Liposomes with pH responsive 'on and off' switches for targeted and intracellular delivery of antibiotics.

pH responsive drug delivery systems are one of the new strategies to address the spread of bacterial resistance to currently used antibiotics. The aim of this study was to formulate liposomes with 'On' and 'Off'' pH responsive switches for infection site targeting. The vancomycin (VCM) loaded liposomes had sizes below 100 nm, at pH 7.4. The QL-liposomes had a negative zeta potential at pH 7.4 that switched to a positive charge at acidic pH. VCM release from the liposome was quicker at pH 6 than pH 7.4. The OA-QL-liposome showed 4-fold lower MIC at pH 7.4 and 8- and 16-fold lower at pH 6.0 against both MSSA and MRSA compared to the bare drug. OA-QL liposome had a 1266.67- and 704.33-fold reduction in the intracellular infection for TPH-1 macrophage and HEK293 cells respectively. In vivo studies showed that the amount of MRSA recovered from mice treated with formulations was 189.67 and 6.33-fold lower than the untreated and bare VCM treated mice respectively. MD simulation of the QL lipid with the phosphatidylcholine membrane (POPC) showed spontaneous binding of the lipid to the bilayer membrane both electrostatic and Van der Waals interactions contributed to the binding. These studies demonstrated that the 'On' and 'Off' pH responsive liposomes enhanced the activity targeted and intracellular delivery VCM.

Novel formulation of antimicrobial peptides enhances antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA).

Antimicrobial peptides (AMPs) have the ability to penetrate as well as transport cargo across bacterial cell membranes, and they have been labeled as exceptional candidates to function in drug delivery. The aim of this study was to investigate the effectiveness of novel formulation of AMPs for enhanced MRSA activity. The strategy was carried out through the formulation of liposomes by thin-layer film hydration methodology, containing phosphatidylcholine, cholesterol, oleic acid, the novel AMP, as well as vancomycin (VCM). Characterization of the AMPs and liposomes included HPLC and LCMS for peptide purity and mass determination; DLS (size, polydispersity, zeta potential), TEM (surface morphology), dialysis (drug release), broth dilution, and flow cytometry (antibacterial activity); MTT assay, haemolysis and intracellular antibacterial studies. The size, PDI, and zeta potential of the drug-loaded AMP2-Lipo-1 were 102.6 ± 1.81 nm, 0.157 ± 0.01, and - 9.81 ± 1.69 mV, respectively, while for AMP3-Lipo-2 drug-loaded formulation, it was 146.4 ± 1.90 nm, 0.412 ± 0.05, and - 4.27 ± 1.25 mV respectively at pH 7.4. However, in acidic pH for both formulations, we observed an increase in size, PDI, and a switch to positive zeta potential, which indicated the pH responsiveness of our liposomal systems. The in vitro drug release studies demonstrated that liposomal formulations released VCM-HCl at a faster rate at pH 6.0 compared to pH 7.4. In vitro antibacterial activity against S. aureus and MRSA revealed that liposomes had enhanced activity at pH 6 compared to pH 7.4. The study revealed that the formulation can potentially be used to enhance activity and penetration of AMPs, thereby improving the treatment of bacterial infections.

Formulation and Molecular Dynamics Simulations of a Fusidic Acid Nanosuspension for Simultaneously Enhancing Solubility and Antibacterial Activity.

The aim of the present study was to formulate a nanosuspension (FA-NS) of fusidic acid (FA) to enhance its aqueous solubility and antibacterial activity. The nanosuspension was characterized using various in vitro, in silico, and in vivo techniques. The size, polydispersity index, and zeta potential of the optimized FA-NS were 265 ± 2.25 nm, 0.158 ± 0.026, and -16.9 ± 0.794 mV, respectively. The molecular dynamics simulation of FA and Poloxamer-188 showed an interaction and binding energy of -74.42 kJ/mol and -49.764 ± 1.298 kJ/mol, respectively, with van der Waals interactions playing a major role in the spontaneous binding. There was an 8-fold increase in the solubility of FA in a nanosuspension compared to the bare drug. The MTT assays showed a cell viability of 75-100% confirming the nontoxic nature of FA-NS. In vitro antibacterial activity revealed a 16- and 18-fold enhanced activity against Staphylococcus aureus (SA) and methicillin-resistant SA (MRSA), respectively, when compared to bare FA. Flowcytometry showed that MRSA cells treated with FA-NS had almost twice the percentage of dead bacteria in the population, despite having an 8-fold lower MIC in comparison to the bare drug. The in vivo skin-infected mice showed a 76-fold reduction in the MRSA load for the FA-NS treated group compared to that of the bare FA. These results show that the nanosuspension of antibiotics can enhance their solubility and antibacterial activity simultaneously.

Novel peptide and hyaluronic acid coated biomimetic liposomes for targeting bacterial infections and sepsis.

Sepsis is a life-threatening syndrome resulting from an imbalanced immune response to severe infections. Despite advances in nanomedicines, effective treatments for sepsis are still lacking. Herein, vancomycin free base (VCM)-loaded dual functionalized biomimetic liposomes based on a novel TLR4-targeting peptide (P3) and hyaluronic acid (HA) (HA-P3-Lipo) were developed to enhance sepsis therapy. The nanocarrier revealed appropriate physicochemical parameters, good stability, and biocompatibility. The release of VCM from HA-P3-Lipo was found to be sustained with 76 % VCM released in 48 h. The biomimicry was elucidated by in silico tools and MST and results confirmed strong binding between the system and TLR4. Furthermore, HA-P3-Lipo revealed 2-fold enhanced antibacterial activity against S. aureus, sustained antibacterial activity against MRSA over 72 h and 5-fold better MRSA biofilm inhibition compared to bare VCM. Bacterial-killing kinetics and flow cytometry confirmed the superiority of HA-P3-Lipo in eliminating MRSA faster than VCM. The in vivo potential of the nanocarrier was elucidated in an MRSA-induced sepsis mice model, and the results confirmed the superiority of HA-P3-Lipo compared to free VCM in eliminating bacteria and down-regulating the proinflammatory markers. Therefore, HA-P3-Lipo exhibits potential as a promising novel multi-functional nanosystem against sepsis and could significantly contribute to the transformation of sepsis therapy.

Engineering hybrid nanosystems for efficient and targeted delivery against bacterial infections.

Hybrid nanoparticles (NPs) are emerging as superior alternatives to conventional nanocarriers for enhancing the delivery of antibiotics and improving their targeting at the infection site, resulting in the eradication of bacterial infections and overcoming antimicrobial resistance. They can specifically control the release of antibiotics when reaching the targeted site of infection, thus enhancing and prolonging their antimicrobial efficacy. In this review, we provide a comprehensive and an up-to-date overview of the recent advances and contributions of lipid-polymer hybrid NPs; organic-inorganic hybrid NPs; metal-organic frameworks; cell membrane-coated hybrid NPs; hybrid NP-hydrogels; and various others, that have been reported in the literature for antibacterial delivery, with emphasis on their design approaches; the nanomaterials constructed; the mechanisms of drug release; and the enhanced antibacterial efficacy of the reported hybrid nanocarriers. This review also highlights future strategies that can be used to improve the potential of hybrid nanosystems to treat bacterial infections and overcome antibiotic resistance.

Synthesis of pH-responsive dimethylglycine surface-modified branched lipids for targeted delivery of antibiotics.

The rampant antimicrobial resistance crisis calls for efficient and targeted drug delivery of antibiotics at the infectious site. Hence, this study aimed to synthesize a pH-responsive dimethylglycine surface-modified branched lipid (DMGSAD-lipid). The structure of the synthesized lipid was fully confirmed. The lipid polymer hybrid nanoparticles (LPHNPs) were formulated using the solvent evaporation method and characterised. Two LPHNPs (VCM_HS15_LPHNPs and VCM_RH40_LPHNPs) were formulated and characterised for size, polydispersity index (PDI), and zeta potential (ZP). Atomistic molecular dynamics simulations revealed that both the systems self-assembled to form energetically stable aggregates. The ZP of RH40_VCM_LPHNPs changed from 0.55 ± 0.14-9.44 ± 0.33 Vm, whereas for SH15_VCM_LPHNPs, ZP changed from - 1.55 ± 0.184 Vm to 9.83 ± 0.52 Vm at pH 7.4 and 6.0, respectively. The encapsulation efficiencies of VCM were above 40% while the drug release was faster at acidic pH when compared to pH 7.4. The antibacterial activity of LPHNPs against MRSA was eight-fold better in MICs at pH 6.0, compared to 7.4, when compared to bare VCM-treated specimens. The study confirms that pH-responsive LPHNPs have the potential for enhancing the treatment of bacterial infections and other diseases characterised by acidic conditions at the target site.

A hyaluronic acid-based nanogel for the co-delivery of nitric oxide (NO) and a novel antimicrobial peptide (AMP) against bacterial biofilms.

Biofilms are a global health concern because they are associated with chronic and recurrent infections as well as resistance to conventional antibiotics. The aim of this study was to prepare a nanogel for the co-delivery of NO and AMPs against bacteria and biofilms. The NO-releasing nanogel was prepared by crosslinking HA solution with divinyl sulfone and extensively characterized. The nanogel was found to be biocompatible, injectable and NO release from the gel was sustained over a period of 24 h. In vitro antibacterial studies showed that the NO-AMP-loaded nanogel exhibited a broad spectrum antibacterial/antibiofilm activity. The NO-releasing nanogel had a greater antibacterial effect when compared to NO alone with MIC values of 1.56, 0.78 and 0.39 µg/ml against Escherichia coli, Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa bacteria respectively. The antibiofilm results showed there was a 12.5 and 24-folds reduction in biofilms of MRSA, and P. aeruginosa respectively for catheters exposed to nanogel loaded with AMP/NO when compared to only NO, while a 7 and 9.4-folds reduction in biofilms of MRSA, and P. aeruginosa respectively was displayed by the nanogel loaded with only NO compared to only NO. The AMP/NO-releasing nanogel showed the potential to combat both biofilms and bacterial infections.

A transferosome-loaded bigel for enhanced transdermal delivery and antibacterial activity of vancomycin hydrochloride.

Transdermal drug delivery is an attractive route of administration relative to other routes as it offers enhanced therapeutic efficacy. However, due to poor skin permeability of certain drugs, their application in transdermal delivery is limited. The ultra-deformable nature of transferosomes makes them suitable vehicles for transdermal delivery of drugs that have high molecular weights and hydrophilicity. However, their low viscosity, which leads to low contact time on the surface of the skin, has restricted their application in transdermal delivery. Therefore, this study aimed to deliver transferosomes loaded with a highly water-soluble and high molecular weight vancomycin hydrochloride (VCM-HCl) via a bigel for systemic delivery and treatment of microbial infections. VCM-HCl-loaded transferosomal formulations (TNFs) were prepared using a reverse-phase evaporation method and then loaded into a bigel. Both the TNFs and TNFs-loaded bigel (TNF-L-B) were characterized by a range of in vitro and ex vivo techniques. TNFs and TNF-L-B were tested for biosafety via the MTT assay and found to be biosafe. Prepared TNFs had sizes, zeta potential and entrapment efficiency of 63.02 ± 5.34 nm, -20.93 ± 6.13 mV and 84.48 ± 1.22% respectively. VCM-HCl release from TNF-L-B showed a prolonged release profile with 39.76 ± 1.6% after 24hrs when compared to bare VCM-HCl loaded in the bigel (74.81 ± 8.84%). Ex-vivo permeation of prepared TNF-L-B showed a higher permeation flux of 0.56 µg/cm2/h compared to the bare VCM-HCl-loaded bigel of 0.23 µg/cm2/h, indicating superior permeation and bioavailability of the drug. Additionally, the prepared TNF-L-B demonstrated improved antimicrobial activity. The TNF-L-B showed minimum inhibitory concentrations (MIC) of 0.97 µg/ml against Staphylococcus aureus (SA) and 1.95 µg/ml against methicillin-resistant SA (MRSA), which were 2-fold lower MIC values than the bare drug. The time-kill assay showed that both TNFs and TNF-L-B systems caused a 5.6-log reduction (100%) in MRSA compared to bare VCM-HCl after 24 hrs of incubation. Furthermore, as opposed to the bare VCM-HCl solution, the degree of biofilm reduction caused by TNFs (55.72%) and TNF-L-B (34.58%) suggests their dominance in eradicating MRSA biofilm. These findings indicate that TNF-L-B is a promising system for transdermal delivery of hydrophilic and high molecular weight drugs.

A self-assembled polymer therapeutic for simultaneously enhancing solubility and antimicrobial activity and lowering serum albumin binding of fusidic acid.

The global antimicrobial resistance crisis has prompted worldwide efforts to develop new and more efficient antimicrobial compounds, as well as to develop new drug delivery strategies and targeting mechanisms. This study aimed to synthesize a novel polyethylene glycol-fusidic acid (PEG-FA) conjugate for self-assembly into nano-sized structures and explore its potential for simultaneously enhancing aqueous solubility and antibacterial activity of FA. In addition, the ability of PEG-FA to bind to HSA with lower affinity than FA is also investigated. Haemolysis and in vitro cytotoxicity studies confirmed superior biosafety of the novel PEG-FA compared to FA. The water solubility of FA after PEG conjugation was increased by 25-fold compared to the bare drug. PEG-FA nanoparticles displayed particle size, polydispersity index and zeta potential of 149.3 ± 0.21 nm, 0.267 ± 0.01 and 5.97 ± 1.03 mV, respectively. Morphology studies using high-resolution transmission electron microscope revealed a homogenous spherical shape of the PEG-FA nanoparticles. In silico studies showed that Van der Waals forces facilitated PEG-FA self-assembly. HSA binding studies showed that PEG-FA had very weak or no interaction with HSA using in silico molecular docking (-2.93 kcal/mol) and microscale thermophoresis (Kd=14999 ± 1.36 µM), which may prevent bilirubin displacement. Conjugation with PEG did not inhibit the antibacterial activity of FA but rather enhanced it by 2.5-fold against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus, compared to the bare FA. These results show that PEG-FA can simultaneously enhance solubility and antibacterial activity of FA, whilst also reducing binding of HSA to decrease its side effects.

Development of niosomes for encapsulating captopril-quercetin prodrug to combat hypertension.

Novel and effective anti-hypertensive agents are required to manage hypertension; therefore, we synthesised a novel antihypertensive drug from captopril and quercetin (cap-que) and explored its antihypertensive potential in a niosomal formulation via molecular hybridisation. The cap-que hybrid was synthesised, and its structure was characterised via NMR, FTIR, and HRMS. Niosomes were then loaded with cap-que using the thin-film hydration method. The particle size, polydispersity index, surface charge and drug entrapment efficiency (EE%) of the formulation were 418.8 ± 4.21 nm, 0.393 ± 0.063, 16.25 ± 0.21 mV, and 87.74 ± 2.82%, respectively. The drug release profile showed a sustained release of the active compound (43 ± 0.09%) from the niosomal formulation, compared to the parent drug (80.7 ± 4.68%), over 24 h. The cell viability study confirmed the biosafety of the formulation. The in vivo study in a rat model showed enhanced antihypertensive activity of the hybrid molecule and niosomal formulation which reduced systolic and diastolic pressure when compared to the individual, bare drugs. The findings of this study concluded that the antihypertensive potential of captopril can be enhanced by its hybridisation with quercetin, followed by niosomal nano drug delivery.

Free radical-releasing systems for targeting biofilms.

The recent rise in antibiotic drug resistance and biofilm formation by microorganisms has driven scientists from different fields to develop newer strategies to target microorganisms responsible for infectious diseases. There is a growing interest in free radicals as therapeutic agents for antimicrobial applications. However, limitations such as short half-life has hindered their usage. Currently, several research groups are exploring various biomaterials that can prolong the half-life, increase storage duration and control the release of the therapeutic ranges of free radicals required for different applications, including biofilm eradication. This review paper initially provides a background to, and theoretical knowledge on, free radicals; and then proceeds to review studies that have employed various free radical-incorporated drug delivery systems as an approach to target biofilm formation and eradication. Some of the free radical releasing systems highlighted include polymers, nanoparticles and hydrogels, with a focus on biofilm eradication, where they impact significantly. The various challenges associated with their application are also discussed. Further, the review identifies future research and strategies that can potentiate the application of free radical-incorporated drug delivery systems for inhibiting biofilm formation and eradicating formed biofilms.

Novel two-chain fatty acid-based lipids for development of vancomycin pH-responsive liposomes against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA).

The development of bacterial resistance against antibiotics is attributed to poor localisation of lethal antibiotic dose at the infection site. This study reports on the synthesis and use of novel two-chain fatty acid-based lipids (FAL) containing amino acid head groups in the formulation of pH-responsive liposomes for the targeted delivery of vancomycin (VAN). The formulated liposomes were characterised for their size, polydispersity index (PDI), surface charge and morphology. The drug-loading capacity, drug release, cell viability, and in vitro and in vivo efficacy of the formulations were investigated. A sustained VAN release profile was observed and in vitro antibacterial studies against S. aureus and MRSA showed superior and prolonged activity over 72 h at both pH 7.4 and 6.0. Enhanced antibacterial activity at pH 6.0 was observed for the DOAPA-VAN-Lipo and DLAPA-VAN-Lipo formulations. Flow cytometry studies indicated a high killing rate of MRSA cells using DOAPA-VN-Lipo (71.98%) and DLAPA-VN-Lipo (73.32%). In vivo studies showed reduced MRSA recovered from mice treated with formulations by four- and two-folds lower than bare VN treated mice, respectively. The targeted delivery of VAN can be improved by novel pH-responsive liposomes from the two-chain (FAL) designed in this study.

A hybrid of mPEG-b-PCL and G1-PEA dendrimer for enhancing delivery of antibiotics.

The development of novel materials is essential for the efficient delivery of drugs. Therefore, the aim of the study was to synthesize a linear polymer dendrimer hybrid star polymer (3-mPEA) comprising of a generation one poly (ester-amine) dendrimer (G1-PEA) and a diblock copolymer of methoxy poly (ethylene glycol)-b-poly(ε-caprolactone) (mPEG-b-PCL) for formulation of nanovesicles for efficient drug delivery. The synthesized star polymer was characterized by FTIR, 1H and 13C NMR, HRMS, GPC and its biosafety was confirmed by MTT assays. Thereafter it was evaluated as a nanovesicle forming polymer. Vancomycin loaded nanovesicles were characterized using in vitro, molecular dynamics (MD) simulations and in vivo techniques. MTT assays confirmed the nontoxic nature of the synthesized polymer, the cell viability was 77.23 to 118.6%. The nanovesicles were prepared with size, polydispersity index and zeta potential of 52.48 ±â€¯2.6 nm, 0.103 ±â€¯0.047, -7.3 ±â€¯1.3 mV respectively, with the encapsulation efficiency being 76.49 ±â€¯2.4%. MD simulations showed spontaneous self-aggregation of the dendritic star polymer and the interaction energy between the two monomers was -146.07 ±â€¯4.92, Van der Waals interactions playing major role for the aggregates stability. Human serum albumin (HSA) binding studies with Microscale Thermophoresis (MST) showed that the 3-mPEA did not have any binding affinity to the HSA, which showed potential for long systemic circulation. The vancomycin (VCM) release from the drug loaded nanovesicles was found to be slower than bare VCM, with an 65.8% release over a period of 48 h. The in vitro antibacterial test revealed that the drug loaded nanovesicles had 8- and 16-fold lower minimum inhibitory concentration (MIC) against methicillin sensitive Staphylococcus aureus and methicillin-resistant S. aureus strains (MRSA) compared to free drug. The flow cytometry study showed 3.9-fold more dead cells of MRSA in the population when samples were treated with the drug loaded nanovesicles than the bare VCM at concentration 0.488 µg/mL. An in vivo skin infection mice model showed a 20-fold reduction in the MRSA load in the drug loaded nanovesicles treated groups compared to bare VCM. These findings confirmed the potential of 3-mPEA as a promising biocompatible effective nanocarrier for antibiotic delivery.

Pegylated oleic acid: A promising amphiphilic polymer for nano-antibiotic delivery.

Vancomycin (VM), a last resort to control methicillin-resistant S. aureus (MRSA) infections, is on the verge of becoming ineffective. Novel nano delivery systems of VM have the potential to combat MRSA. The search for novel materials for nanoantibiotic development is therefore an active research area. In this study, oleic acid (OA) was coupled with monomethoxy polyethylene glycol (mPEG) to obtain a novel bio-safe amphiphilic polymer, mPEG-OA. The critical micelle concentration of mPEG-OA, was found to be 4.5×10-8m/L. VM-loaded polymersomes were prepared from mPEG-OA and evaluated for size, polydispersity index (PDI), zeta potential (ZP), surface morphology, drug release, in vitro and in vivo antibacterial activity. The size, PDI and ZP of VM-loaded polymersomes were 142.9±7.5nm, 0.228±0.03 and -18.3±3.55mV respectively. Transmission electron microscopy images revealed the spherical shape of polymersomes. The encapsulation efficiency was 53.64±1.86%. The drug release from polymersomes was sustained and in vitro antibacterial activity was 42- and 5-fold more against S. aureus and MRSA, compared with plain VM. An in vivo BALB/c mice, skin infection models revealed that treatment with VM-loaded polymersomes significantly reduced the MRSA burden compared with plain VM and blank polymersomes. There was a 183 and a 25-fold reduction in the MRSA colony finding units load in mice skin treated with VM-loaded polymersomes compared to that treated with blank polymersomes and bare VM respectively. In summary, the developed VM-loaded polymersomes from novel mPEG-OA polymer were found to be a promising nanoantibiotic against MRSA.