Membrane-targeted mechanism for amphiphilic Vitamin C compounds as methicillin-resistant Staphylococcus aureus biofilm eradicating agents.

Staphylococcus aureus infections and its biofilm removal is an important concern in health care management. Methicillin-resistant S. aureus is responsible for severe morbidity and mortality worldwide. The extensive use of disinfectants against biofilms has led to negative environmental impacts. Developing new and more potent biofilm eradication agents with minimal detrimental effects on human and environmental health is currently on the agenda. The alkyl esters of L-ascorbic acid (ASCn) are antioxidant amphiphiles, which show antimicrobial capacity against methicillin-sensitive and resistant S. aureus strains. ASC12 and ASC14 formulations are able to kill the persister cells of the deepest layers of the biofilm. We tested the hypothesis that the antimicrobial and antibiofilm capacity found for the ASCn emerges from a combined effect of its amphiphilic and their redox capacity. This mechanism appears related to: I) a larger diffusion capacity of the ASC12 micelles than ASC14 and ASC16 microstructures; II) the neutralization of the ASCn acid hydroxyl when the amphiphile reaches the surface of an anionic surface, followed by a rapid insertion; III) the disruption of cell membrane by alteration of membrane tension and structure and IV) ASCn accumulation in the cell membrane or biofilm extracellular matrix surfaces, reducing functional chemical groups and affecting its biological function.

Supramolecular assemblies of amphiphilic donor-acceptor Stenhouse adducts as macroscopic soft scaffolds.

In the design of photoharvesting and photoresponsive supramolecular systems in aqueous medium, the fabrication of amphiphilic photoswitches enables a noninvasive functional response through photoirradiation. Although most aqueous supramolecular assemblies are driven by high-energy and biodamaging UV light, we have previously reported a design of amphiphilic donor-acceptor Stenhouse adducts (DASAs) controlled by white light. Herein, we present a series of DASA amphiphiles (DAs) with minor structural modifications on the alkyl linker chain length connecting the DASA motif with the hydrophilic moiety. The excellent photoswitchability in organic medium and the photoresponsiveness in aqueous medium, driven by visible light, were investigated by UV-vis absorption spectroscopy. The assembled supramolecular nanostructures were confirmed by electron microscopy, while the supramolecular packing was revealed by X-ray diffraction analysis. Upon visible-light irradiation, significant transformations of the DA geometry enabled transformations of the supramolecular assemblies on a microscopic scale, subsequently disassembling macroscopic soft scaffolds of DAs. The current work shows promising use for the fabrication of visible-light-controlled macroscopic scaffolds, offering the next generation of biomedical materials with visible-light-controlled microenvironments and future soft-robotic systems.

An Amphiphilic Surface with Improved Thermal Radiation for Water Harvesting.

Water scarcity poses a significant challenge for people living in arid areas. Despite the effectiveness of many bioinspired surfaces in promoting vapor condensation, their water-harvesting efficiency is insufficient. This is often exacerbated by overheating, which decreases the performance in terms of the micro-droplet concentration and movement on surfaces. In this study, we used a spotted amphiphilic surface to enhance the surfaces' water-harvesting efficiency while maintaining their heat emissivity. Through hydrophilic particle screening and hydrophobic groove modifying, the coalescence and sliding characteristics of droplets on the amphiphilic surfaces were improved. The incorporation of boron nitride (BN) nanoparticles further enhanced the surfaces' ability to harvest energy from condensation. To evaluate the water-harvesting performance of these amphiphilic surfaces, we utilized a real-time recording water-harvesting platform to identify microscopic weight changes on the surfaces. Our findings indicated that the inclusion of glass particles in hydrophobic grooves, combined with 1.0 wt.% BN nanoparticles, enhanced the water-harvesting efficiency of the amphiphilic surfaces by more than 20%.

Self-Sorting vs Coassembly in Peptide Amphiphile Supramolecular Nanostructures.

The functionality of supramolecular nanostructures can be expanded if systems containing multiple components are designed to either self-sort or mix into coassemblies. This is critical to gain the ability to craft self-assembling materials that integrate functions, and our understanding of this process is in its early stages. In this work, we have utilized three different peptide amphiphiles with the capacity to form ß-sheets within supramolecular nanostructures and found binary systems that self-sort and others that form coassemblies. This was measured using atomic force microscopy to reveal the nanoscale morphology of assemblies and confocal laser scanning microscopy to determine the distribution of fluorescently labeled monomers. We discovered that PA assemblies with opposite supramolecular chirality self-sorted into chemically distinct nanostructures. In contrast, the PA molecules that formed a mixture of right-handed, left-handed, and flat nanostructures on their own were able to coassemble with the other PA molecules. We attribute this phenomenon to the energy barrier associated with changing the handedness of a ß-sheet twist in a coassembly of two different PA molecules. This observation could be useful for designing biomolecular nanostructures with dual bioactivity or interpenetrating networks of PA supramolecular assemblies.

Polymer-DNA Carriers Co-Deliver Photosensitizer and siRNA for Light-Promoted Gene Transfection and Hypoxia-Relieved Photodynamic Therapy.

Photochemical internalization is an efficient strategy relying on photodynamic reactions to promote siRNA endosomal escape for the success of RNA-interference gene regulation, which makes gene-photodynamic combined therapy highly synergistic and efficient. However, it is still desired to explore capable carriers to improve the delivery efficiency of the immiscible siRNA and organic photosensitizers simultaneously. Herein, we employ a micellar nanostructure (PSNA) self-assembled from polymer-DNA molecular chimeras to fulfill this task. PSNA can plentifully load photosensitizers in its hydrophobic core simply by the nanoprecipitation method. Moreover, it can organize siRNA self-assembly by the densely packed DNA shell, which leads to a higher loading capacity than the typical electrostatic condensation method. The experimental results prove that this PSNA carrier can greatly facilitate siRNA escape from the endosome/lysosome and enhance transfection. Accordingly, the PSNA-administrated therapy exhibits a significantly improved anti-tumor efficacy owing to the highly efficient co-delivery capability.

ON-OFF Control of Marangoni Self-propulsion via A Supra-amphiphile Fuel and Switch.

Marangoni self-propulsion refers to motion of liquid or solid driven by a surface tension gradient, and has applications in soft robots/devices, cargo delivery, self-assembly etc. However, two problems remain to be addressed for motion control (e.g., ON-OFF) with conventional surfactants as Marangoni fuel: (1) limited motion lifetime due to saturated interfacial adsorption of surfactants; (2) in- situ motion stop is difficult once Marangoni flows are triggered. Instead of covalent surfactants, supra-amphiphiles with hydrophilic and hydrophobic parts linked noncovalently, hold promise to solve these problems owing to its dynamic and reversible surface activity responsively. Here, we propose a new concept of 'supra-amphiphile fuel and switch' based on the facile synthesis of disodium-4-azobenzene-amino-1,3-benzenedisulfonate (DABS) linked by a Schiff base, which has amphiphilicity for self-propulsion, hydrolyzes timely to avoid saturated adsorption, and provides pH-responsive control over ON-OFF motion. The self-propulsion lifetime is extended by 50-fold with DABS and motion control is achieved. The mechanism is revealed with coupled interface chemistry involving two competitive processes of interfacial adsorption and hydrolysis of DABS based on both experiments and simulation. The concept of 'supra-amphiphile fuel and switch' provides an active solution to prolong and control Marangoni self-propulsive devices for the advance of intelligent material systems.

Novel natural osthole-inspired amphiphiles as membrane targeting antibacterials against methicillin-resistant Staphylococcus aureus (MRSA).

Methicillin-resistant Staphylococcus aureus (MRSA) is a widespread pathogen causing clinical infections and is multi-resistant to many antibiotics, making it urgent need to develop novel antibacterials to combat MRSA. Herein, we designed and prepared a series of novel osthole amphiphiles 6a-6ad by mimicking the structures and function of antimicrobial peptides (AMPs). Antibacterial assays showed that osthole amphiphile 6aa strongly inhibited S. aureus and 10 clinical MRSA isolates with MIC values of 1-2 µg/mL, comparable to that of the commercial antibiotic vancomycin. Additionally, 6aa had the advantages of rapid bacteria killing without readily developing drug resistance, low toxicity, good membrane selectivity, and good plasma stability. Mechanistic studies indicated that 6aa possesses good membrane-targeting ability to bind to phosphatidylglycerol (PG) on the bacterial cell membranes, thereby disrupting the cell membranes and causing an increase in intracellular ROS as well as leakage of proteins and DNA, and accelerating bacterial death. Notably, in vivo activity results revealed that 6aa exhibits strong anti-MRSA efficacy than vancomycin as well as a substantial reduction in MRSA-induced proinflammatory cytokines, including TNF-α and IL-6. Given the impressive in vitro and in vivo anti-MRSA efficacy of 6aa, which makes it a potential candidate against MRSA infections.

Aqueous Supramolecular Transformations of Motor Bola-Amphiphiles at Multiple Length-Scale.

Molecular motor amphiphiles have already been widely attempted for dynamic nanosystems across multiple length-scale for developments of small functional materials, including controlling macroscopic foam properties, amplifying motion as artificial molecular muscles, and serving as extracellular matrix mimicking cell scaffolds. However, limiting examples of bola-type molecular motor amphiphiles are considered for constructing macroscopic biomaterials. Herein, this work presents the designed two second generation molecular motor amphiphiles, motor bola-amphiphiles (MBAs). Aside from the photoinduced motor rotation of MBAs achieved in both organic and aqueous media, the rate of recovering thermal helix inversion step can be controlled by the rotor part with different steric hindrances. Dynamic assembled structures of MBAs are observed under (cryo)-transmission electron microscopy (TEM). This dynamicity assists MBAs in further assembling as macroscopic soft scaffolds by applying a shear-flow method. Upon photoirradiation, the phototropic bending function of MBA scaffolds is observed, demonstrating the amplification of molecular motion into macroscopic phototropic bending functions at the macroscopic length-scale. Since MBAs are confirmed with low cytotoxicity, human bone marrow-derived mesenchymal stem cells (hBM-MSCs) can grow on the surface of MBA scaffolds. These results clearly demonstrate the concept of designing MBAs for developing photoresponsive dynamic functional materials to create new-generation soft robotic systems and cell-material interfaces.

Reversing the Trend: Deciphering Self-Assembly of Unconventional Amphiphiles Having Both Alkyl-Chain and PEG.

In the field of molecular self-assembly, the core of an assembly is always made up of hydrophobic moiety like a long alkyl chain, whereas the outer part has always been a hydrophilic moiety such as poly(ethylene glycol) (PEG), or charged species. Hence, reversing the trend to manifest self-assembled structures with a PEG core and a surface consisting of alkyl chains in aqueous system is incredibly challenging. Herein, we architected a unique class of cationic bolaamphiphiles containing low molecular weight PEG and alkyl chains of different lengths. The bolaamphiphiles spontaneously form vesicles without external stimuli. These vesicles are unprecedented because PEG makes up the vesicle core, while the alkyl chains appear on the vesicles' exterior. Hence, this particular design reverses the usual trend of self-assembly formation. The vesicle size increases with the increase in alkyl chain-length. To our great surprise, we obtained large micelles for longest alkyl-chain amphiphile, which in turn act as a gemini amphiphile. The shift from a particular bolaamphiphile to gemini amphiphile with the variation of alkyl chain is also unexplored. Therefore, this specific class of self-assembled structure would compound a new paradigm in molecular self-assembly and supramolecular chemistry.

A Self-Assembled 3D Model Demonstrates How Stiffness Educates Tumor Cell Phenotypes and Therapy Resistance in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense and stiff extracellular matrix (ECM) associated with tumor progression and therapy resistance. To further the understanding of how stiffening of the tumor microenvironment (TME) contributes to aggressiveness, a three-dimensional (3D) self-assembling hydrogel disease model is developed based on peptide amphiphiles (PAs, PA-E3Y) designed to tailor stiffness. The model displays nanofibrous architectures reminiscent of native TME and enables the study of the invasive behavior of PDAC cells. Enhanced tuneability of stiffness is demonstrated by interacting thermally annealed aqueous solutions of PA-E3Y (PA-E3Yh) with divalent cations to create hydrogels with mechanical properties and ultrastructure similar to native tumor ECM. It is shown that stiffening of PA-E3Yh hydrogels to levels found in PDAC induces ECM deposition, promotes epithelial-to-mesenchymal transition (EMT), enriches CD133+/CXCR4+ cancer stem cells (CSCs), and subsequently enhances drug resistance. The findings reveal how a stiff 3D environment renders PDAC cells more aggressive and therefore more faithfully recapitulates in vivo tumors.

Direct Membrane Penetration and Cytosolic Delivery of Nanoparticles via Electrostatically Bound Amphiphiles.

Nanoparticles usually enter cells through energy-dependent endocytosis that involves their cytosolic entry via biomembrane-coated endosomes. In contrast, direct translocation of nanoparticles with straight access to cytosol/subcellular components without any membrane coating is limited to very selective conditions/approaches. Here we show that nanoparticles can switch from energy-dependent endocytosis to energy-independent direct membrane penetration once an amphiphile is electrostatically bound to their surface. Compared to endocytotic uptake, this direct cell translocation is faster and nanoparticles are distributed inside the cytosol without any lysosomal trafficking. We found that this direct cell translocation option is sensitive to the charges of both the nanoparticles and the amphiphile. We propose that an electrostatically bound amphiphile induces temporary opening of the cell membrane, which allows direct cell translocation of nanoparticles. This approach can be adapted for efficient subcellular targeting of nanoparticles and nanoparticle-based drug delivery application, bypassing the endosomal trapping and lysosomal degradation.

A Fluorous Peptide Amphiphile with Potent Antimicrobial Activity for the Treatment of MRSA-induced Sepsis and Chronic Wound Infection.

The rising prevalence of global antibiotic resistance evokes the urgent need for novel antimicrobial candidates. Cationic lipopeptides have attracted much attention due to their strong antimicrobial activity, broad-spectrum and low resistance tendency. Herein, a library of fluoro-lipopeptide amphiphiles was synthesized by tagging a series of cationic oligopeptides with a fluoroalkyl tail via a disulfide spacer. Among the lipopeptide candidates, R6F bearing six arginine moieties and a fluorous tag shows the highest antibacterial activity, and it exhibits an interesting fluorine effect as compared to the non-fluorinated lipopeptides. The high antibacterial activity of R6F is attributed to its excellent bacterial membrane permeability, which further disrupts the respiratory chain redox stress and cell wall biosynthesis of the bacteria. By co-assembling with lipid nanoparticles, R6F showed high therapeutic efficacy and minimal adverse effects in the treatment of MRSA-induced sepsis and chronic wound infection. This work provides a novel strategy to design highly potent antibacterial peptide amphiphiles for the treatment of drug-resistant bacterial infections.

Poly(vinylpyridine-co-vinylpyridine N-oxide) Excipients Mediate Rapid Dissolution and Sustained Supersaturation of Posaconazole Amorphous Solid Dispersions.

The chemical structure of excipients molecularly mixed in an amorphous solid dispersion (ASD) has a significant impact on properties of the ASD including dissolution behavior, physical stability, and bioavailability. Polymers used in ASDs require a balance between hydrophobic and hydrophilic functionalities to ensure rapid dissolution of the amorphous dispersion as well as sustained supersaturation of the drug in solution. This work demonstrates the use of postpolymerization functionalization of poly(vinylpyridine) excipients to elucidate the impact of polymer properties on the dissolution behavior of amorphous dispersions containing posaconazole. It was found that N-oxidation of pyridine functionalities increased the solubility of poly(vinylpyridine) derivatives in neutral aqueous conditions and allowed for nanoparticle formation which supplied posaconazole into solution at concentrations exceeding those achieved by more conventional excipients such as hydroxypropyl methylcellulose acetate succinate (HPMCAS) or Eudragit E PO. By leveraging these functional modifications of the parent poly(vinylpyridine) excipient to increase polymer hydrophilicity and minimize the effect of polymer on pH, a new polymeric excipient was optimized for rapid dissolution and supersaturation maintenance for a model compound.

Red-light-controlled supramolecular assemblies of indigo amphiphiles at multiple length scales.

Amphiphilic molecules functionalized with photoresponsive motifs have attractive prospects for applications in smart functional bio-material ranging from cell-material interfaces to drug delivery systems owing to the precisely controllable functionality of self-assembled hierarchical supramolecular structures in aqueous media by a non-invasive light stimulation with high temporal- and spatial-resolution. However, most of reported photoresponsive amphiphiles are triggered by bio-damaging UV-light, which greatly limits the potential in bio-related applications. Herein, we present newly designed red-light controlled N,N'-diaryl-substituted indigo amphiphiles (IA), exhibiting excellent photoswitchablity and photostability with dual red-/green-light in organic media. Meanwhile, aqueous solutions of IA assembled into supramolecular structures in both microscopic and macroscopic length-scale, though the photoresponsiveness of IA is slightly compromised in aqueous media. At macroscopic length-scale, morphological changes of IA macroscopic scaffold prepared by a shear-flow method can be fine adjusted upon red-light irradiation. Moreover, the preferential attachment of live h-MSCs to IA macroscopic scaffold surface also indicates a good biocompatibility of IA macroscopic scaffold. These results provide the potential for developing the next generation of red-light controlled soft functional materials with good biocompatibility.

Structure-Activity Relationship Study to Develop Peptide Amphiphiles as Species-Specific Antimicrobials.

Antimicrobial peptide amphiphiles (PAs) are a promising class of molecules that can disrupt the bacterial membrane or act as drug nanocarriers. In this study, we prepared 33 PAs to establish supramolecular structure-activity relationships. We studied the morphology and activity of the nanostructures against different Gram-positive and Gram-negative bacterial strains (such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii). Next, we used principal component analysis (PCA) to determine the key contributors to activity. We found that for S. aureus, the zeta potential was the major contributor to the activity while Gram-negative bacteria were more influenced by the partition coefficient (LogP) with the following order P. aeruginosa>E. coli>A. baumannii. We also performed a study of the mechanism of action of selected PAs on the bacterial membrane assessing the membrane permeability and depolarization, changes in zeta potential and overall integrity. We studied the toxicity of the nanostructures against mammalian cells. Finally, we performed an in vivo study using the wax moth larvae to determine the therapeutic efficacy of the active PAs. This study shows cationic PA nanostructures can be an intriguing platform for the development of nanoantibacterials.

Development of a Novel Covalently Bonded Conjugate of Caprylic Acid Tripeptide (Isoleucine-Leucine-Aspartic Acid) for Wound-Compatible and Injectable Hydrogel to Accelerate Healing.

Third-degree burn injuries pose a significant health threat. Safer, easier-to-use, and more effective techniques are urgently needed for their treatment. We hypothesized that covalently bonded conjugates of fatty acids and tripeptides can form wound-compatible hydrogels that can accelerate healing. We first designed conjugated structures as fatty acid-aminoacid1-amonoacid2-aspartate amphiphiles (Cn acid-AA1-AA2-D), which were potentially capable of self-assembling into hydrogels according to the structure and properties of each moiety. We then generated 14 novel conjugates based on this design by using two Fmoc/tBu solid-phase peptide synthesis techniques; we verified their structures and purities through liquid chromatography with tandem mass spectrometry and nuclear magnetic resonance spectroscopy. Of them, 13 conjugates formed hydrogels at low concentrations (≥0.25% w/v), but C8 acid-ILD-NH2 showed the best hydrogelation and was investigated further. Scanning electron microscopy revealed that C8 acid-ILD-NH2 formed fibrous network structures and rapidly formed hydrogels that were stable in phosphate-buffered saline (pH 2-8, 37 °C), a typical pathophysiological condition. Injection and rheological studies revealed that the hydrogels manifested important wound treatment properties, including injectability, shear thinning, rapid re-gelation, and wound-compatible mechanics (e.g., moduli G″ and G', ~0.5-15 kPa). The C8 acid-ILD-NH2(2) hydrogel markedly accelerated the healing of third-degree burn wounds on C57BL/6J mice. Taken together, our findings demonstrated the potential of the Cn fatty acid-AA1-AA2-D molecular template to form hydrogels capable of promoting the wound healing of third-degree burns.

Tailoring Multicontrolled Supramolecular Assemblies of Stiff-Stilbene Amphiphiles into Macroscopic Soft Scaffolds as Cell-Material Interfaces.

Biocompatible synthetic supramolecular systems have shed light on biomedical and tissue-regenerative material applications. The intrinsic functional applicability, tunability, and stimuli-responsiveness of synthetic supramolecular systems allow one to develop various multicontrolled supramolecular assemblies in aqueous media. However, it remains highly challenging to use state-of-the-art supramolecular assemblies of photoresponsive amphiphiles controlled by multiple stimulations in fabricating macroscopic materials. Herein, we demonstrate a stiff-stilbene amphiphile (SA) multicontrolled supramolecular assembling system that comprises two different charged end groups. The excellent photoswitchabilities of SA in both organic and aqueous media are demonstrated. Furthermore, multiple stimuli, i.e., light, pH, and counterions, are applied to control the supramolecular assembling behaviors, which are monitored by circular dichroism spectroscopy and electron microscopies. This multicontrolled supramolecular system can be systematically assembled into macroscopic soft functional scaffolds, whose structural parameters are investigated by electron microscopies and X-ray diffraction techniques, suggesting the large aspect ratio of SA nanostructures assembled into macroscopic soft scaffolds. The fabricated soft functional scaffold is highly biocompatible for photocontrolled biotarget encapsulation/release selectively, as well as a cell-material interface for diverse cells' attachment. This new synthetic multicontrolled soft functional material provides a new strategy toward the development of next-generation controllable and biocompatible soft functional materials.

Synergetic Self-Assembly of Liquid Crystalline Block Copolymer with Amphiphiles for Fabrication of Hierarchical Assemblies.

Novel functions and advanced structure, where each single component could not be produced individually, can exhibit from the collective and synergistic behavior of component systems. This synergetic strategy has been successfully demonstrated for co-assembly of polymer-polymer to construct hierarchical nanomaterials. However, differences in the natures of polymer and small molecules impose challenges in the construction of sophisticated co-assemblies with geometrical and compositional control. Herein, a synergetic self-assembly strategy is proposed to prepare organic-organic hybrid colloidal mesostructures by blending a liquid crystalline block copolymer (LC-BCP) with small molecular amphiphiles. Through a classic solvent-exchange process, amphiphiles embedded with LC-BCP realize multi-component nucleation and hierarchical assembly driven by anisotropic interaction from the LC ordering alignment of the core-forming block. 1D nanofibers with a periodic striped structure are formed by further LC component fusion and refinement. In addition, LC ordering effect of LC-BCP can be regulated by selecting appropriate solvents and leads to the formation of vesicular co-micelles. By means of the thermal-responsive behavior of amphiphiles, hexagonal pore arrays are finally generated on the surface of those vesicles.

Design, synthesis and biological evaluation of novel cationic liposomes loaded with melphalan for the treatment of cancer.

Therapeutically active lipids in drug delivery systems offer customization for enhanced pharmaceutical and biological effects, improving safety and efficacy. Biologically active N, N-didodecyl-3,4-dimethoxy-N-methylbenzenaminium lipid (Q) was synthesized and employed to create a liposome formulation (FQ) encapsulating melphalan (M) through a thin film hydration method. Synthesized cationic lipids and their liposomal formulation underwent characterization and assessment for additive anti-cancer effects on myeloma and melanoma cancer cell lines. These effects were evaluated through various studies, including cytotoxicity assessments, cell cycle arrest analysis, apoptosis measurements, mitochondrial membrane potential depolarization, DNA fragmentation, and a significant reduction in tumorigenic potential, as evidenced by a decrease in both the number and percentage area of cancer spheroids.

Resveratrol and Resveratrol-Loaded Galactosylated Liposomes: Anti-Adherence and Cell Wall Damage Effects on Staphylococcus aureus and MRSA.

Antibiotic resistance due to bacterial biofilm formation is a major global health concern that makes the search for new therapeutic approaches an urgent need. In this context,, trans-resveratrol (RSV), a polyphenolic natural substance, seems to be a good candidate for preventing and eradicating biofilm-associated infections but its mechanism of action is poorly understood. In addition, RSV suffers from low bioavailability and chemical instability in the biological media that make its encapsulation in delivery systems necessary. In this work, the anti-biofilm activity of free RSV was investigated on Staphylococcus aureus and, to highlight the possible mechanism of action, we studied the anti-adherence activity and also the cell wall damage on a MRSA strain. Free RSV activity was compared to that of RSV loaded in liposomes, specifically neutral liposomes (L = DOPC/Cholesterol) and cationic liposomes (LG = DOPC/Chol/GLT1) characterized by a galactosylated amphiphile (GLT1) that promotes the interaction with bacteria. The results indicate that RSV loaded in LG has anti-adherence and anti-biofilm activity higher than free RSV. On the other side, free RSV has a higher bacterial-growth-inhibiting effect than encapsulated RSV and it can damage cell walls by creating pores; however, this effect can not prevent bacteria from growing again. This RSV ability may underlie its bacteriostatic activity.