Understanding the mechanistic uptake and exploring the applications of novel amino acid based ion pair complexes

Student thesis: Doctoral ThesisDoctor of Philosophy


High throughput screening has resulted in a significant increase in the rate at which new chemical entities are discovered. However, major hurdles such as poor solubility, permeability and efflux limit their application. Not only is it difficult to get a drug into the body from the most convenient routes, once inside, challenges such as increasing resistance to drugs in cancer cells, fungi, bacteria and viruses present significant drug delivery challenges. To address these challenges, we have investigated the role and application of amino acids for solubility enhancement and in increasing drug uptake. Tetracycline (TC), ciprofloxacin (Cip) and digoxin were chosen as model drugs along with acidic and basic amino acids as counterions.
Saturated solubility was studied in different concentrations of the acidic (L-aspartic acid and L-glutamic acid) and basic (L-arginine, L-lysine and L-histidine) amino acids. We have shown that solubility of both TC and digoxin can be enhanced using amino acids, with the extent depending on the ionisation ability of the solution. Whilst both acidic and basic amino acids led to increased TC solubility, acidic amino acids were considered suitable counterions due better stability of TC in solution. Likewise, basic amino acids were determined to be suitable for digoxin compared to acidic since the latter led to drug degradation. Permeability of digoxin was studied using caco-2 cell line. Basic amino acids L-arginine and L-lysine led to a decrease in efflux ratio of digoxin. In parallel, the same counterions enhanced the permeability of digoxin through caco-2 monolayers via mechanisms which are likely to involve tight junction modulation triggered para-cellular transport, transcellular transport through ion-pair formation or reduced efflux thorough P-glycoprotein inhibition.
Use of amino acids as antimicrobial agents on their own as well as counterions to antibiotics, in a bid to investigate the potential optimisation of antibiotic activity, was investigated in planktonic cells as well as biofilms. For this, accumulation assays using ethidium bromide as an efflux competitor were done, showing that acidic L-amino acids were able to enhance both TC and Cip accumulation within Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). This translated into specific combinations of the amino acids and the antibiotics exhibiting synergistic hindrance in the growth of both bacteria. Pyocyanin and pyoverdine pigment production was also inhibited in P. aeruginosa; both with amino acids alone and synergistically when the amino acids were combined with the antibiotics. Antimicrobial activity of acidic D-amino acids was also investigated on S. aureus biofilms, specifically on their effect on biofilm density (both D- and L-amino acids), colony forming units (CFU), rate of cell attachment to surface and biofilm architecture. A concentration dependent ability to inhibit S. aureus biofilm formation and disperse already formed ones was observed with these amino acids; effecting both biofilm density and CFU. Synergy with Cip in the anti-biofilm activity of acidic amino acids was witnessed at specific combinations. Most interestingly, confocal imaging with the nucleic acid dyes SYTO 9 and propidium iodide, revealed that the amino acids disrupt the honeycomb-like extracellular DNA (eDNA) meshwork whilst also preventing its formation; a finding which was not observed with the use of Cip on its own. It is likely that the acidic amino acids act through mechanisms which modulate the acid base interactions involved in anchoring eDNA to cells. Being very non-specific, mechanisms such as modulation of acid-base interactions or ionic interactions may hold a key in evading evolutionary challenges, since overcoming these would require, if at all possible, drastic changes within bacteria. Our work has concluded that the amino acids present a wide functionality, ranging from solubility and permeability enhancement to overcoming antimicrobial resistance.
Date of Award2020
Original languageEnglish
SupervisorAfzal-Ur-Rahman Mohammed (Supervisor), Ayesha S Rahman (Supervisor), Yvonne Perrie (Supervisor) & Majad Hussain (Supervisor)


  • solubility
  • permeability
  • efflux
  • antimicrobial resistance
  • biofilms

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