IC50 values 0.5, 0.35, and 0.1 mg/mL, respectively. Phytochemicals in MEFGS increased membrane permeabilization in all three bacteria, as indicated by 23, 17, and 17% increase in the uptake of crystal violet, respectively. MEFGS enhanced membrane damage, resulting in a 3–5 fold increase in leakage of cytosolic ions, 0.5–2 fold increase in leakage of PO4–, and 15–20% increase in loss of cellular proteins. MEFGS synergistically increased the efficacy of curcumin, amoxillin, ampicillin, and cefotaxime on S. aureus probably by enhancing their permeability into the bacterium. For the first time, our study reveals that phytochemicals from G. sylvestre enhance the permeability of the bacterial plasma membrane by facilitating flip-flop of membrane lipids. Lipid-flipping phytochemicals from G. sylvestre can be used as adjuvant therapeutics to enhance the efficacy of antibacterials by increasing their bioavailability in the target bacteria.

Endosomal Escape: Amphiphilic Polyelectrolyte Graft Copolymers Enhance the Activity of Cyclic Dinucleotide STING Agonists (Adv. Healthcare Mater. 2/2021)

Endosomal escape is a rate limiting step for many promising therapeutics, including cyclic dinucleotide (CDN) agonists of the stimulator of interferon genes (STING) pathway. In article number 2001056, John T. Wilson and co-workers synthesize and screen a novel library of polymers with pH-responsive, membrane-destabilizing activity and evaluate their capacity to enhance cytosolic delivery of CDNs, resulting in identification of new drug carrier technologies for potent STING activation.

Amphiphilic Polyelectrolyte Graft Copolymers Enhance the Activity of Cyclic Dinucleotide STING Agonists.

Cyclic dinucleotide (CDN) agonists of stimulator of interferon genes (STING) hold great therapeutic potential, but their activity is hindered by poor drug-like properties that restrict cytosolic bioavailability. Here, this challenge is addressed through the synthesis and evaluation of a novel series of PEGMA-co-DEAEMA-co-BMA copolymers with pH-responsive, membrane-destabilizing activity to enhance intracellular delivery of the CDN, cGAMP. Copolymers are synthesized with PEGMA of two different molecular weights (300 and 950Da) and over a range of PEG mass fraction and polymer molecular weight, and relationships between copolymer structure, self-assembly, endosomal escape, and cGAMP activity are elucidated. A subset of polymers that self-assembled into 50-800nm nanoparticles is identified, which can be loaded with cGAMP via a simple mixing strategy, resulting in significantly enhanced immunostimulatory activity. Increased cGAMP activity is found to be highly correlated with the capacity of carriers to enhance intracellular CDN uptake and to promote endosomal destabilization, findings that establish efficient cytosolic delivery as a criterion for CDN carriers. Additionally, it is demonstrated that a lead CDN carrier formulation can enhance STING activation in vivo in a model of intratumoral immunotherapy. Collectively, these investigations demonstrate the utility of PEGMA-co-DEAEMA-co-BMA copolymers as carriers for CDNs and potentially other cytosolically-acting drug cargo.

Development of an SS-Cleavable pH-Activated Lipid-Like Material (ssPalm) as a Nucleic Acid Delivery Device.

Gene and nucleic acid-based medication is an ultimate strategy in the field of personalized medicine. A gene or short interference RNA (siRNA) molecule needs to be delivered to the appropriate organelle (i.e., nucleus and cytoplasm, respectively). We recently focused on improving the intrinsic activity of my original material (ssPalm) in terms of endosomal/lysosomal membrane destabilization activity by chemically modifying the tertiary amine structure. In parallel, I have been expanding the range of applications of ssPalms. The first application is a DNA or RNA vaccine. My crucial finding is that the vitamin E-scaffold ssPalm (ssPalmE) is highly immune-stimulative when combined with DNA. Thereafter, I redesigned the hydrophobic scaffold structure, and found that an oleic acid-scaffold ssPalm (ssPalmO) can confer anti-inflammatory characteristics. Based on this result, I further upgraded the ssPalmO, by inserting a newly designed linker with self-degradable properties.

An Anti-microbial Terpenoid Fraction from Gymnema sylvestre Induces Flip-flop of Fluorescent-Phospholipid Analogs in Model Membrane.

Therapeutic potential of Gymnema sylvestre on diverse cell types is predominantly due to a variety of terpenoids and their derivatives. However, their bioavailability becomes limited due to poor solubility and lower lipophilic properties, provoking the search for novel membranotropic terpenoids and their mechanism of action. A terpenoid fraction purified from Gymnema sylvestre exhibited broad spectrum antimicrobial activity against both Gram positive and Gram negative bacteria with IC50 ˂ 0.1mg/ml. Evaluation of its membranotropic effect in vitro on reconstituted model membrane revealed that the fraction induced flip-flop of fluorescent phospholipid analogs across the lipid bilayer. The terpenoid-induced lipid flipping was biphasic with a fast linear phase (rate constant (k1) = 3 to 5S-1) and a second slow exponential phase (rate constant (k2) = (4 to 9) × 10-3S-1). The lipid-flippase activity of the terpenoid fraction showed concentration and incubation-dependent cooperativity, indicating their lipophilic nature and membrane-destabilizing activity that facilitated lipid translocation. For the first time, our study reveals the flippase activity of a terpenoid fraction of Gymnema sylvestre that could be further explored for their membrane-mediated pharmacological properties. Graphical Abstract.

Development of a lipoplex-type mRNA carrier composed of an ionizable lipid with a vitamin E scaffold and the KALA peptide for use as an ex vivo dendritic cell-based cancer vaccine

A dendritic cells (DCs)-based vaccine (DC-vaccine) system is an attractive technology for eliciting antigen-specific immune responses that can protect subjects from infectious diseases and for curing various types of cancers. For the insertion of a foreign antigen to DCs, the transfection of an antigen-coding mRNA to the cells is a promising approach. In order to introduce an antigen, a carrier for mRNA transfection is required, since the mRNA molecule per se is unstable in serum-containing medium. We previously reported on an ionizable lipid-like material with vitamin E-scaffolds (ssPalmE) as a material for a lipid nanoparticle (LNP)-based carrier for nucleic acids. In the present study, we report on the development of a lipoplex-type mRNA carrier for use as a DC-vaccine by using a combination of an ssPalmE-LNP and an α-helical cationic peptide “KALA” (ssPalmE-KALA). The transfection of mRNAs complexed with the ssPalmE-KALA achieved a significantly higher protein expression and the production of proinflammatory cytokines from murine bone marrow derived DCs (BMDCs) in comparison with a lipoplex that was prepared with an ssPalm with fatty acid-scaffolds (myristic acid; ssPalmM-KALA). A cellular uptake process and a pH-responsive membrane-destabilization activity cannot explain the preferred protein expression and immune-stimulation caused by the ssPalmE-KALA. Proteomic analyses suggest that transfection with the ssPalmM-KALA stimulates a down-regulatory pathway of translation, while the transfection with the ssPalmE-KALA does not stimulate it. In the vaccination with the BMDCs that were preliminarily transfected with an ovalbumin (OVA)-encoding mRNA elicited the induction OVA specific cytotoxic T-lymphocyte activity in vivo. In parallel, the vaccination induced significant prophylactic anti-tumor effects against a model tumor that stably expressed the OVA protein. Based on the above findings, the ssPalmE-KALA appears to be a potent ex vivo DCs-based RNA vaccine platform.

Exposure to Sub-inhibitory Concentrations of the Chemosensitizer 1-(1-Naphthylmethyl)-Piperazine Creates Membrane Destabilization in Multi-Drug Resistant Klebsiella pneumoniae.

Antimicrobial efflux is one of the important mechanisms causing multi-drug resistance (MDR) in bacteria. Chemosensitizers like 1-(1-naphthylmethyl)-piperazine (NMP) can inhibit an efflux pump and therefore can overcome MDR. However, secondary effects of NMP other than efflux pump inhibition are rarely investigated. Here, using phenotypic assays, phenotypic microarray and transcriptomic assays we show that NMP creates membrane destabilization in MDR Klebsiella pneumoniae MGH 78578 strain. The NMP mediated membrane destabilization activity was measured using β-lactamase activity, membrane potential alteration studies, and transmission electron microscopy assays. Results from both β-lactamase and membrane potential alteration studies shows that both outer and inner membranes are destabilized in NMP exposed K. pneumoniae MGH 78578 cells. Phenotypic Microarray and RNA-seq were further used to elucidate the metabolic and transcriptional signals underpinning membrane destabilization. Membrane destabilization happens as early as 15 min post-NMP treatment. Our RNA-seq data shows that many genes involved in envelope stress response were differentially regulated in the NMP treated cells. Up-regulation of genes encoding the envelope stress response and repair systems show the distortion in membrane homeostasis during survival in an environment containing sub-inhibitory concentration of NMP. In addition, the lsr operon encoding the production of autoinducer-2 responsible for biofilm production was down-regulated resulting in reduced biofilm formation in NMP treated cells, a phenotype confirmed by crystal violet-based assays. We postulate that the early membrane disruption leads to destabilization of inner membrane potential, impairing ATP production and consequently resulting in efflux pump inhibition.

The efficiency of cytosolic drug delivery using pH-responsive endosomolytic polymers does not correlate with activation of the NLRP3 inflammasome.

Inefficient cytosolic delivery has limited the development of many promising biomacromolecular drugs, a long-standing challenge that has prompted extensive development of drug carriers that facilitate endosomal escape. Although many such carriers have shown considerable promise for cytosolic delivery of a diversity of therapeutics, the rupture or destabilization of endo/lysosomal membranes has also been associated with activation of the inflammasome with attendant risk of inflammation and toxicity. In this study, we investigated relationships between pH-dependent membrane destabilization, cytosolic drug delivery, and inflammasome activation using a series of well-defined poly[(ethylene glycol)-block-[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)] copolymers of variable second block composition and pH-responsive properties. We found that polymers that demonstrated the most potent membrane-destabilizing activity at early endosomal pH values in an erythrocyte hemolysis assay were most efficient at delivery of siRNA, yet tended to be associated with the least amount of NOD-like related protein 3 (NLRP3) inflammasome activation. By contrast, polymers that displayed minimal hemolysis activity and poor siRNA knockdown, and instead mediated lysosomal rupture likely due to a proton sponge mechanism, strongly induced NLPR3 inflammasome activation in a caspase- and cathepsin-dependent manner. Collectively, these findings reinforce the importance of early endosomal escape in minimizing inflammasome activation and also demonstrate the ability to tune the degree inflammasome activation via control of polymer structure with potential implications for design of vaccine adjuvants and immunotherapeutics.

Rigorous control of vesicle-forming lipid pKa by fluorine-conjugated bioisosteres for gene-silencing with siRNA

While the influence of pKa provided by amine-containing materials in siRNA delivery vectors for use in gene-silencing has been widely studied, there are little reports in which amine pKa is controlled rigorously by using bioisosteres and its effect on gene-silencing. Here, we report that amine pKa could be rigorously controlled by replacement of hydrogen atom(s) with fluorine atom(s). A series of mono- and di-amine lipids with a different number of fluorine atoms were synthesized. The pKa of the polyamine lipids was shifted to a lower value with an increase in the number of fluorine atoms. The optimal pKa for high gene-silencing efficiency varied according to the number of amine residues in the polyamine lipid. Whereas the endosomal escape ability of mono-amine lipid-containing lipid vesicles (LVs) depended on the pKa, that of all tested di-amine lipid-containing LVs showed equal membrane-destabilizing activity. LVs showing moderately weak interactions with siRNA facilitated cytoplasmic release of siRNA, resulting in strong gene-silencing. These findings indicate that appropriate amine pKa engineering depending on the number of amines is important for the induction of effective RNA interference.

Unveiling the Multifaceted Mechanisms of Antibacterial Activity of Buforin II and Frenatin 2.3S Peptides from Skin Micro-Organs of the Orinoco Lime Treefrog (Sphaenorhynchus lacteus).

Amphibian skin is a rich source of natural compounds with diverse antimicrobial and immune defense properties. Our previous studies showed that the frog skin secretions obtained by skin micro-organs from various species of Colombian anurans have antimicrobial activities against bacteria and viruses. We purified for the first time two antimicrobial peptides from the skin micro-organs of the Orinoco lime treefrog (Sphaenorhynchus lacteus) that correspond to Buforin II (BF2) and Frenatin 2.3S (F2.3S). Here, we have synthesized the two peptides and tested them against Gram-negative and Gram-positive bacteria, observing an effective bactericidal activity at micromolar concentrations. Evaluation of BF2 and F2.3S membrane destabilization activity on bacterial cell cultures and synthetic lipid bilayers reveals a distinct membrane interaction mechanism. BF2 agglutinates E. coli cells and synthetic vesicles, whereas F2.3S shows a high depolarization and membrane destabilization activities. Interestingly, we found that F2.3S is able to internalize within bacterial cells and can bind nucleic acids, as previously reported for BF2. Moreover, bacterial exposure to both peptides alters the expression profile of genes related to stress and resistance response. Overall, these results show the multifaceted mechanism of action of both antimicrobial peptides that can provide alternative tools in the fight against bacterial resistance.

Polymer-augmented liposomes enhancing antibiotic delivery against intracellular infections.

Pulmonary intracellular infections, such as tuberculosis, anthrax, and tularemia, have remained a significant challenge to conventional antibiotic therapy. Ineffective antibiotic treatment of these infections can lead not only to undesired side effects, but also to the emergence of antibiotic resistance. Aminoglycosides (e.g., streptomycin) have long been part of the therapeutic regiment for many pulmonary intracellular infections. Their bioavailability for intracellular bacterial pools, however, is limited by poor membrane permeability and rapid elimination. To address this challenge, polymer-augmented liposomes (PALs) were developed to provide improved cytosolic delivery of streptomycin to alveolar macrophages, an important host cell for intracellular pathogens. A multifunctional diblock copolymer was engineered to functionalize PALs with carbohydrate-mediated targeting, pH-responsive drug release, and endosomal release activity with a single functional polymer that replaces the pegylated lipid component to simplify the liposome formulation. The pH-sensing functionality enabled PALs to provide enhanced release of streptomycin under endosomal pH conditions (70% release in 6 hours) with limited release at physiological pH 7.4 (16%). The membrane-destabilizing activity connected to endosomal release was characterized in a hemolysis assay and PALs displayed a sharp pH profile across the endosomal pH development target range. The direct connection of this membrane-destabilizing pH profile to model drug release was demonstrated in an established pyranine/p-xylene bispyridinium dibromide (DPX) fluorescence dequenching assay. PALs displayed similar sharp pH-responsive release, whereas PEGylated control liposomes did not, and similar profiles were then shown for streptomycin release. The mannose-targeting capability of the PALs was also demonstrated with 2.5 times higher internalization compared to non-targeted PEGylated liposomes. Finally, the streptomycin-loaded PALs were shown to have a significantly improved intracellular antibacterial activity in a Francisella-macrophage co-culture model, compared with free streptomycin or streptomycin delivered by control PEGylated liposomes (13× and 16×, respectively). This study suggests the potential of PALs as a useful platform to deliver antibiotics for the treatment of intracellular macrophage infections.

Factors Influencing the Chaperone-Like Activity of Major Proteins of Mammalian Seminal Plasma, Equine HSP-1/2 and Bovine PDC-109: Effect of Membrane Binding, pH and Ionic Strength.

HSP-1/2 and PDC-109 belong to a family of fibronectin type II proteins, present in high concentrations in bovine and equine seminal plasma, respectively. These proteins act as extracellular small heat shock proteins and protect target/client proteins against various kinds of stress. They also exhibit characteristic binding to choline phospholipids present on the sperm plasma membrane and cause efflux of choline phospholipids and cholesterol, resulting in sperm capacitation. The current study demonstrates that hypersaline conditions decrease the chaperone-like activity (CLA) of HSP-1/2. On the other hand, lipoprotein aggregates formed by the binding of choline phospholipids to this protein exhibit higher CLA than HSP-1/2 alone in vitro; the increased CLA can be correlated to the increased surface hydrophobicity of the lipoprotein aggregates. Presence of cholesterol in the membrane was found to decrease such enhancement in the CLA. We have also observed that salinity of the medium affects the chaperone activity by altering the polydisperse nature of the HSP-1/2. Together these results indicate that hydrophobicity and polydispersity are important for the chaperone-like activity of HSP-1/2 and factors that can alter these properties of HSP-1/2 can modulate its CLA. Further, studies on PDC-109 show that the chaperone-like and membrane-destabilizing activities of this protein are differentially affected by change in pH.

Membrane-Anchoring, Comb-Like Pseudopeptides for Efficient, pH-Mediated Membrane Destabilization and Intracellular Delivery.

Endosomal release has been identified as a rate-limiting step for intracellular delivery of therapeutic agents, in particular macromolecular drugs. Herein, we report a series of synthetic pH-responsive, membrane-anchoring polymers exhibiting dramatic endosomolytic activity for efficient intracellular delivery. The comb-like pseudopeptidic polymers were synthesized by grafting different amounts of decylamine (NDA), which act as hydrophobic membrane anchors, onto the pendant carboxylic acid groups of a pseudopeptide, poly(l-lysine iso-phthalamide). The effects of the hydrophobic relatively long alkyl side chains on aqueous solution properties, cell membrane destabilization activity, and in-vitro cytotoxicity were investigated. The optimal polymer containing 18 mol % NDA exhibited limited hemolysis at pH 7.4 but induced nearly complete membrane destabilization at endosomal pH within only 20 min. The mechanistic investigation of membrane destabilization suggests the polymer-mediated pore formation. It has been demonstrated that the polymer with hydrophobic side chains displayed a considerable endosomolytic ability to release endocytosed materials into the cytoplasm of various cell lines, which is of critical importance for intracellular drug delivery applications.

Modifying Antigen-Encapsulating Liposomes with KALA Facilitates MHC Class I Antigen Presentation and Enhances Anti-tumor Effects

For a successful anti-cancer vaccine, antigen presentation on the major histocompatibility complex (MHC) class I is a requirement. To accomplish this, an antigen must be delivered to the cytoplasm by overcoming the endosome/lysosome. We previously reported that a lipid nanoparticle modified with a KALA peptide (WEAKLAKALAKALAKHLAKALAKALKA), an α-helical cationic peptide, permits the encapsulated pDNA to be efficiently delivered to the cytoplasm in bone marrow-derived dendritic cells (BMDCs). Herein, we report on the use of KALA-modified liposomes as an antigen carrier, in an attempt to induce potent antigen-specific cellular immunity. The subcutaneous injection of KALA-modified ovalbumin (OVA)-encapsulating liposomes (KALA-OVA-LPs) elicited a much more potent OVA-specific cytotoxic T lymphocyte activity and anti-tumor effect in comparison with particles that were modified with octa-arginine (R8), a cell-penetrating peptide (R8-OVA-LPs). In addition, the numbers of OVA-specific CD8+ Tcells were increased by immunization the KALA-OVA-LPs. The treatment of BMDCs with KALA-OVA-LPs induced a substantial MHC class I antigen presentation. Furthermore, the acidic pH-dependent membrane destabilization activity of KALA-OVA-LPs strongly suggests that they are able to escape from endosomes/lysosomes and thereby deliver their cargos to the cytoplasm. Collectively, the KALA-modified liposome is a potential antigen delivery platform for use as a protein vaccine.

Molecular Tuning of a Vitamin E-Scaffold pH-Sensitive and Reductive Cleavable Lipid-like Material for Accelerated in Vivo Hepatic siRNA Delivery.

A lipid nanoparticle (LNP) composed of a series of SS-cleavable and pH-activated lipid-like materials (ssPalm) was previously developed as a platform of a gene delivery system. A tertiary amine and disulfide bonding were employed to destabilize the endosomal membrane and for intracellular collapse. We report herein on the development of a hepatocyte-targeting siRNA carrier by the molecular tuning of the hydrophobic scaffold, and tertiary amine structures. The gene knockdown activity against a hepatocyte-specific marker (factor VII: FVII) was improved when a more fat-soluble vitamin (vitamin E) was employed as a hydrophobic scaffold. Moreover, to allow the tertiary amines to accept protons by sensing a slight change in endosomal acidification, its structural flexibility was minimized by fixing it in a piperidine structure, and the distance between the surface of the particle to the ternary amine was increased. As a result, the pKa value was increased to the approximately 6.18 depending on its distance, while the pKa reached plateau when the tertiary amine was linked by an excess number of linear carbon chains. The pH-dependent membrane destabilization activity, as assessed by a hemolysis assay, was increased in parallel with the pKa value. Moreover, the gene knockdown activity was improved in parallel with hemolytic activity. Finally, further optimization of the lipid/siRNA ratio, and the use of chemically (2'-fluoro) modified siRNA synergistically improved the gene knockdown efficacy to an effective dose (ED50) of 0.035 mg/kg. The developed ssPalm represents a promising platform for use as a hepatocyte-targeting siRNA carrier.

Nanoparticles incorporating pH-responsive surfactants as a viable approach to improve the intracellular drug delivery.

The pH-responsive delivery systems have brought new advances in the field of functional nanodevices and might allow more accurate and controllable delivery of specific cargoes, which is expected to result in promising applications in different clinical therapies. Here we describe a family of chitosan-TPP (tripolyphosphate) nanoparticles (NPs) for intracellular drug delivery, which were designed using two pH-sensitive amino acid-based surfactants from the family N(α),N(ε)-dioctanoyl lysine as bioactive compounds. Low and medium molecular weight chitosan (LMW-CS and MMW-CS, respectively) were used for NP preparation, and it was observed that the size distribution for NPs with LMW-CS were smaller (~168 nm) than that for NPs prepared with MMW-CS (~310 nm). Hemolysis assay demonstrated the pH-dependent biomembrane disruptional capability of the constructed NPs. The nanostructures incorporating the surfactants cause negligible membrane permeabilization at pH7.4. However, at acidic pH, prevailing in endosomes, membrane-destabilizing activity in an erythrocyte lysis assay became evident. When pH decreased to 6.6 and 5.4, hemolytic capability of chitosan NPs increased along with the raise of concentration. Furthermore, studies with cell culture showed that these pH-responsive NPs displayed low cytotoxic effects against 3T3 fibroblasts. The influence of chitosan molecular weight, chitosan to TPP weight ratio, nanoparticle size and nature of the surfactant counterion on the membrane-disruptive properties of nanoparticles was discussed in detail. Altogether, the results achieved here showed that by inserting the lysine-based amphiphiles into chitosan NPs, pH-sensitive membranolytic and potentially endosomolytic nanocarriers were developed, which, therefore, demonstrated ideal feasibility for intracellular drug delivery.

Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli.

The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high degree of homology with the normally silent AcrEF system. Exposure of a strain with acrAB deleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine β-naphthylamide (PAβN) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PAβN and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PAβN, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PAβN. Strong action of PAβN in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PAβN acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PAβN acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PAβN in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PAβN on membrane integrity are compounded in cells unable to extrude PAβN. The increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance of employing real-time fluorescence-based assays in differentiating multidrug efflux-inhibitory and outer membrane-destabilizing activities of antibacterial compounds.

Enhancement of MHC-I antigen presentation via architectural control of pH-responsive, endosomolytic polymer nanoparticles.

Protein-based vaccines offer a number of important advantages over organism-based vaccines but generally elicit poor CD8(+) T cell responses. We have previously demonstrated that pH-responsive, endosomolytic polymers can enhance protein antigen delivery to major histocompatibility complex class I (MHC-I) antigen presentation pathways thereby augmenting CD8(+) T cell responses following immunization. Here, we describe a new family of nanocarriers for protein antigen delivery assembled using architecturally distinct pH-responsive polymers. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize linear, hyperbranched, and core-crosslinked copolymers of 2-(N,N-diethylamino)ethyl methacrylate (DEAEMA) and butyl methacrylate (BMA) that were subsequently chain extended with a hydrophilic N,N-dimethylacrylamide (DMA) segment copolymerized with thiol-reactive pyridyl disulfide (PDS) groups. In aqueous solution, polymer chains assembled into 25 nm micellar nanoparticles and enabled efficient and reducible conjugation of a thiolated protein antigen, ovalbumin. Polymers demonstrated pH-dependent membrane-destabilizing activity in an erythrocyte lysis assay, with the hyperbranched and cross-linked polymer architectures exhibiting significantly higher hemolysis at pH ≤ 7.0 than the linear diblock. Antigen delivery with the hyperbranched and cross-linked polymer architecture enhanced in vitro MHC-I antigen presentation relative to free antigen, whereas the linear construct did not have a discernible effect. The hyperbranched system elicited a four- to fivefold increase in MHC-I presentation relative to the cross-linked architecture, demonstrating the superior capacity of the hyperbranched architecture in enhancing MHC-I presentation. This work demonstrates that the architecture of pH-responsive, endosomolytic polymers can have dramatic effects on intracellular antigen delivery, and offers a promising strategy for enhancing CD8(+) T cell responses to protein-based vaccines.