Abdelwahab Omri

The Effect of Different Lipid Components on the In Vitro Stability and Release Kinetics of Liposome Formulations

Liposomes are colloidal carriers that form when certain (phospho)lipid molecules are hydrated in an aqueous media with some energy input. The ideal liposome formulation with optimum stability will improve drug delivery by decreasing the required dose and increasing the efficacy of the entrapped drug at the target organ or tissue. The most important parameter of interest in this article was to compare the efficacy of three different liposomes formulated with DSPC, DMPC, and DPPC, all saturated neutral phospholipids with different acyl chain lengths and transition temperatures. DMPC has a phase transition temperature (Tc) below 37°C, whereas the other two phospholipids possess Tcs above the physiological temperature. These lipids were then added to a cholesterol concentration of 21% to optimize the stability of the vesicles. The liposomes were prepared by a sonication and incubated in phosphate buffered saline (PBS) at 4°C and 37°C. The encapsulation efficiency, initial size, and drug retention of the vesicles were tested over a 48-hr period employing radiolabeled inulin as a model drug. The phase transition temperature of liposomes, which depends on the Tc of the constituent lipids, was an important factor in liposome stability. Of all the liposomes tested, the greatest encapsulation efficiency was found for the DSPC liposomes (2.95%) that also had the greatest drug retention over 48 hr at both 4°C (87.1 ± 6.8%) and 37°C (85.2 ± 10.1%), none of these values being significantly different from time zero. The lowest drug retention was found for DMPC liposomes for which a significant difference in drug retention was seen after only 15 min at both 4°C (47.3 ± 6.9%) and 37°C (53.8 ± 4.3%). The DPPC liposomes showed a significant difference in drug retention after 3 hr at 4°C (62.1 ± 8.2%) and after 24 hr at 37°C (60.8 ± 8.9%). Following the initial drop at certain time intervals a plateau was reached for all of the liposome formulations after which no significant difference in drug retention was observed. In conclusion, liposomes with higher transition temperatures appear to be more stable in PBS either at 4°C or 37°C, indicating that the increase in acyl chain length (and therefore transition temperature) is directly proportional to stability.

Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa

OBJECTIVES This study evaluated the potential of DNase, alginate lyase (AlgL) and N-acetylcysteine (NAC) in enhancing the in vitro bactericidal activity of conventional (free) and vesicle-entrapped (liposomal) gentamicin, amikacin and tobramycin. METHODS The MICs and biofilm eradication for two clinical isolates of Pseudomonas aeruginosa (a mucoid strain and a non-mucoid strain) were determined in the presence and absence of AlgL. The co-activity of aminoglycosides with DNase and/or AlgL against endogenous P. aeruginosa in cystic fibrosis (CF) sputum was also measured. The inhibitory effects of mucin in the presence and absence of the mucolytic agent NAC on aminoglycosidic activity were also examined. RESULTS The MIC values of the liposomal aminoglycosides were similar to or lower than those of free aminoglycosides. Biofilm formation increased the bactericidal concentrations of these drugs by 8- to 256-fold and treatment with AlgL improved killing of the mucoid strain. The activity of some aminoglycosides against the sputum was increased by the addition of DNase or AlgL (P < 0.05), and was increasingly evident with concurrent DNase and AlgL administration. Addition of mucin inhibited liposomal aminoglycosidic activity (up to 32-fold) evidently more than the free aminoglycosides (up to 8-fold). The addition of NAC did not improve activity significantly (P > 0.05). Tobramycin was the most effective aminoglycoside to reduce biofilms and sputum. CONCLUSIONS Liposomal aminoglycosides do not fare better than conventional forms. The co-administration of DNase and AlgL is essential for enhanced activity in reducing biofilm growth and sputum bacterial counts. While mucin retards bactericidal activity, NAC does not improve aminoglycosidic activity.

Enhanced activity of liposomal polymyxin B against Pseudomonas aeruginosa in a rat model of lung infection

The bactericidal effectiveness of liposomal polymyxin B against Pseudomonas aeruginosa was investigated in an animal model of pulmonary infection. Polymyxin B was incorporated into liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (Chol) (2:1). Lung infection was induced in rats following intratracheal instillation of 10(7) colony-forming units (CFU) of P. aeruginosa (ATCC 27853) embedded in agar beads. Starting on day 3 post-infection, animals were treated daily, for 3 consecutive days, with saline, empty liposomes, free polymyxin B, or liposomal polymyxin B (2mg polymyxin B/kg body weight) by intratracheal instillation; animals were killed 24hr after the third drug instillation. Treatment of infected animals with liposomal polymyxin B significantly reduced the pulmonary bacterial counts (3.7+/-0.4log CFU/paired lungs) as compared with that of free polymyxin B (5.1+/-0.2log CFU/paired lungs). Treatment of infected animals with empty liposomes gave pulmonary bacterial counts similar to those obtained from the saline-treated group. Pulmonary infection with P. aeruginosa also resulted in lung injury as evidenced by increases in wet lung weight and decreases in angiotensin converting enzyme activity as well as increases in myeloperoxidase activity, an index of the inflammatory response. Treatment with free polymyxin B ameliorated the lung injuries induced by the microorganism, a protective effect that was more pronounced in the liposomal polymyxin B-treated group. The levels of polymyxin B in the lungs of the infected animals treated with the liposomal suspension were significantly higher (42.8+/-6.2 microg/paired lungs) compared with those treated with the free drug (8.2+/-0.4 microg/paired lungs). These data suggest that direct delivery of liposomal polymyxin B to the lung can be effective in the treatment of pulmonary infection with P. aeruginosa by enhancing retention of the antibiotic in the lung.

Formulation Strategies to Improve the Bioavailability of Poorly Absorbed Drugs with Special Emphasis on Self-Emulsifying Systems

Poorly water-soluble drug candidates are becoming more prevalent. It has been estimated that approximately 60–70% of the drug molecules are insufficiently soluble in aqueous media and/or have very low permeability to allow for their adequate and reproducible absorption from the gastrointestinal tract (GIT) following oral administration. Formulation scientists have to adopt various strategies to enhance their absorption. Lipidic formulations are found to be a promising approach to combat the challenges. In this review article, potential advantages and drawbacks of various conventional techniques and the newer approaches specifically the self-emulsifying systems are discussed. Various components of the self-emulsifying systems and their selection criteria are critically reviewed. The attempts of various scientists to transform the liquid self-emulsifying drug delivery systems (SEDDS) to solid-SEDDS by adsorption, spray drying, lyophilization, melt granulation, extrusion, and so forth to formulate various dosage forms like self emulsifying capsules, tablets, controlled release pellets, beads, microspheres, nanoparticles, suppositories, implants, and so forth have also been included. Formulation of SEDDS is a potential strategy to deliver new drug molecules with enhanced bioavailability mostly exhibiting poor aqueous solubility. The self-emulsifying system offers various advantages over other drug delivery systems having potential to solve various problems associated with drugs of all the classes of biopharmaceutical classification system (BCS).

Antimicrobial effectiveness of liposomal polymyxin B against resistant Gram-negative bacterial strains.

Polymyxin B is a polycationic antibiotic effective in the treatment of Gram-negative bacterial infections. Systemic use of polymyxin B has been limited due to its toxicity, most notably nephrotoxicity, ototoxicity, and neuromuscular blockade. Entrapment of antibiotics in liposomes is known to enhance their antimicrobial activities while minimizing their toxic effects. In the present study, polymyxin B was incorporated into liposomes composed of either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol (Chol) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and Chol. The entrapment efficiency of sonicated liposomes containing DPPC/Chol (32.1+/-2.43%) was six-fold higher than that of liposomes containing POPC/Chol (5.35+/-0.32%). On the other hand, the entrapment efficiency of extruded DPPC/Chol liposomes (3.23+/-0.46%) was about 30% less than that of liposomes composed of POPC/Chol (5.10+/-0.37%). Incubation of extruded DPPC/Chol liposomes containing polymyxin B in serum at 37 degrees C resulted in a complete release of the antibiotic into the supernatant after 3h as compared to 6h in the case of POPC/Chol liposomes. Spontaneous release of polymyxin B from DPPC/Chol liposomes incubated in saline was significantly higher (66%) than that from POPC/Chol liposomes (24%) after 48h at 37 degrees C. With respect to the antimicrobial activities of the liposomal polymyxin B formulations, the MICs of sonicated DPPC/Chol liposomes against Gram-negative strains were generally lower when compared to free polymyxin B. Immunocytochemistry and electron transmission microscopic studies revealed that the penetration of polymyxin B into a resistant strain of Pseudomonas aeruginosa was higher following its administration as a liposomal formulation as compared to its conventional form. The combination of free drug and plain liposomes had an antibacterial activity similar to that of free antibiotic. These data suggest that incorporation of polymyxin B in liposomes could be useful in the management of Gram-negative infections induced by these microorganisms.

Co-encapsulation of gallium with gentamicin in liposomes enhances antimicrobial activity of gentamicin against Pseudomonas aeruginosa

OBJECTIVES The aim of this study was to enhance the antimicrobial efficacy of a liposomal gentamicin formulation with gallium metal (Lipo-Ga-GEN) against clinical isolates of Pseudomonas aeruginosa. METHODS Sputum isolates of P. aeruginosa from cystic fibrosis patients were used to determine the MIC and MBC of Lipo-Ga-GEN. P. aeruginosa biofilms were formed and used to compare the minimum biofilm eradication concentration of the conventional drugs with that of Lipo-Ga-GEN. Quorum sensing (QS) molecule reduction of P. aeruginosa was determined by monitoring N-acyl homoserine lactone production using Agrobacterium tumefaciens reporter strain (A136). Viability of the cultured human lung epithelial cells (A549) was determined by Trypan Blue assay in order to assess Ga toxicity. RESULTS MIC and MBC values indicated that gentamicin was more effective against a highly resistant strain of P. aeruginosa (PA-48913) when delivered as a Lipo-Ga-GEN formulation (256 mg/L free gentamicin versus 2 mg/L Lipo-Ga-GEN). Lipo-Ga-GEN was the only formulation that completely eradicated biofilms and blocked QS molecules at a very low concentration (0.94 mg/L gentamicin). The decrease in cell viability was less in A549 cells exposed to Lipo-Ga, suggesting that encapsulated Ga is safer. CONCLUSIONS The results clearly indicate that the Lipo-Ga-GEN formulation is more effective than gentamicin alone in eradicating antibiotic-resistant P. aeruginosa isolates growing in a planktonic or biofilm community.

Bismuth–thiol incorporation enhances biological activities of liposomal tobramycin against bacterial biofilm and quorum sensing molecules production by Pseudomonas aeruginosa

Recurrent pulmonary infection and inflammation are major risk factors for high morbidity and mortality in patients with cystic fibrosis (CF). As such, frequent antibiotic use and drug resistant bacterial strains are main concerns in individuals with CF. Bacterial virulence and resistance are influenced by unique CF airways fluid lining and Pseudomonas aeruginosa quorum sensing (QS) and biofilm formation. We have developed a novel liposome formulation consist of bismuth-thiol and tobramycin (LipoBiEDT-TOB) that is non-toxic and highly effective against planktonic bacteria. In this study, we examined the effect of LipoBiEDT-TOB on QS molecule N-acyl homoserine lactone (AHL) secretion by P. aeruginosa isolates in the presence of Agrobacterium tumefaciens reporter strain (A136). LipoBiEDT-TOB activity against biofilm forming P. aeruginosa was compared to free tobramycin using the Calgary Biofilm Device (CBD). Our data indicate that LipoBiEDT-TOB prevents AHL production at low tobramycin concentration (as low as 0.012 mg/l) and stops biofilm forming P. aeruginosa growth at 64 mg/l. The formulation is stable in different biological environments (biofilm, sputum, and bronchoalveolar lavage) and is able to penetrate CF sputum. Taken together, co-encapsulation of bismuth-thiol metal with tobramycin in liposome improves its antimicrobial activities in vitro.

Efficacy and Safety of Liposomal Clarithromycin and Its Effect on Pseudomonas aeruginosa Virulence Factors

ABSTRACT We investigated the efficacy and safety of liposomal clarithromycin formulations with different surface charges against clinical isolates of Pseudomonas aeruginosa from the lungs of cystic fibrosis (CF) patients. The liposomal clarithromycin formulations were prepared by the dehydration-rehydration method, and their sizes were measured using the dynamic-light-scattering technique. Encapsulation efficiency was determined by microbiological assay, and the stabilities of the formulations in biological fluid were evaluated for a period of 48 h. The MICs and minimum bactericidal concentrations (MBCs) of free and liposomal formulations were determined with P. aeruginosa strains isolated from CF patients. Liposomal clarithromycin activity against biofilm-forming P. aeruginosa was compared to that of free antibiotic using the Calgary Biofilm Device (CBD). The effects of subinhibitory concentrations of free and liposomal clarithromycin on bacterial virulence factors and motility on agar were investigated on clinical isolates of P. aeruginosa. The cytotoxicities of the liposome preparations and free drug were evaluated on a pulmonary epithelial cell line (A549). The average diameter of the formulations was >222 nm, with encapsulation efficiencies ranging from 5.7% to 30.4%. The liposomes retained more than 70% of their drug content during the 48-h time period. The highly resistant strains of P. aeruginosa became susceptible to liposome-encapsulated clarithromycin (MIC, 256 mg/liter versus 8 mg/liter; P < 0.001). Liposomal clarithromycin reduced the bacterial growth within the biofilm by 3 to 4 log units (P < 0.001), significantly attenuated virulence factor production, and reduced bacterial twitching, swarming, and swimming motilities. The clarithromycin-entrapped liposomes were less cytotoxic than the free drug (P < 0.001). These data indicate that our novel formulations could be a useful strategy to enhance the efficacy of clarithromycin against resistant P. aeruginosa strains that commonly affect individuals with cystic fibrosis.

Role of nanocarrier systems in cancer nanotherapy

Cancer continues to be a major cause of morbidity and mortality worldwide. While discovery of new drugs and cancer chemotherapy opened a new era for the treatment of tumors, optimized concentration of drug at the target site is only possible at the expense of severe side effects. Nanoscale carrier systems have the potential to limit drug toxicity and achieve tumor localization. When linked with tumor-targeting moieties, such as tumor-specific ligands or monoclonal antibodies, the nanocarriers can be used to target cancer-specific receptors, tumor antigens, and tumor vasculatures with high affinity and precision. This article is an overview of advances and prospects in the applications of nanocarrier technology in cancer therapy. Applications of nanoliposomes, dendrimers, and nanoparticles in cancer therapy are explained, along with their preparation methods and targeting strategies.

Attenuation of Pseudomonas aeruginosa virulence factors and biofilms by co-encapsulation of bismuth–ethanedithiol with tobramycin in liposomes

OBJECTIVES This study examined the activities of tobramycin and bismuth against quorum sensing, virulence factors and biofilms of Pseudomonas aeruginosa by co-encapsulating the agents in liposomes in order to achieve greater delivery of the agents. METHODS The inhibitory effects of the agents, in either their conventional (free) or vesicle-entrapped (liposomal) formulations, were assessed by measuring the changes in the quorum-sensing signal molecule N-acyl homoserine lactone, pyoverdine, pyocyanin, elastase, protease, chitinase, bacterial attachment and biofilms in vitro. RESULTS The effectiveness of tobramycin and bismuth was superior when they were co-administered as a liposomal formulation as measured by their ability to attenuate the production of N-acyl homoserine lactone, elastase (P < 0.01), protease (P < 0.05) and chitinase (P < 0.01). In the presence of non-lethal concentrations of free and liposomal tobramycin and bismuth, bacterial attachment was attenuated. Biofilm formation was also attenuated with free tobramycin and bismuth, yet, in the presence of liposomal tobramycin and bismuth, biofilm complexes could form but contained mostly dead bacteria. When established biofilms were treated with higher concentrations, free tobramycin and bismuth killed and detached bacteria, while the liposomal tobramycin and bismuth penetrated and killed bacteria in the cores of the biofilms. CONCLUSIONS These data suggest that treatment of P. aeruginosa with tobramycin and bismuth, as measured by the changes in quorum sensing, virulence factors and biofilms, is most effective when delivered as a liposomal formulation at a lower concentration compared with the free formulation.