Formulation of pH responsive multilamellar vesicles for targeted delivery of hydrophilic antibiotics.

Fight against antimicrobial resistance calls for innovative strategies that can target infection sites and enhance activity of antibiotics. Herein is a pH responsive multilamellar vesicles (MLVs) for targeting bacterial infection sites. The vancomycin (VCM) loaded MLVs had 62.25 ± 8.7 nm, 0.15 ± 0.01 and -5.55 ± 2.76 mV size, PDI and zeta potential, respectively at pH 7.4. The MLVs had a negative ZP at pH 7.4 that switched to a positive charge and faster release of the drug at acidic pH. The encapsulation efficiency was found to be 46.34 ± 3.88 %. In silico studies of the lipids, interaction suggested an energetically stable system. Studies to determine the minimum inhibitory concentration studies (MIC) showed the MLVs had 2-times and 8-times MIC against Staphylococcus aureus (SA) and Methicillin resistance SA respectively at physiological pH. While at pH 6.0 there was 8 times reduction in MICs for the formulation against SA and MRSA in comparison to the bare drug. Fluorescence-activated Cell Sorting (FACS) studies demonstrated that even with 8-times lower MIC, MLVs had a similar elimination ability of MRSA cells when compared to the bare drug. Fluorescence microscopy showed MLVs had the ability to clear biofilms while the bare drug could not. Mice skin infection models studies showed that the colony finding units (CFUs) of MRSA recovered from groups treated with MLVs was 4,050 and 525-fold lower than the untreated and bare VCM treated groups, respectively. This study demonstrated pH-responsive multilamellar vesicles as effective system for targeting and enhancing antibacterial agents.

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.

AB2-type amphiphilic block copolymer containing a pH-cleavable hydrazone linkage for targeted antibiotic delivery.

A novel AB2-type amphiphilic block copolymer [OA-CN-NH-(PEG)2] with hydrazone linkage was synthesized and explored for pH-triggered antibiotic delivery. Vancomycin (VCM) loaded micelles of the polymer [OA-CN-NH-(PEG)2-VCM] were spherical in shape with size, polydispersity index, zeta potential and entrapment efficiency of 130.33 ± 7.36 nm, 0.163 ± 0.009, -4.33 ± 0.55 mV and 39.61 ± 4.01% respectively. The dilution stability study exhibited no significant change in the size distribution of OA-CN-NH-(PEG)2-VCM micelles up to 320-fold dilution. An in vitro drug release assay confirmed greater release of VCM from OA-CN-NH-(PEG)2-VCM at pH 6, compared to pH 7.4. An in vitro antibacterial activity evaluation of OA-CN-NH-(PEG)2-VCM showed 2-fold enhancement in antibacterial activity of VCM after 54 h of incubation against Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) at acidic pH compared to physiological pH. An in vivo antibacterial activity of OA-CN-NH-(PEG)2-VCM further proved the enhanced activity of OA-CN-NH-(PEG)2-VCM against MRSA. In conclusion, micelles from pH-responsive OA-CN-NH-(PEG)2 could be utilized for site-specific delivery of VCM at the infection site.

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.

Synthesis of an oleic acid based pH-responsive lipid and its application in nanodelivery of vancomycin.

Stimuli-responsive nano-drug delivery systems can optimize antibiotic delivery to infection sites. Identifying novel lipids for pH responsive delivery to acidic conditions of infection sites will enhance the performance of nano-drug delivery systems. The aim of the present investigation was to synthesize and characterize a biosafe novel pH-responsive lipid for vancomycin delivery to acidic conditions of infection sites. A pH-responsive solid lipid, N-(2-morpholinoethyl) oleamide (NMEO) was synthesized and used to prepare vancomycin (VCM)-loaded solid lipid nanoparticles (VCM_NMEO SLNs). The particle size (PS), polydispersity index (PDI), zeta potential (ZP) and entrapment efficiency (EE) of the formulation were 302.8 ±â€¯0.12 nm, 0.23 ±â€¯0.03, -6.27 ±â€¯0.017 mV and 81.18 ±â€¯0.57% respectively. The study revealed that drug release and antibacterial activity were significantly greater at pH 6.0 than at pH 7.4, while the in silico studies exposed the molecular mechanisms for improved stability and drug release. Moreover, the reduction of MRSA load was 4.14 times greater (p < 0.05) in the skin of VCM_NMEO SLNs treated mice than that of bare VCM treated specimens. Thus, this study confirmed that NMEO can successfully be used to formulate pH-responsive SLNs with potential to enhance the treatment of bacterial infections.

Novel lipids with three C18-fatty acid chains and an amino acid head group for pH-responsive and sustained antibiotic delivery.

The acidic environment at bacterial infection sites is a potential external stimulus for targeted antibiotic delivery. This paper reports new biocompatible pH-sensitive lipids (PSLs) with three hydrocarbon tails, and a head group with a secondary amine and carboxylate function for site-specific nano delivery of vancomycin (VCM). PSLs formed stable liposomes with mean vesicle diameters and polydispersity indices between 99.38 ±â€¯6.59 nm to 105.60 ±â€¯5.38 nm and 0.161 ±â€¯0.003 to 0.219 ±â€¯0.05 respectively. The zeta potential values were negative at physiological pH (7.4) and shifted towards positivity with a decrease in pH. The encapsulation efficiency and loading capacity were in the range of 29-45% and 2.8-4.5% respectively. The VCM release increased and was more sustained at acidic pH than at the physiological pH. The molecular modeling studies revealed that structural changes in lipids at acidic pH could have caused the deformation of liposome structure and subsequent fast release. In vitro antibacterial activity revealed that the minimum inhibitory concentrations (MICs) of prepared liposomes at pH 6.5 were lower than the MICs at pH 7.4 against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) respectively. In addition, in vivo antibacterial activity study performed on two of the most active formulations showed that log10 CFU/mL of MRSA recovered from TOAPA-VCM-Lipo and the TLAPA-VCM-Lipo treated mice were 1.5- and 1.8-fold lower than that found in bare VCM treated ones respectively.

Identifying the Interaction of Vancomycin With Novel pH-Responsive Lipids as Antibacterial Biomaterials Via Accelerated Molecular Dynamics and Binding Free Energy Calculations.

Nano-drug delivery systems have proven to be an efficient formulation tool to overcome the challenges with current antibiotics therapy and resistance. A series of pH-responsive lipid molecules were designed and synthesized for future liposomal formulation as a nano-drug delivery system for vancomycin at the infection site. The structures of these lipids differ from each other in respect of hydrocarbon tails: Lipid1, 2, 3 and 4 have stearic, oleic, linoleic, and linolenic acid hydrocarbon chains, respectively. The impact of variation in the hydrocarbon chain in the lipid structure on drug encapsulation and release profile, as well as mode of drug interaction, was investigated using molecular modeling analyses. A wide range of computational tools, including accelerated molecular dynamics, normal molecular dynamics, binding free energy calculations and principle component analysis, were applied to provide comprehensive insight into the interaction landscape between vancomycin and the designed lipid molecules. Interestingly, both MM-GBSA and MM-PBSA binding affinity calculations using normal molecular dynamics and accelerated molecular dynamics trajectories showed a very consistent trend, where the order of binding affinity towards vancomycin was lipid4 > lipid1 > lipid2 > lipid3. From both normal molecular dynamics and accelerated molecular dynamics, the interaction of lipid3 with vancomycin is demonstrated to be the weakest (∆Gbinding = -2.17 and -11.57, for normal molecular dynamics and accelerated molecular dynamics, respectively) when compared to other complexes. We believe that the degree of unsaturation of the hydrocarbon chain in the lipid molecules may impact on the overall conformational behavior, interaction mode and encapsulation (wrapping) of the lipid molecules around the vancomycin molecule. This thorough computational analysis prior to the experimental investigation is a valuable approach to guide for predicting the encapsulation ability, drug release and further development of novel liposome-based pH-responsive nano-drug delivery system with refined structural and chemical features of potential lipid molecule for formulation development.

pH-responsive chitosan nanoparticles from a novel twin-chain anionic amphiphile for controlled and targeted delivery of vancomycin.

The design and synthesis of novel pH-responsive nanoantibiotics is an emerging research area to address the antibiotic resistance crisis. The purpose of this study was therefore to synthesize a new anionic gemini surfactant (AGS) that could result in the formulation of pH-responsive chitosan nanoparticles (CSNPs) to treat methicillin-resistant Staphylococcus aureus (MRSA) infections. The coupling of oleic acid with 2,2-dimethyl-5,5-bis(hydroxymethyl)-1,3-dioxane and subsequent deprotection followed by a reaction with succinic anhydride and sodium bicarbonate yielded AGS. Critical micelle concentration (CMC) was determined using conductometry and in vitro cytotoxicity was performed using a MTT assay. Vancomycin loaded CSNPs containing AGS (DL_CSSNPs) were prepared by ionotropic gelation of chitosan with pentasodium tripolyphosphate. CSNPs were characterized for size, polydispersity index (PDI), zeta potential (ZP), entrapment efficiency, surface morphology, in vitro drug release and in vitro antibacterial activity (at pH 6.5 and 7.4). Results from the in vitro antibacterial activity were further supported by an in vivo study using a mice skin infection model. The CMC of AGS was found to be 1.3mM/L and it was non-toxic. The DL_CSSNPs were spherical with size, PDI and ZP of 220.57±5.9nm, 0.299±0.004 and 21.9±0.9mV respectively. An increase in the vancomycin release from the DL_CSSNPs was observed at pH 6.5 compared to pH 7.4. The minimum inhibitory concentration values at pH 6.5 and 7.4 against MRSA were 7.81 and 62.5µg/ml respectively. In vivo antibacterial activity showed that the MRSA burden in mice treated with DL_CSSNPs was reduced by almost 8-fold compared to those treated with pure vancomycin.

Exploring unsaturated fatty acid cholesteryl esters as transdermal permeation enhancers.

The intrinsic protective barrier property of skin, one of the major challenges in the design of transdermal drug delivery systems, can be overcome through the use of chemical permeation enhancers (CPEs). Herein, we explore the potential of unsaturated fatty acid (UFA) esters of cholesterol (Chol) viz., oleate, linoleate and linolenate, as transdermal CPEs using tenofovir (TNF) as a model drug. All Chol UFA esters at 1% w/w were found to be more effective enhancers when compared to their respective parent fatty acids (FAs) and saturated FA counterparts. Cholesteryl linolenate (Chol-LLA) showed the most superior performance (enhancement ratio (ER) = 3.71). The greatest ER for Chol-LLA (5.93) was achieved at a concentration of 2% w/w. The histomorphological and transepithelial electrical resistance (TEER) evaluations supported the results of the permeability studies. These findings showed no significant loss in the integrity of the epidermis, with drug and enhancer treatment having temporary effects on the barrier property of the epidermis. Chol UFA esters can therefore be considered as new CPEs for exploitation in topical formulations for various classes of drugs.

Preparation and Optimization of Meropenem-Loaded Solid Lipid Nanoparticles: In Vitro Evaluation and Molecular Modeling.

Encapsulation of antibiotics into nanocarriers has the potential to overcome resistance and disadvantages associated with conventional dosage forms as well as increase half-life of an antibiotic. Encapsulation of meropenem (MRPN) into solid lipid nanoparticles (SLNs) remains unexplored among the limited work reported on nanoformulation incorporating MRPN. The study aimed to use an experimental design, to optimize MRPN-loaded SLNs, and to undertake in vitro and in silico evaluations. A Box-Behnken design (BBD) was used to optimize manufacturing conditions of glycerol monostearate (GMS) SLNs loaded with MRPN. The SLNs were prepared using hot homogenization and ultrasonication method. Optimized MRPN-SLNs showed particle size, zeta potential, and entrapment efficiency of 112.61 ± 0.66 nm, -20.43 ± 0.99 mV, and 89.94 ± 1.26%, respectively. The morphology of the SLNs revealed nearly spherical shaped particles. Differential scanning calorimetry and X-ray diffraction analysis showed that meropenem was present in amorphous form in the SLNs. Controlled in vitro MRPN release from SLNs was achieved and followed the Korsmeyer-Peppas model (R 2 = 0.9679). Prolonged in vitro antibacterial activity against Escherichia coli was also observed. The molecular modeling showed that both hydrophobic interactions and hydrogen bonding led to a stable MRPN-GMS complex formation, which was confirmed by its low heat of formation (-5536.13 kcal/mol). This stable complex could have contributed to the controlled release of MRPN from the SLNs and subsequent sustained antibacterial activity.

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.

Co-encapsulation of multi-lipids and polymers enhances the performance of vancomycin in lipid-polymer hybrid nanoparticles: In vitro and in silico studies.

Nano-drug delivery systems are being widely explored to overcome the challenges with existing antibiotics to treat bacterial infections [1]. Lipid-polymer hybrid nanoparticles (LPNs) display unique advantages of both liposomes and polymeric nanoparticles while excluding some of their limitations, particularly the structural integrity of the polymeric particles and the biomimetic properties of the liposome [1]. The use of helper lipids and polymers in LPNs has not been investigated, but has shown potential in other nano-drug delivery systems to improve drug encapsulation, antibacterial activity and drug release. Therefore, LPNs using co-excipients were prepared using vancomycin (VCM), glyceryl triplamitate and Eudragit RS100 as the drug, lipid and polymer respectively. Oleic acid (OA), Chitosan (CHT) and Sodium alginate (ALG) were explored as co-excipients. Results indicated rod-shaped LPNs with suitable size, PDI and zeta potential, while encapsulation efficiency (%EE) increased from 27.8% to 41.5%, 54.3% and 69.3% with the addition of OA, CHT and ALG respectively. Drug release indicated that VCM-CHT had the best performance in sustained drug release of 36.1 ± 5.35% after 24h. The EE and drug release were further corroborated by in silico and release kinetics data. In vitro antibacterial studies of all formulations exhibited better activity against bare VCM and sustained activity up to day 5 against both Staphylococcus aureus and MRSA, with VCM-OA and VCM-CHT showing better activity against MRSA. Therefore, this LPN proves to be a promising system for delivery of VCM as well as other antibiotics.

Solid lipid nanoparticles of clotrimazole silver complex: An efficient nano antibacterial against Staphylococcus aureus and MRSA.

New and effective strategies to transform current antimicrobials are required to address the increasing issue of microbial resistance and declining introduction of new antibiotic drugs. In this context, metal complexes of known drugs and nano delivery systems for antibiotics are proving to be promising strategies. The aim of the study was therefore to synthesize a silver complex of clotrimazole and formulate it into a nano delivery system for enhanced and sustained antibacterial activity against susceptible and resistant Staphylococcus aureus. A silver complex of clotrimazole was synthesized, characterized and further encapsulated into solid lipid nanoparticles to evaluate its antibacterial activity against S. aureus and methicillin-resistant S. aureus (MRSA). An in vitro cytotoxicity study was performed on HepG2 cell lines to assess the overall biosafety of the synthesized clotrimazole silver complex to mammalian cells, and was found to be non-toxic to mammalian cells (cell viability >80%). The minimum inhibitory concentrations (MIC) of clotrimazole and clotrimazole-silver were 31.25 and 9.76 µg/mL against S. aureus, and 31.25 and 15.62 against MRSA, respectively. Clotrimazole SLNs exhibited MIC values of 104 and 208 µg/mL against both MSSA and MRSA at the end of 18 and 36 h, respectively, but thereafter completely lost its antibacterial activity. Clotrimazole-silver SLNs had an MIC value of 52 µg/mL up to 54 h, after which the MIC value was 104 µg/mL against both strains at the end of 72 h. Thus, clotrimazole-silver SLNs was found to be an efficient nanoantibiotic.