Declaration 2 Publications
4.4 Abstract
Vancomycin (VCM) loaded liposomes with Oleic acid based ‘On’ and ‘Off’” pH responsive switches for infection site and intracellular bacteria targeting were formulated using thin layer rehydration technic and found to have size, of 98.88±01.92 at pH 7.4. They showed surface charge switching from negative at pH 7.4 to positive charge at acidic pH accompanied by faster drug release at pH 6.0. Molecular dynamic studies of lipids forming the switches showed of spontaneous opening and closing of the gates at protonated and deprotonated states. Liposomes had 4-fold lower minimum inhibitory concentration (MIC) at pH 7.4 and 8- and 16-fold lower MICs at pH 6.0 compared to bare VCM against both Methicillin susceptible (MSSA) and resistant Staphylococcus aureus (MRSA) respectively. When tested for the intracellular infection in TPH-1 macrophage and HEK293 cells the liposome had a 1266.67- and 704.33- fold reduction in the intracellular MRSA respectively. In vivo studies showed a 189.67 and 6.33-fold lower MRSA burden from mice treated with formulations than the untreated and bare VCM treated groups respectively.
These studies demonstrated that the ‘On’ and ‘Off’ pH responsive liposomes enhanced the activity and targeted delivery of the loaded drug.
Keywords: Oleic acid, liposomes, pH response, vancomycin, intracellular infection.
4.5 Introduction
The success of antibiotics since the middle of the 20th century to manage infectious diseases has been immense, however bacterial infections continue to cause significant challenges worldwide [1]. Since the discovery of antibiotics, resistant bacterial strains have emerged against almost all the introduced newly antibiotics within a short period of time. [2, 3]. This is further compounded by the drying up of the antibiotic pipeline [4] and a short half-life between the introduction of new antibiotics and the development of resistant strains [5]. There is therefore a need for approaches to safeguard and enhance antibacterial activity of the existing antibiotics to greatly extend the period between the introduction of antibiotics and the development of resistance.
Since their introduction, antibiotics have been delivered via conventional dosage forms.
Limitations of conventional dosage forms, such as sub-lethal concentrations at the infection site, lack of targeting that results in exposure to uninfected sites, elimination of the normal flora and the inability to extend the half-life of drugs, have been well documented [6-9]. Although antimicrobial resistance is a multifaced problem, the limitations of conventional dosage forms have been one of the underlining contributors to antimicrobial resistance. Recently, researchers have focused on the development of novel strategies, such as nano based drug delivery systems, which are showing potential in overcoming the limitations of conventional dosage forms while protecting the current antibiotics in the market by enhancing their activity and overcoming the resistance mechanisms of bacteria [10, 11].
Liposomes are one of the most widely used nano drug delivery systems, particularly for the delivery of antibiotics, due to their attractive attributes, such as membrane fusogenic ability [12-
14], intracellular delivery and lowering toxicity of antibiotics [15-17]. They are usually composed of phospholipids, which assemble to form lipid bilayers vesicles with an aqueous core [18]. Drugs with different lipophilicities can be encapsulated into liposomes, which i.e. lipophilic
drugs can be entrapped in the lipid bilayer, while hydrophilic drugs can be encapsulated in the aqueous core. However, depending on the log P of the drug, it can partition between the lipid and aqueous phases [19]. Due to the versatility in the formulation of liposomes, stimuli- responsive biomaterials can be incorporated in them for the site-specific delivery of drugs.
Liposomes have been engineered to possess distinctive properties, such as long systemic circulation, to target specific cells and receptors, and to respond to various stimuli, such as environmental pH and redox, and changes in temperature [20, 21].
pH is one of the common biomarkers for a number of diseases, such as Huntington's Disease
[22], cancer [23], diabetes [24] and bacterial infections [25]. Nano drug delivery systems have been devised to achieve the programmable release of drugs due to pH changes at a target disease site with great success [26, 27] via micelles [28, 29], polymersomes[30, 31], dendrimer based systems[32,
33], peptides [34, 35] and liposomes [36, 37]. A survey of the literature shows that most of the reports of pH responsive liposomes have been in the field of cancer. However, there are limited reports regarding pH responsive liposomes to deliver antibiotics. The reported pH responsive liposomal systems for antibiotics delivery have been for gentamycin, [38] which employed phospholipid-cholesterol hemisuccinate as a pH responsive material, and for vancomycin, where intramolecular protonation and deprotonation of zwitterionic lipids were responsible for pH response, as reported by our group and other researchers [39]. With the current global crisis of antibiotic resistance, delivering antibiotics with pH responsive liposomes could prove to be a valuable tool. There is therefore a need for more studies to evaluate pH responsive systems for the targeted delivery of antibiotics.
Treatment of Staphylococcus aureus infections is often problematic due to the slow response to therapy and the high frequency of infection recurrence [40]. The intracellular persistence of staphylococci has been recognized as the reason for chronic and recurrence infections.
Moreover, it is associated with a reduced expression of virulence factors that are important for
acute infection, which leads to the persistent and long-term survival of pathogens within their hosts. This intracellular reservoir is linked with treatment difficulties, such as a slow response to antibiotic treatment, an extended duration of antimicrobial therapy and treatment failure [41]. The presence of intracellular bacteria could offer a slow response or an inability of the antibiotics to clear this reservoir, as the bacteria might be shielded from the effects of antibiotics that have low intracellular penetration [42]. Moreover, bacteria have shown to localize the endosomes and phagolysosomes of the cells that are acidic and making them their reservoirs
[43] which forms small colony variants in persistent infections (SCVs). Therefore, designing systems with pH stimuli responsive and having cell wall penetration could prove to be useful in eliminating the SCVs.
Compared to those reported in the literature so far, we herein report a liposome with a different mechanism of pH responsiveness by inserting ‘On’ and ‘Off’ switches within the bilayer membrane. The liposomes will be incorporated with a newly designed oleic acid derived quaternary lipid (QL) and parent Oleic acid (OA) as pH responsive components for site specific antibiotic delivery. We envisage that the novel QL can form a supra-molecular complex with OA in the lipid bilayer membrane of the liposome. Depending on the pH environment, the formed complex will form an “On and Off” switch to release the drug from the liposome. Under basic pH, the oleic acid will deprotonate and acquire a negative charge, thus forming a supramolecular complex with the quaternary nitrogen in the novel QL molecules (Off position of the liposome), and the negative charge will dominate, while in an acidic environment, oleic acid will be protonated, losing its charge, which will result in a slight repulsion between the positively charged QL and rearrangements (On position of the liposome) and the net charge of the liposomes will be positive. Thus, surface charge switching and increased drug release will be expected in an acidic pH, which will increase targeted drug release at the infection site,
while the positive charge will also potentiate the binding of the liposome to the negatively charged bacterial membrane.
To the best of our knowledge, this is the first report on delivery of antibiotics using liposomes having a pH triggered dependent “On and Off” switches. This kind of trigger mechanism can be of a better response and more sensitive in the acidic environment due to the noncovalent bond between the QL lipid and OA. This paper also reports, for the first time, on the delivery of any class of drug with the synthesized QL lipid, and the delivery of antibiotics with acid sensitive smart switches liposomes. This work could assist in helping to solve the current global antimicrobial crisis and SCVs, as the synthesized material will widen the pool of available material for drug delivery. The results and succinct discussions obtained from the synthesis of the QL lipid, in vitro, in silico and in vivo findings from the formulated liposome are herein reported in this paper.
4.6. Materials and Methods