Antimicrobial Peptide Synergies for Fighting Infectious Diseases.

Antimicrobial peptides (AMPs) are essential elements of thehost defense system. Characterized by heterogenous structures and broad-spectrumaction, they are promising candidates for combating multidrug resistance. Thecombined use of AMPs with other antimicrobial agents provides a new arsenal ofdrugs with synergistic action, thereby overcoming the drawback of monotherapiesduring infections. AMPs kill microbes via pore formation, thus inhibitingintracellular functions. This mechanism of action by AMPs is an advantage overantibiotics as it hinders the development of drug resistance. The synergisticeffect of AMPs will allow the repurposing of conventional antimicrobials andenhance their clinical outcomes, reduce toxicity, and, most significantly,prevent the development of resistance. In this review, various synergies ofAMPs with antimicrobials and miscellaneous agents are discussed. The effect ofstructural diversity and chemical modification on AMP properties is firstaddressed and then different combinations that can lead to synergistic action,whether this combination is between AMPs and antimicrobials, or AMPs andmiscellaneous compounds, are attended. This review can serve as guidance whenredesigning and repurposing the use of AMPs in combination with other antimicrobialagents for enhanced clinical outcomes.

Novel CA(1-7)M(2-9) Analogs: Synthesis, Characterization, and Antibacterial Evaluation.

Hybrid peptides from cecropin A and melittin have attracted the interest of the research community for decades. Here we synthesized several new analogs of the pentadecapeptide CA(1-7)M(2-9) and studied their antibacterial and hemolytic activity and tryptic stability. Single substitution of the Lys residues by Arg did not have a significant impact on the antibacterial activity of these analogs, but the substitution of the five Lys residues by Arg resulted in an increment in hemolytic activity. In contrast, the substitution of Lys residues by Orn conserved the antibacterial activity, with even lower hemolysis, and improved the enzymatic stability. The disulfide cyclic version of CA(1-7)M(2-9) was obtained by adding a Cys residue to each end of the peptide and carrying out a chemoselective thiol-disulfide interchange using sec-isoamylmecaptan as protecting group of one of these residues. This cyclic peptide showed good antibacterial activity with low hemolysis and improved enzymatic stability.

Novel Biomimetic Human TLR2-Derived Peptides for Potential Targeting of Lipoteichoic Acid: An In Silico Assessment.

Antimicrobial resistance is one of the most significant threats to health and economy around the globe and has been compounded by the emergence of COVID-19, raising important consequences for antimicrobial resistance development. Contrary to conventional targeting approaches, the use of biomimetic application via nanoparticles for enhanced cellular targeting, cell penetration and localized antibiotic delivery has been highlighted as a superior approach to identify novel targeting ligands for combatting antimicrobial resistance. Gram-positive bacterial cell walls contain lipoteichoic acid (LTA), which binds specifically to Toll-like receptor 2 (TLR2) on human macrophages. This phenomenon has the potential to be exploited for the design of biomimetic peptides for antibacterial application. In this study, we have derived peptides from sequences present in human TLR2 that bind to LTA with high affinity. In silico approaches including molecular modelling, molecular docking, molecular dynamics, and thermodynamics have enabled the identification of these crucial binding amino acids, the design of four novel biomimetic TLR2-derived peptides and their LTA binding potential. The outcomes of this study have revealed that one of these novel peptides binds to LTA more strongly and stably than the other three peptides and has the potential to enhance LTA targeting and bacterial cell penetration.

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.

Supramolecular amphiphiles of Beta-cyclodextrin and Oleylamine for enhancement of vancomycin delivery.

The global threat of antimicrobial resistant strains calls for innovative strategies to utilize nano drug delivery systems to enhance the delivery of antibiotics, thus reducing the development of resistance. Supramolecular amphiphiles that can self-assemble into nanostructures are one such nano delivery system, that are showing potential for effective drug delivery. The aim of this study was to synthesize and formulate a novel sugar-based cationic amphiphile (BCD-OLA) derivative from a Beta-cyclodextrin (BCD) head and long C18 carbon chain with a terminal amine; oleylamine (OLA), using inclusion complexation for application in antibiotic delivery. A suspension method was used for preparing the BCD-OLA amphiphile, which was then utilized for the formulation of nanovesicles. The complexation of BCD-OLA was confirmed by FTIR, 1H NMR, 2D NMR NOESY spectrum and molecular dynamic (MD) simulations. Thereafter, biosafety was evaluated using the in vitro MTT cytotoxicity assay. Size, zeta potential (ZP), polydispersity index (PDI), entrapment efficiency, in vitro drug release and antimicrobial activity of BCD-OLA-loaded nanovesicles was also evaluated. MD of the BCD-OLA simulation showed that the mechanism responsible for amphiphile formation was through hydrophobic inclusion of OLA in BCD. MTT results showed cell viability of 75-100%, thus affirming biosafety of BCD-OLA complex. TEM images showed the self-assembled structures to be vesicles. The formulated nanovesicles size was shown to be 125.1 ± 8.30 nm with a PDI of 0.231 ± 0.05, and ZP of 19.3 ± 9.20mv. The encapsulation efficiency of vancomycin was 40.2 ± 4.5%. Vancomycin release from the nanovesicles was found to be sustained, with an 80% release over a 48 h period. The in vitro antibacterial test showed that the BCD-OLA had a 2- and 4-fold lower MIC against Staphylococcus aureus (SA) and Methicillin-resistant Staphylococcus aureus (MRSA), respectively, compared to bare vancomycin. Further, intracellular and macrophage studies showed that the system had a 459-fold reduction of intracellular bacteria using infected human embryotic kidney cells (HEK), and an 8-fold reduction in infected macrophages, contrast with bare vancomycin. These discoveries affirmed the potential of the BCD-OLA complex as a promising biosafe effective nanocarrier for antibiotic delivery.

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.

Delivery of novel vancomycin nanoplexes for combating methicillin resistant Staphylococcus aureus (MRSA) infections.

The development of novel antibiotic systems is needed to address the methicillin-resistant Staphylococcus aureus (MRSA) infections. The aim of the study was to explore the novel nanoplex delivery method for vancomycin (VCM) against MRSA using dextran sulfate sodium salt (DXT) as a polyelectrolyte complexing agent. Nanoplexes were formulated by the self-assembling amphiphile polyelectrolyte complexation method and characterized. The size, polydispersity index (PDI), and zeta potential (ZP) of the optimized VCM nanoplexes were 84.6 ±â€¯4.248 nm, 0.449 ±â€¯0.024 and -33.0 ±â€¯4.87 mV respectively, with 90.4 ±â€¯0.77% complexation efficiency (CE %) and 62.3 ±â€¯0.23% drug loading. The in vitro (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)tetrazolium (MTT) studies of the nanoplexes were found to be non-toxic against different mammalian cell lines tested and may confirm its biosafety. While the in vitro drug release studies demonstrated sustained slower release. The in silico study confirmed the spontaneous interaction of VCM with DXT in the presence of sodium chloride. A 6.24-fold enhancement was observed for VCM nanoplexes via in vitro antibacterial studies. Flow-cytometric analysis showed effective cell killing of 67% from VCM nanoplexes compared to 32.98% from the bare vancomycin at the minimum inhibitory concentration (MIC) of 1.25 µg/mL. The in vivo studies using BALB/c mouse skin infection model revealed that nanoplexes reduced MRSA burden by 2.3-folds compared to bare VCM. The novel nanoplexes have potential to be a promising delivery system to combat MRSA infections for improved treatment of bacterial infections.

Grafted hyaluronic acid N-acetyl-l-methionine for targeting of LAT1 receptor: In-silico, synthesis and microscale thermophoresis studies.

Neutral amino acids can be delivered into cells through the l-type amino acid transporter-1 (LAT1), which is a sodium independent transporter. The LAT1 protein is expressed in different tissues, including kidney, blood brain barrier and intestinal wall hence LAT1 can be used as a target in diseases associated with its overexpression. In-silico interactions between different ligands, including methionine (Met), N-acetyl-l-methionine (AcMet), hyaluronic acid (HA), grafted hyaluronic-acid l-methionine (HA-ADH-Met) and a novel grafted hyaluronic acid-N-acetyl-l-methionine (HA-ADH-AcMet), which are at the active site of the LAT1 transporter, were studied and the binding energies calculated. The HA-ADH-AcMet complex demonstrated binding energy and solvation energy of -74.84 and 81.46 kcal/mol, respectively, thus validating its potential to be synthesized. The structural conformation of the HA-ADH-AcMet was confirmed using 1H NMR, FTIR, DSC and PXRD. Microscale thermophoresis was employed to study the binding affinity between the different ligands and LAT1. The binding affinity was expressed in terms of a dissociation constant (Kd), where that of HA-ADH-AcMet was found to be 408 nM which was considered the strongest among the different ligands tested. HA-ADH-AcMet can be used as a targeting moiety for development of medicines to treat different diseases and processes that express LAT1 protein.

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.

Chitosan-Modified Cationic Amino Acid Nanoparticles as a Novel Oral Delivery System for Insulin.

In this study, chitosan-modified basic amino acid derivatives were explored as novel absorption enhancers and nanocarriers for oral insulin delivery. N-Arginine-chitosan (ACS) and N-histidine-chitosan (HCS) were successfully synthesized, and their polyelectrolyte complexes (PECs) with insulin were formed by the ordinary self-assembly method. The obtained PECs exhibited a spherical morphology with a narrow size of 205-303 nm, positive surface charge (ζ potential + 14- + 27 mV) and encapsulation efficiency of approximately 80%. The electrostatic interactions between chitosan derivatives and insulin were confirmed by molecular modeling simulation. In vitro studies demonstrated that PECs could partially protect insulin from proteolysis and degradation at 50 degrees C for at least 6 h. Compared with the insulin solution, internalization of PECs into Caco-2 cells was increased by up to 20.7-fold. Moreover, permeability was enhanced as the degrees of substitution of arginine and histidine increased. The PECs had in vivo pharmacological activities of 2.29%-5.39%, with a significant reduction of blood glucose levels in diabetic rats. These results suggested that ACS and HCS PECs hold promising potential for the oral delivery of insulin, peptides and proteins.

N-octyl-N-arginine-chitosan (OACS) micelles for gambogic acid oral delivery: preparation, characterization and its study on in situ intestinal perfusion.

CONTEXT: Gambogic acid (GA) can inhibit the growth of various cancer cells. However, the low bioavailability caused by insolubility, limits its clinical application. L-arginine is always used with GA to form a complex to obtain the higher solubility. Moreover, guanidyl group from arginine, which can facilitate the cellular uptake, was identified. OBJECTIVE: In this study, L-arginine and chitosan (CS) were used for the first time to prepare N-octyl-N-arginine CS (OACS), a novel amphiphilic carrier for GA with solubility- and absorption-enhancing functions; the characterization of the GA loaded OACS micelles (GA-OACS) and its absorption-enhancing effect were also investigated. MATERIALS AND METHODS: GA-OACS were prepared by the dialysis method. The formed micelles were characterized and evaluated by atomic force microscope (AFM), dynamic light scattering, differential scanning calorimeter (DSC), solubility test, in vitro release and in situ intestinal perfusion. RESULTS: The GA-OACS micelles were successfully prepared attaining a 35.3% drug loading and 82.2% entrapment efficiency. GA-OACS had a homogeneous particle size of 160.3 nm; +21.8 mv zeta potential with smooth continuous surface was observed by using AFM. DSC diagram suggested that GA was encapsulated in the micelles. Meanwhile, GA encapsulated in micelles exhibited a desirable slow release in vitro experiment. The solubility of GA in OACS micelles was increased up to 3.16 ± 0.13 mg/mL, 2320 times than that of free GA. The single pass perfusion showed that the absorption of GA-OACS micelles was enhanced 3.6-fold, 2.1-fold and 2.2-fold for jejunum, ileum and colon, respectively. DISCUSSION AND CONCLUSION: OACS provided excellent ability of drug loading, increasing solubility and enhanced absorption for GA, which indicated that OACS micelles as an oral drug delivery carrier may have potential research and application values.

Micelles of TPGS modified apigenin phospholipid complex for oral administration: preparation, in vitro and in vivo evaluation.

Mixed micelles were designed to increase oral bioavailability of Apigenin (Ap). The phospholipid (Ph) complex technology was exploited alongside TPGS' stabilizing effect by PEG chain sterical hindrance of the phase II enzymes. This prevented extensive metabolism of Ap while inhibiting P-glycoprotein's exocytosis. TPGS modified micelles of Ap-Ph complex (TPGS-Ap-Ph) were prepared by thin film hydration method. Ap-Ph complex was confirmed by FTIR and NMR spectroscopy while Ap, Ph and TPGS interactions were studied by surface tensiometry. TPGS-Ap-Ph micelles achieved 87.35% drug encapsulation and 12.6% drug loading showing spherical morphology 137.1 +/- 3.4 nm particle size and -12.94 mV surface charge. The negative zeta potential confirmed computer simulation predictions that PEG moieties of TPGS were at micelles surface, while hydrophobic part inserted to the phospholipid hydrophobic core by electrostatic interactions. TPGS-Ap-Ph micelles were found to be stable for more than 90 days after lyophilization. Comparing to free drug, the micelles increased intestinal absorption of Ap 2.4 fold, illustrating apparent permeation (P(app)) and absorption constant (K(a)) of 7.9 x 10(-4) and 2.05 x 10(-4) (p < 0.001) respectively. Moreover, cell culture studies showed high cellular uptake with sufficient intracellular trafficking in A549 cells. MTT assays revealed a significant cytotoxic effect by TPGS-Ap-Ph micelles. In vivo, an effective inhibition of 72.9% was achieved upon oral administration to S180 carcinoma mice compared to 19.5% by Ap-Ph complex. Altogether reflect that orally administered mixed micelles of TPGS-Ap-Ph could effectively inhibit cancer. The results present the designed micelles as a new way to improve oral bioavailability of sparingly soluble and poorly absorbed drugs.

Formulation, characterization and pharmacokinetics of Morin hydrate niosomes prepared from various non-ionic surfactants.

Morin hydrate (MH), a bioflavonoid with antioxidant and anticancer activity as well as the ability to improve the bioavailability of other drugs on their concurrent use. Three differently optimized niosomal formulations using three different non-ionic surfactants (Span 60, Span 80 and Tween 60) were achieved by (L9 (3(4))) Taguchi orthogonal array (TOA). The analysis of TOA revealed that Tween 60 Niosomes had the highest entrapment efficiency (93.4%) compared to other optimized Niosomal formulations (71-79%). In terms of MH remaining %, Tween 60 Niosomes were found to be the most stable (89%) at 4 °C over one month compared to Span 60 (56%) and Span 80 (57%) Niosomes. The release pattern in all Niosomal formulations was found to follow the Weibull model and Tween 60 Niosomes had the highest release rate. The molecular modeling simulation explained the binding of MH to the human serum albumin (HSA) by hydrogen bonds during the in vitro release process. As for the bioavailability, the AUC0-8 showed 1.3-2.7 fold increase compared to the MH solution. Ex vivo images of the excised organs showed that MH could accumulate in brain which indicates that MH-Tween 60 Niosomes might be a possible candidate to deliver hydrophobic drugs and overcome the blood-brain barrier (BBB) penetration.

N-octyl-N-Arginine chitosan micelles as an oral delivery system of insulin.

N-octyl-N-Arginine chitosan (OACS) was synthesized in an attempt to combine the permeation enhancing effects of arginine-rich peptides and the drug loading capacity of the amphipathic polymers for insulin oral delivery. OACS self-assembled micelles of insulin were prepared by the conventional stirring technique, which were characterized by Dynamic light scattering, transmission electron microscopy and differential scanning calorimetry. Molecular docking by Discovery studio software confirmed that the interactions between OACS and insulin were mostly electrostatic in nature. In vitro, the result of the degradation experiment by enzyme showed that the OACS has a relative protective effect for insulin from proteolyses. Compared to the insulin solution, OACS micelles increased the Caco-2 cell's internalization by up to 22.3 folds. In vivo, the pharmacological activity PA% of series OACS-insulin micelles ranged from 7.7%-16.8%. Meanwhile by increasing arginine degree of the substitution both the uptake in Caco-2 cells and the hypoglycemic effect in diabetic rats were enhanced. Therefore, it is concluded that using arginine polymeric micelles for the enhancement of oral insulin delivery is a promising approach for the oral peptide delivery.

[Study on intestinal absorption kinetics of gambogic acid in rats].

OBJECTIVE: To investigate the intestinal absorption kinetics of gambogic acid (GA) in rats. METHOD: In situ single-way intestinal perfusion model was established to study the intestinal absorption kinetics of GA in different absorption segments, and the concentration of GA in the perfusate was determined by HPLC. The effect of drug concentrations on intestinal absorption was also detected. RESULT: GA showed a higher absorption rate than other intestinal segments (P < 0.05) and kept unchanged in duodenum after addition in drug concentration. CONCLUSION: GA can be absorbed in all intestinal segments in rats with the higher absorption rate in duodenum. Its mechanism is passive diffusion.

Preparation, physicochemical characteristics and bioavailability studies of an atorvastatin hydroxypropyl-beta-cyclodextrin complex.

The aim of this study was to improve the solubility, stability and bioavailability of amorphous atorvastatin calcium (AT) by complexing it with hydroxypropyl-beta-cyclodextrin. The formation of the inclusion complexation was identified by molecular modeling, phase solubility diagrams, differential scanning calorimetry and X-ray powder diffractometry. Orally Disintegrating Tablets (ODT) were then manufactured by direct compression. Apart from improved stability compared to pure AT, disintegration time of 27s, hardness of 5 kg and favorable mouth feel were achieved. In vitro dissolution tests of the ODT of AT inclusion complex exhibited higher dissolution rates than those with pure drug and the commercial tablet Lipitor. In vivo bioavailability studies in rats also showed shorter T(max), higher C(max) and increased AUC of 4.42 and 1.86 fold compared to the plain drug ODT and Lipitor. These results strongly suggest to use HP-beta-CD to improve the physicochemical characteristics and bioavailability of AT.

Redox-sensitive micelles self-assembled from amphiphilic hyaluronic acid-deoxycholic acid conjugates for targeted intracellular delivery of paclitaxel.

A targeted intracellular delivery system of paclitaxel (PTX) was successfully developed based on redox-sensitive hyaluronic acid-deoxycholic acid (HA-ss-DOCA) conjugates. The conjugates self-assembled into nano-size micelles in aqueous media and exhibited excellent drug-loading capacities (34.1%) and entrapment efficiency (93.2%) for PTX. HA-ss-DOCA micelles were sufficiently stable at simulated normal physiologic condition but fast disassembled in the presence of 20 mm reducing agent, glutathione. In vitro drug release studies showed that the PTX-loaded HA-ss-DOCA micelles accomplished rapid drug release under reducing condition. Intracellular release of fluorescent probe nile red indicated that HA-ss-DOCA micelles provide an effective approach for rapid transport of cargo into the cytoplasm. Enhanced cytotoxicity of PTX-loaded HA-ss-DOCA micelles further confirmed that the sensitive micelles are more potent for intracellular drug delivery as compared to the insensitive control. Based on flow cytometry and confocal microscopic analyses, observations revealed that HA-ss-DOCA micelles were taken up to human breast adenocarcinoma cells (MDA-MB-231) via HA-receptor mediated endocytosis. In vivo investigation of micelles in tumor-bearing mice confirmed that HA-ss-DOCA micelles possessed much higher tumor targeting capacity than the insensitive control. These results suggest that redox-sensitive HA-ss-DOCA micelles hold great potential as targeted intracellular delivery carriers of lipophilic anticancer drugs.