Mechanism of Antibacterial Activity of Liposomal Linolenic Acid against <i>Helicobacter pylori</i>

<div><p><i>Helicobacter pylori</i> infects approximately half of the world population and is a major cause of gastritis, peptic ulcer, and gastric cancer. Moreover, this bacterium has quickly developed resistance to all major antibiotics. Recently, we developed a novel liposomal linolenic acid (LipoLLA) formulation, which showed potent bactericidal activity against several clinical isolated antibiotic-resistant strains of <i>H</i>. <i>pylori</i> including both the spiral and coccoid form. In addition, LipoLLA had superior <i>in vivo</i> efficacy compared to the standard triple therapy. Our data showed that LipoLLA associated with <i>H</i>. <i>pylori</i> cell membrane. Therefore, in this study, we investigated the possible antibacterial mechanism of LipoLLA against <i>H</i>. <i>pylori</i>. The antibacterial activity of LipoLLA (C18:3) was compared to that of liposomal stearic acid (LipoSA, C18:0) and oleic acid (LipoOA, C18:1). LipoLLA showed the most potent bactericidal effect and completely killed <i>H</i>. <i>pylori</i> within 5 min. The permeability of the outer membrane of <i>H</i>. <i>pylori</i> increased when treated with LipoOA and LipoLLA. Moreover, by detecting released adenosine triphosphate (ATP) from bacteria, we found that bacterial plasma membrane of <i>H</i>. <i>pylori</i> treated with LipoLLA exhibited significantly higher permeability than those treated with LipoOA, resulting in bacteria cell death. Furthermore, LipoLLA caused structural changes in the bacterial membrane within 5 min affecting membrane integrity and leading to leakage of cytoplasmic contents, observed by both transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Our findings showing rapid bactericidal effect of LipoLLA suggest it is a very promising new, effective anti-<i>H</i>. <i>pylori</i> agent.</p></div

Comparison of hydrodynamic size and surface zeta potential of bare liposome, liposomal stearic acid (LipoSA), liposomal oleic acid (LipoOA) and liposomal linolenic acid acid (LipoLLA).

<p>All characterizations were performed using dynamic light scattering and each measurement was repeated three times at 25°C.</p

Release of ATP from <i>H</i>. <i>pylori</i> by liposomal fatty acids.

<p><i>H</i>. <i>pylori</i> were treated for 5 min with the indicated compounds, and bacterial cells and supernatants separated by centrifugation. Supernatants were analyzed for their ATP content as described in Methods. *Significantly different from control (<i>P</i> < 0.01); **Significantly different between bacterial cells exposed to LipoOA and LipoLLA (<i>P</i> < 0.01).</p

TEM (A and B) and SEM (C and D) images of <i>H</i>. <i>pylori</i> bacteria exposed to LipoLLA.

<p>Bacteria were treated for 5 min with PBS control (A and C) or LipoLLA (B and D). The concentration of bacteria used was 5 × 10⁶ CFU/mL and the drug concentration was 400 μg/mL.</p

Schematic illustration of (A) <i>H</i>. <i>pylori</i> incubated with LipoFFA; (B) Structure and composition of LipoFFA consisting of phospholipid, cholesterol and FFA; (C) LipoFFA fuses with bacterial membrane for antibacterial activity.

<p>Schematic illustration of (A) <i>H</i>. <i>pylori</i> incubated with LipoFFA; (B) Structure and composition of LipoFFA consisting of phospholipid, cholesterol and FFA; (C) LipoFFA fuses with bacterial membrane for antibacterial activity.</p

Time-dependent bactericidal activity of LipoLLA against <i>H</i>. <i>pylori</i>.

<p>5×10⁶ CFU/mL <i>H</i>. <i>pylori</i> was incubated with 200, 300 or 400 g/mL LipoLLA at 37°C under microaerobic conditions for 5, 10, 20, or 30 min followed by inoculation onto Columbia agar plates for colony observation.</p

Uptake of N-phenylnapthylamine (NPN) by <i>H</i>. <i>pylori</i> in the presence of bare liposome, LipoSA, LipoOA, or LipoLLA.

<p>^a Significantly different from controls (<i>P</i> < 0.001).</p><p>^b Significantly different from cells treated with LipoOA (<i>P</i> = 0.02).</p><p>RFU, relative fluorescence unit; SD, standard deviation</p><p>Uptake of N-phenylnapthylamine (NPN) by <i>H</i>. <i>pylori</i> in the presence of bare liposome, LipoSA, LipoOA, or LipoLLA.</p

Concentration-dependent bactericidal activity of LipoLLA, LipoOA, and LipoSA against <i>H</i>. <i>pylori</i>.

<p>5×10⁶ CFU/mL <i>H</i>. <i>pylori</i> was incubated with LipoLLA, LipoOA, or LipoSA at 37°C under microaerobic conditions for 30 min followed by inoculation onto Columbia agar plates for colony observation.</p

Artificial Micromotors in the Mouse’s Stomach: A Step toward <i>in Vivo</i> Use of Synthetic Motors

Artificial micromotors, operating on locally supplied fuels and performing complex tasks, offer great potential for diverse biomedical applications, including autonomous delivery and release of therapeutic payloads and cell manipulation. Various types of synthetic motors, utilizing different propulsion mechanisms, have been fabricated to operate in biological matrices. However, the performance of these man-made motors has been tested exclusively under <i>in vitro</i> conditions (outside the body); their behavior and functionalities in an <i>in vivo</i> environment (inside the body) remain unknown. Herein, we report an <i>in vivo</i> study of artificial micromotors in a living organism using a mouse model. Such <i>in vivo</i> evaluation examines the distribution, retention, cargo delivery, and acute toxicity profile of synthetic motors in mouse stomach via oral administration. Using zinc-based micromotors as a model, we demonstrate that the acid-driven propulsion in the stomach effectively enhances the binding and retention of the motors as well as of cargo payloads on the stomach wall. The body of the motors gradually dissolves in the gastric acid, autonomously releasing their carried payloads, leaving nothing toxic behind. This work is anticipated to significantly advance the emerging field of nano/micromotors and to open the door to <i>in vivo</i> evaluation and clinical applications of these synthetic motors

Hydrogel Containing Nanoparticle-Stabilized Liposomes for Topical Antimicrobial Delivery

Adsorbing small charged nanoparticles onto the outer surfaces of liposomes has become an effective strategy to stabilize liposomes against fusion prior to “seeing” target bacteria, yet allow them to fuse with the bacteria upon arrival at the infection sites. As a result, nanoparticle-stabilized liposomes have become an emerging drug delivery platform for treatment of various bacterial infections. To facilitate the translation of this platform for clinical tests and uses, herein we integrate nanoparticle-stabilized liposomes with hydrogel technology for more effective and sustained topical drug delivery. The hydrogel formulation not only preserves the structural integrity of the nanoparticle-stabilized liposomes, but also allows for controllable viscoeleasticity and tunable liposome release rate. Using Staphylococcus aureus bacteria as a model pathogen, we demonstrate that the hydrogel formulation can effectively release nanoparticle-stabilized liposomes to the bacterial culture, which subsequently fuse with bacterial membrane in a pH-dependent manner. When topically applied onto mouse skin, the hydrogel formulation does not generate any observable skin toxicity within a 7-day treatment. Collectively, the hydrogel containing nanoparticle-stabilized liposomes hold great promise for topical applications against various microbial infections