Ahmed Amer

• Amer, Ahmed; Charnock, Colin & Nguyen, Sanko (2025). The impact of surface charge on the interaction of cholesterol-free fusogenic liposomes with planktonic microbial cells and biofilms. International Journal of Pharmaceutics. ISSN 0378-5173. 669. doi: 10.1016/j.ijpharm.2024.125088. Full text in Research Archive Show summary

  This study focused on the development of cholesterol-free fusogenic liposomes with different surface charge with the aim of improving biofilm penetration. In vitro assessments of the liposomes included physical stability, biocompatibility, fusion with microbial cells, and the ability to penetrate established biofilms. Using dynamic light scattering, cholesterol-free, fusogenic liposomes were found to be < 200 nm in size with small size distribution (PDI < 0.1) and physically stable for a year when stored at 4◦C. Transmission electron microscopy (TEM) images confirmed vesicular sizes for selected liposomal formulations. Liposomal ability to fuse with microbial cells was assessed using lipid mixing and flow cytometer assays. Fusion levels were found to be higher with Escherichia coli compared to Staphylococcus aureus and Candida albicans regardless of the liposomal charge. Neutral liposomes exhibited highest fusion, followed by cationic and anionic liposomes, respectively. Our investigations demonstrated that fusion is a multifactorial process influenced by the chemical composition of the liposomes, the liposomal surface charge, and components of the microbial cell envelope. Penetration and retention within preformed S. aureus biofilms were assessed for liposomes with various surface charges. All liposomes, regardless of surface charge, were capable of penetrating and diffusing through the biofilm matrix. However, cationic liposomes displayed greatest interaction and retention. Biocompatibility was confirmed through haemolysis and cytotoxicity studies. The cholesterol-free fusogenic liposomes developed in this study demonstrated promising potential as drug delivery systems for incorporating antimicrobial agents for biofilm treatment.

• Amer, Ahmed; Charnock, Colin; Ovchinnikov, Kirill V.; Thorstensen, Tage & Nguyen, Sanko (2025). Phospholipid acyl chain length modulation: a strategy to enhance liposomal drug delivery of the hydrophobic bacteriocin Micrococcin P1 to biofilms. European Journal of Pharmaceutical Sciences. ISSN 0928-0987. 211. doi: 10.1016/j.ejps.2025.107149. Full text in Research Archive Show summary

  This study describes the development of fusogenic liposomes as a drug delivery system for the hydrophobic antimicrobial peptide micrococcin P1 (MP1). The liposomes were formulated using phospholipids with varying acyl chain lengths, with the goal of improving biofilm eradication. Entrapment of MP1 in liposomes effectively improved its stability in solution, as demonstrated by liquid chromatography-mass spectrometry monitoring over a two-month period. Liposomal entrapment lowered the minimum inhibitory concentration of MP1 against several Staphylococcus aureus strains, including clinical isolates, by 4- to 16-folds. Increasing the phospholipid acyl chain length (16-carbon to 20-carbon) in the liposomal composition, resulted not only in an improved entrapment of MP1, but also higher antibiofilm activity. Confocal laser scanning microscopy imaging revealed that the MP1-loaded liposomal effect was likely due to disruption of the biofilm matrix. At a concentration of 0.25 µg/mL, MP1 loaded in 1,2-diarachidoyl-sn‑glycero-3-phosphocholine (DAPC)-based fusogenic liposomes reduced biofilm cell viability by approximately 55 %, compared to only 15 % with free MP1 equivalents. However, the increased liposomal bilayer hydrophobicity via the longer acyl chains compromised the physical stability of the fusogenic liposomes. While MP1-loaded liposomes based on the shorter 16-carbon acyl chain 1,2-dipalmitoyl-sn‑glycero-3-phosphocholine (DPPC) remained stable for two months, the DAPC liposomes were only stable for two weeks. The physical stability was improved by increasing the concentration of the cationic phospholipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), from 25 mol % to 50 mol % in the liposomal composition. Overall, these findings highlight the potential of liposomal systems for delivering hydrophobic peptides like MP1 to Staphylococcus aureus biofilms, offering promise for improving the treatment of biofilm-associated infections.

• Amer, Ahmed; Naqvi, Maria & Charnock, Colin (2024). Genomics of Staphylococcus aureus and Enterococcus faecalis isolated from the ocular surface of dry eye disease sufferers. Experimental Eye Research. ISSN 0014-4835. 248. doi: 10.1016/j.exer.2024.110071. Full text in Research Archive Show summary

  Ocular surface inflammatory disorders, such as dry eye, are becoming increasingly prevalent. Developing new treatment strategies targeting harmful bacteria could provide significant therapeutic benefits. The purpose of this study was to characterize the common ocular pathogen Staphylococcus aureus and the rarer endophthalmitis- associated species Enterococcus faecalis isolated from the ocular surface of dry eye disease patients in Norway. Together the 7 isolates (5 S. aureus and 2 E. faecalis) comprise the complete set of members of each species isolated in our previous study of the ocular microbiome of 61 dry eye sufferers. We aimed to investigate the pathogenic potential of these isolates in relation to ocular surface health. To this end, we used whole genome sequencing, multiplex PCR directed at virulence genes and antibiotic susceptibility tests encompassing clinically relevant agents. The E. faecalis isolates showed resistance to only gentamicin. S. aureus isolates displayed susceptibility to most of the tested antibiotics, except for two isolates which showed resistance to trimethoprim/ sulfamethoxazole and three isolates which were resistant to ampicillin. Susceptibilities included sensitivity to several first-line antibiotics for treatment of ocular infections by these species. Thus, treatment options would be available if required. However, spontaneous resistance development to gentamicin and rifampicin occurred in some S. aureus which could be a cause for concern. Whole genome sequencing of the isolates showed genome sizes ranging from 2.74 to 2.83 Mbp for S. aureus and 2.86 Mbp for E. faecalis, which is typical for these species. Multilocus sequence typing and phylogenetic comparisons with previously published genomes, did not suggest the presence of eye-specific clusters for either species. Genomic analysis indicated a high probability of pathogenicity among all isolates included in the study. Resistome analysis revealed the presence of the beta-lactamase blaZ gene in all S. aureus isolates and the dfrG gene in two of them; while E. faecalis isolates carried the lsa(A) gene which confers intrinsic resistance to lincosamides and streptogramin A in this species. Screening for virulence factors revealed the presence of various pathogenicity associated genes in both S. aureus and E. faecalis isolates. These included genes coding for toxin production and factors associated with evading the host immune system. Some of the identified genes (tst, hylA & hylB) are suggested to be linked to the pathophysiology of dry eye disease. Lastly, the presence of specific S. aureus virulence genes was confirmed through multiplex PCR analysis.

• Amer, Ahmed; Charnock, Colin & Nguyen, Sanko (2024). Novel Teixobactin Analogues Show Promising In Vitro Activity on Biofilm Formation by Staphylococcus aureus and Enterococcus faecalis. Current Microbiology. ISSN 0343-8651. 81(10). doi: 10.1007/s00284-024-03857-9. Full text in Research Archive Show summary

  The treatment of infections caused by biofilm-forming organisms is challenging. The newly discovered antibiotic teixobactin shows activity against a wide range of biofilm-forming bacteria. However, the laborious and low-yield chemical synthesis of teixobactin complicates its further development for clinical application. The use of more easily synthesized teixobactin analogues may offer promise in this regard. In this article, three newly developed analogues were tested for efficacy against Staphylococcus aureus and Enterococcus faecalis. Minimum inhibitory and -bactericidal concentrations were investigated. MIC values for S. aureus and E. faecalis ranged from 0.5–2 and 2–4 μg/mL, respectively. Moreover, the ability of the analogues to prevent biofilm formation and to inactivate bacterial cells in already established S. aureus biofilm on medical grade materials (PVC and PTFE) used in the production of infusion tubing and catheters were also tested. The analogues showed an ability to prevent biofilm formation and inactivate bacterial cells in established biofilms at concentrations as low as 1–2 μg/mL. Confocal laser scanning microscopy showed that the most promising analogue (TB3) inactivated S. aureus cells in a preformed biofilm and gave a reduction in biovolume. The relative ease of synthesis of the analogues and their in vitro efficacy, makes them promising candidates for pharmaceutical development.

• Eassa, Heba A.; Helal, Nada A.; Amer, Ahmed M.; Fouad, Aliaa; Bedair, Asser F. & Nagib, Reem [Show all 12 contributors for this article] (2022). 3D-Printed Microfluidics Potential in Combating Future and Current Pandemics (COVID-19). Recent advances in drug delivery and formulation. ISSN 2667-3878. doi: 10.2174/2667387816666220727101214. Full text in Research Archive Show summary

  Coronavirus disease (COVID-19) emerged in China in December 2019. In March 2020, the WHO declared it a pandemic leading to worldwide lockdowns and travel restrictions. By May, it infected 4,789,205 and killed 318,789 people. This led to severe shortages in the medical sector besides devastating socio-economic effects. Many technologies such as artificial intelligence (AI), virtual reality (VR), microfluidics, 3D printing, and 3D scanning can step into contain the virus and hinder its extensive spread. This article aims to explore the potentials of 3D printing and microfluidic in accelerating the diagnosis and monitoring of the disease and fulfilling the shortages of personal protective equipment (PPE) and medical equipment. It highlights the main applications of 3D printers and microfluidics in providing PPE (masks, respirators, face shields, goggles, and isolation chambers/hoods), supportive care (respiratory equipment) and diagnostic supplies (sampling swabs &amp; lab-on-chip) to ease the COVID-19 pressures. Also, the cost of such technology and regulation considerations are addressed. We conclude that 3D printing provided reusable and low-cost solutions to mitigate the shortages. However, safety, sterility, and compatibility with environmental protection standards need to be guaranteed through standardization and assessment by regulatory bodies. Finally, lessons learned from this pandemic can also help the world prepare for upcoming outbreaks.

• Amer, Ahmed M.; Allam, Ahmed N. & Abdallah, Ossama Y. (2019). Preparation, characterization and ex vivo–in vivo assessment of candesartan cilexetil nanocrystals via solid dispersion technique using an alkaline esterase activator carrier. Drug Development and Industrial Pharmacy. ISSN 0363-9045. 45(7), p. 1148–1148. doi: 10.1080/03639045.2019.1600533. Full text in Research Archive Show summary

  The objective of this study was to improve candesartan cilexetil (CC) efficacy by formulating nanocrystals via solid dispersion (SD) technique using tromethamine (Tris). SD was prepared by solvent evaporation at different drug carrier ratios, evaluated for particle size, vitro dissolution studies, TEM, FTIR, and X-ray powder diffraction. Ex vivo, in vivo pharmacokinetic parameters were conducted on selected formulae compared to drug suspension and marketed product. Size analysis demonstrated formation of particles in the nanorange lower than 300 nm. A burst drug release followed by an improved dissolution was observed indicating instant formation of nanocrystals along with amorphization as confirmed by X-ray diffraction. FTIR studies suggested the absence of chemical interaction between Tris and CC. TEM revealed formation of irregular oval nanoparticles. SD-1:5 has higher apparent permeability coefficient compared to CC suspension. Furthermore, the pharmacokinetic results proved the ability of the formed nanoparticles to enhance the efficacy of CC compared to drug suspension and marketed product. In conclusion, using of Tris as alkaline esterase activator carrier could be a promising tool to bypass the controversial effect of esterase enzymes that may be a source for inter-individual variations affecting ester prodrug candidates’ efficacy.

• Helal, Nada A; Elnoweam, Ola; Eassa, Heba Abdullah; Amer, Ahmed M.; Eltokhy, Mohamed Ashraf & Helal, Mohamed A [Show all 8 contributors for this article] (2019). Integrated Continuous Manufacturing in Pharmaceutical Industry: Current Evolutionary Steps Toward Revolutionary Future. Pharmaceutical Patent Analyst. 8(4), p. 161–161. doi: 10.4155/ppa-2019-0011. Full text in Research Archive Show summary

  Continuous manufacturing (CM) has the potential to provide pharmaceutical products with better quality, improved yield and with reduced cost and time. Moreover, ease of scale-up, small manufacturing footprint and on-line/in-line monitoring and control of the process are other merits for CM. Regulating authorities are supporting the adoption of CM by pharmaceutical manufacturers through issuing proper guidelines. However, implementation of this technology in pharmaceutical industry is encountered by a number of challenges regarding the process development and quality assurance. This article provides a background on the implementation of CM in pharmaceutical industry, literature survey of the most recent state-of-the-art technologies and critically discussing the encountered challenges and its future prospective in pharmaceutical industry.

• Helal, Nada A.; Eassa, Heba A.; Amer, Ahmed M.; Eltokhy, Mohamed A.; Edafiogho, Ivan & Nounou, Mohamed I. (2019). Nutraceuticals’ Novel Formulations: The Good, the Bad, the Unknown and Patents Involved. Recent Patents on Drug Delivery and Formulation. doi: 10.2174/1872211313666190503112040. Full text in Research Archive Show summary

  : Traditional nutraceuticals and cosmeceuticals hold pragmatic nature with respect to their definitions, claims, purposes and marketing strategies. Their definitions are not well established worldwide. They also have different regulatory definitions and registration regulatory processes in different parts of the world. Global prevalence of nutraceuticals and cosmeceuticals is noticeably high with large market share with minimal regulation compared to traditional drugs. The global market is flooded with nutraceuticals and cosmeceuticals claiming to be of natural origin and sold with a therapeutic claim by major online retail stores such as Amazon and eBay. Apart from the traditional formulations, many manufacturers and researchers use novel formulation technologies in nutraceutical and cosmeceutical formulations for different reasons and objectives. Manufacturers tend to differentiate their products with novel formulations to increase market appeal and sales. On the other hand, researchers use novel strategies to enhance nutraceuticals and cosmeceuticals activity and safety. : The objective of this review is to assess the current patents and research adopting novel formulation strategies in nutraceuticals and cosmeceuticals. Patents and research papers investigating nutraceutical and cosmeceutical novel formulations were surveyed for the past 15 years. Various nanosystems and advanced biotechnology systems have been introduced to improve the therapeutic efficacy, safety and market appeal of nutraceuticals and cosmeceuticals, including liposomes, polymeric micelles, quantum dots, nanoparticles, and dendrimers. This review provides an overview of nutraceuticals and cosmeceuticals current technologies, highlighting their pros, cons, misconceptions, regulatory definitions and market. This review also aims in separating the science from fiction in the nutraceuticals and cosmeceuticals development, research and marketing.

• Amer, Ahmed M.; Allam, Ahmed N. & Abdallah, Ossama Y. (2018). Evaluation of the Discriminatory Power of USP Dissolution Method for Candesartan Cilexetil Tablets through Testing of Marketed Products in Egypt. Dissolution Technologies. 25(4), p. 46–46. doi: 10.14227/dt250418p40. Full text in Research Archive Show summary

  The development of dissolution testing conditions for drugs exhibiting low aqueous solubility like candesartan cilexetil is a challenging task for pharmaceutical scientists and regulatory organizations. The purpose of this study was to evaluate the discriminatory power of the dissolution testing medium specified by USP, which is 0.35% polysorbate 20 in 0.05 M potassium dihydrogen phosphate pH 6.5, for 4-, 8-, and 16-mg candesartan cilexetil tablets. We analyzed the drug release pattern and similarity factors of some generic products marketed in Egypt in comparison to the reference product (Atacand). It was found that, despite the ability of the method to effectively discriminate between the different 16-mg tablets, it failed to demonstrate any discriminative ability of the 4- and 8-mg doses without any formula alteration. Therefore, an attempt was made to develop a new medium for the 4- and 8-mg doses that could provide a reasonable sink condition and discriminatory power by reducing the percentage of polysorbate 20 in the medium from 0.35% to 0.1%. The alternative dissolution medium demonstrated an acceptable sink condition and satisfactory discrimination power between the different marketed products, suggesting the use of this medium (0.1% polysorbate 20 in 0.05 M potassium dihydrogen phosphate, pH 6.5) to aid in product development and testing of finished product quality.

• Amer, Ahmed M.; Allam, Ahmed N. & Abdallah, Ossama Y. (2017). Comparative Pharmaceutical Evaluation of Candesartan and Candesartan Cilexetil: Physicochemical Properties, In Vitro Dissolution and Ex Vivo In Vivo Studies. AAPS PharmSciTech. 19(2), p. 667–667. doi: 10.1208/s12249-017-0879-x. Full text in Research Archive Show summary

  The aim of the present work is to answer the question is it possible to replace the ester prodrug candesartan cilexetil (CC) by its active metabolite candesartan (C) to bypass the in vivo variable effect of esterase enzymes. A comparative physicochemical evaluation was conducted through solubility, dissolution, and stability studies; additionally, ex vivo permeation and in vivo studies were assessed. C demonstrated higher solubility over CC at alkaline pH. Moreover, dissolution testing using the pharmacopeial method showed better release profile of C even in the absence of surfactant in the testing medium. Both drugs demonstrated a slight degradation in acidic pH after short-term stability. Instead, shifting to alkaline pH of 6.5 and 7.4 showed superiority of C solution stability compared to CC solution. The ex vivo permeation results demonstrated that the parent compound C has a significant (P < 0.05) enhanced permeation compared to its prodrug from CC, that agreed with in vivo results in which C suspension reached significantly (P < 0.05) higher C max of 1.39 ± 0.59 μg/mL at T max of 0.66 ± 0.11 h, while CC suspension reached C max of 0.47 ± 0.22 μg/mL at T max of 2.00 ± 0.27 h, a lag period of 40 min is needed prior to detection of any absorbed CC in plasma. Those findings are not in agreement with the previously reported rationale on the prodrug formation owing to the poor permeability of the parent compound, suggesting the possibility of marketing the parent drug candesartan for clinical use similarly to azilsartan and its prodrug.

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• Amer, Ahmed; Nguyen, Sanko & Charnock, Colin (2025). Antibiofilm Activity of Novel Antimicrobial Peptides and Liposomal Encapsulation as a Promising Approach to Enhance Their Efficacy. OsloMet - storbyuniversitetet. ISSN 9788283646955. Full text in Research Archive Show summary

  Biofilms are structured communities of microorganisms embedded within a self-produced extracellular matrix. Biofilm-associated infections are difficult to treat, as the extracellular matrix acts as both a physical and chemical barrier that hinders the penetration of antimicrobial agents. Treatment challenges are further compounded by the emergence of antimicrobial resistant strains. Together, these factors highlight the urgent need for novel antimicrobial agents suitable for clinical use, as well as effective delivery systems capable of targeting microbes within the biofilm. Antimicrobial peptides (AMPs) offer a promising alternative due to their diverse mechanisms of action and ability to target drug-resistant microbes. However, their clinical application is hindered by suboptimal physicochemical and pharmacokinetic properties. This thesis explores the antimicrobial and antibiofilm activity of novel AMPs, namely teixobactin analogues and micrococcin P1 (MP1), and liposomal encapsulation as a strategy to enhance their delivery and efficacy against biofilm-forming pathogens. Initially, cholesterol-free fusogenic liposomes with varying surface charges were developed, and their ability to fuse with microbial cells and penetrate the biofilm matrix was assessed. All liposomal formulations demonstrated fusion with different microbial cells, highlighting their potential for AMP delivery. Moreover, cationic liposomes showed the strongest interaction and retention within biofilms. Subsequently, MP1 was incorporated into cholesterol-free fusogenic liposomes formulated using phospholipids with varying acyl chain lengths. Liposomes with longest acyl chains exhibited higher MP1 entrapment efficiency and enhanced the peptide’s antimicrobial and antibiofilm activity. Furthermore, MP1-loaded liposomes effectively disrupted Staphylococcus aureus biofilms formed on biomedical surfaces. Finally, this thesis evaluated the antimicrobial and antibiofilm activities of three teixobactin analogues. The natural teixobactin compound represents a novel class of antibiotics with a unique mechanism of action and has attracted considerable interest as a potential drug candidate. While all three analogues demonstrated promising efficacy against biofilm-forming pathogens, one showed comparatively stronger effects and is proposed as a lead candidate for further liposomal encapsulation. In conclusion, this work highlights the potential of fusogenic liposomes as an advanced delivery system for novel AMPs targeting biofilms, offering a promising strategy for combating biofilmassociated infections. Biofilmer er strukturerte samfunn av mikroorganismer som er omsluttet i en egenprodusert ekstracellulær matriks. Infeksjoner assosiert med biofilm er utfordrende å behandle, ettersom den ekstracellulære matriksen fungerer både som en fysisk og kjemisk barriere som hemmer penetrasjon av antimikrobielle midler. Behandlingsutfordringene forsterkes ytterligere av fremveksten av antimikrobiell resistens. Samlet sett understreker disse faktorene det akutte behovet for nye antimikrobielle midler som egner seg for klinisk bruk, samt effektive leveringssystemer som kan målstyres mot mikrober inni biofilmen. Antimikrobielle peptider (AMP-er) representerer et lovende alternativ grunnet deres varierte virkningsmekanismer og evne til å angripe resistente mikroorganismer. Deres kliniske anvendelse begrenses imidlertid av suboptimale fysikalskkjemiske og farmakokinetiske egenskaper. Denne avhandlingen undersøker den antimikrobielle og antibiofilmaktiviteten til nye AMP-er, nærmere bestemt teixobactin-analoger og micrococcin P1 (MP1), samt liposomal innkapsling som en strategi for å forbedre leveringen og effekten deres mot biofilmdannende patogener. Innledningsvis ble det utviklet kolesterolfrie fusogene liposomer med ulik overflateladning, og deres evne til å fusjonere med mikrobielle celler og penetrere biofilmmatrisen ble evaluert. Alle liposomformuleringene demonstrerte fusjon med ulike mikrobielle celler, noe som understreker deres potensiale som legemiddelleveringssystem for AMP-er. Kationiske liposomer utviste sterkest interaksjon med og retensjon i biofilmen. Deretter ble MP1 innkapslet i kolesterolfrie fusogene liposomer formulert med fosfolipider med varierende acylkjedelengde. Liposomer med lengstacylkjeder viste høyere innkapslingseffektivitet for MP1 og forbedret både den antimikrobielle og antibiofilmaktiviteten til peptidet. Videre viste MP1-liposomer effektiv forstyrrelse av biofilmer dannet av Staphylococcus aureus på biomedisinske overflater. I denne avhandlingen ble den antimikrobielle og antibiofilmaktiviteten til tre teixobactin-analoger også evaluert. Det naturlige stoffet teixobactin? representerer en ny klasse antibiotika med en unik virkningsmekanisme og har vakt betydelig interesse som lovende legemiddelkandidat. Alle tre analogene viste lovende effekt mot biofilmdannende patogener, men én av dem utpekte seg med relativ sterkere effekt og foreslås som en mulig kandidat for videre formulering med liposomer. Dette arbeidet har løftet frem potensialet til fusogene liposomer som et avansert legemiddelleveringssystem for nye AMP-er rettet mot biofilmer. Dette kan væreen lovende strategi for å bekjempe biofilmassosierte infeksjoner.

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