Yufan Ma

Enhanced bactericidal potency of nanoliposomes by modification of the fusion activity between liposomes and bacterium

Background Pseudomonas aeruginosa represents a good model of antibiotic resistance. These organisms have an outer membrane with a low level of permeability to drugs that is often combined with multidrug efflux pumps, enzymatic inactivation of the drug, or alteration of its molecular target. The acute and growing problem of antibiotic resistance of Pseudomonas to conventional antibiotics made it imperative to develop new liposome formulations to overcome these mechanisms, and investigate the fusion between liposome and bacterium. Methods The rigidity, stability and charge properties of phospholipid vesicles were modified by varying the cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and negatively charged lipids 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol sodium salt (DMPG), 1,2-dimyristoyl-sn-glycero-3-phopho-L-serine sodium salt (DMPS), 1,2-dimyristoyl-sn-glycero-3-phosphate monosodium salt (DMPA), nature phosphatidylserine sodium salt from brain and nature phosphatidylinositol sodium salt from soybean concentrations in liposomes. Liposomal fusion with intact bacteria was monitored using a lipid-mixing assay. Results It was discovered that the fluid liposomes-bacterium fusion is not dependent on liposomal size and lamellarity. A similar degree of fusion was observed for liposomes with a particle size from 100 to 800 nm. The fluidity of liposomes is an essential pre-request for liposomes fusion with bacteria. Fusion was almost completely inhibited by incorporation of cholesterol into fluid liposomes. The increase in the amount of negative charges in fluid liposomes reduces fluid liposomes-bacteria fusion when tested without calcium cations due to electric repulsion, but addition of calcium cations brings the fusion level of fluid liposomes to similar or higher levels. Among the negative phospholipids examined, DMPA gave the highest degree of fusion, DMPS and DMPG had intermediate fusion levels, and PI resulted in the lowest degree of fusion. Furthermore, the fluid liposomal encapsulated tobramycin was prepared, and the bactericidal effect occurred more quickly when bacteria were cultured with liposomal encapsulated tobramycin. Conclusion The bactericidal potency of fluid liposomes is dramatically enhanced with respect to fusion ability when the fusogenic lipid, DOPE, is included. Regardless of changes in liposome composition, fluid liposomes-bacterium fusion is universally enhanced by calcium ions. The information obtained in this study will increase our understanding of fluid liposomal action mechanisms, and help in optimizing the new generation of fluid liposomal formulations for the treatment of pulmonary bacterial infections.

Influence of polymer size, liposomal composition, surface charge, and temperature on the permeability of pH-sensitive liposomes containing lipid-anchored poly(2-ethylacrylic acid).

Background Liposomes containing pH-sensitive polymers are promising candidates for the treatment of tumors and localized infection. This study aimed to identify parameters influencing the extent of contents release from poly(ethylacrylic acid) (PEAA) vesicles, focusing on the effects of polymer size, lipid composition, vesicle surface charge, and temperature. Methods Anchored lipid pH-sensitive PEAA was synthesized using PEAA with a molecular weight of 8.4 kDa. PEAA vesicles were prepared by insertion of the lipid-anchored PEAA into preformed large unilamellar vesicles. The preformed liposomes were manipulated by varying the phosphocholine and cholesterol content, and by adding negative or positive charges to the liposomes. A calcein release assay was used to evaluate the effects of polymer size, liposome composition, surface charge, and temperature on liposomal permeability. Results The release efficiency of the calcein-entrapped vesicles was found to be dependent on the PEAA polymer size. PEAA vesicles containing a phosphatidylcholine to cholesterol ratio of 60:40 (mol/mol) released more than 80% of their calcein content when the molecular weight of PEAA was larger than 8.4 kDa. Therefore, the same-sized polymer of 8.4 kDa was used for the rest of study. The calcein release potential was found to decrease as the percentage of cholesterol increased and with an increase in the phosphocholine acyl chain length (DMPC DPPC DSPC). Negatively charged and neutral vesicles released similar amounts of calcein, whereas positively charged liposomes released a significant amount of their contents. pH-sensitive release was dependent on temperature. Dramatic content release was observed at higher temperatures. Conclusion The observed synergistic effect of pH and temperature on release of the contents of PEAA vesicles suggests that this pH-sensitive liposome might be a good candidate for intracellular drug delivery in the treatment of tumors or localized infection.

Designer Functionalized Self-Assembling Peptide Scaffolds for Adhesion, Proliferation, and Differentiation of MC3T3-E1

Induction of some specific cell signaling molecules in peptide scaffold play a critical role in developing of functional substitutes for damaged tissue. In this study, a new peptide ELK8 (Ac-LELELKLK-CONH2) was designed by replacing Ala in EAK16 (Ac-AEAEAKAKAEAEAKAK-CONH2) with more hydrophobic residue Leu in order to enhance the strength of peptide. The functionalized peptides were obtained through directly coupling three functional motifs to C-terminal of ELK8, such as osteogenic growth peptide, osteopontin cell adhesion motif, and 2-unit RGD binding sequence. Then, the new self-assembling peptide scaffolds were fabricated by mixing three functionalized peptides severally with pure ELK8 at the ratio of 1:1 (v:v) and the nanostructures of self-assembling peptides were investigated with AFM and DLS. The osteogenic efficacy of designed peptides on mouse pre-osteoblast MC3T3-E1 were examined after cells were incubated in scaffolds for 14 d. The results showed that these functionalized peptides, ALK mix particularly, promoted the attachment, proliferation, and osteogenic differentiation of MC3T3-E1 cells by increasing in cell numbers, ALP activities and osteocalcin concentrations.

Ethinylestradiol liposome preparation and its effects on ovariectomized rats’ osteoporosis

Ethinylestradiol liposome (EEL) was prepared by thin film evaporation–extrusion technique and its effects on ovariectomized rat osteoporosis were investigated. The liposomes were characterized with clear homogeneous lamellar vesicles and had the average size of 200 ± 1.5 nm. The long term stability of liposome formulations were tested over a 28-day period at 4°C and ethinylestradiol retention was approximately 80% up to 28 day. Release data of ethinylestradiol was tested in PBS pH 7.4 at 37°C using a dialysis method and its release profiles were biphasic, showing a relatively large burst effect over the first four hours, followed by a slower release phase. Ovariectomized rat model was used to investigate EEL effects on osteoporosis. After three months post-surgery, the rats were divided into two groups and injected intraperitoneally (IP) with 2 μg per kilogram per day of either free ethinylestradiol (EE) or encapsulated ethinlyestradiol (EEL). At termination of one month IP injection, the rats were killed and the bone mineral density (BMD) and alkaline phosphatase (ALP) were measured. BMD value in free EE group increased by 20.3 %, while it increased by 37.8 % in EEL group. According to ALP value, the two treatment groups increased by 28.6 % and 42.0 %, respectively. These data indicated that ethinlyetradiol liposome had better effect than that of free ethinylestradiol did in treatment of the ovariectomized rats’ osteoporosis. But the metabolites and biodistribution of ethinylestradiol liposome in vivo need to investigate in the further research work.

Fusion between fluid liposomes and intact bacteria: study of driving parameters and in vitro bactericidal efficacy

Background Pseudomonas aeruginosa represents a good model of antibiotic resistance. These organisms have an outer membrane with a low level of permeability to drugs that is often combined with multidrug efflux pumps, enzymatic inactivation of the drug, or alteration of its molecular target. The acute and growing problem of antibiotic resistance of bacteria to conventional antibiotics made it imperative to develop new liposome formulations for antibiotics, and investigate the fusion between liposome and bacterium. Methods In this study, the factors involved in fluid liposome interaction with bacteria have been investigated. We also demonstrated a mechanism of fusion between liposomes (1,2-dipa lmitoyl-sn-glycero-3-phosphocholine [DPPC]/dimyristoylphosphatidylglycerol [DMPG] 9:1, mol/mol) in a fluid state, and intact bacterial cells, by lipid mixing assay. Results The observed fusion process is shown to be mainly dependent on several key factors. Perturbation of liposome fluidity by addition of cholesterol dramatically decreased the degree of fusion with P. aeruginosa from 44% to 5%. It was observed that fusion between fluid liposomes and bacteria and also the bactericidal activities were strongly dependent upon the properties of the bacteria themselves. The level of fusion detected when fluid liposomes were mixed with Escherichia coli (66%) or P. aeruginosa (44%) seems to be correlated to their outer membrane phosphatidylethanolamine (PE) phospholipids composition (91% and 71%, respectively). Divalent cations increased the degree of fusion in the sequence Fe2+ > Mg2+ > Ca2+ > Ba2+ whereas temperatures lower than the phase transition temperature of DPPC/DMPG (9:1) vesicles decreased their fusion capacity. Acidic as well as basic pHs conferred higher degrees of fusion (54% and 45%, respectively) when compared to neutral pH (35%). Conclusion Based on the results of this study, a possible mechanism involving cationic bridging between bacterial negatively charged lipopolysaccharide and fluid liposomes DMPG phospholipids was outlined. Furthermore, the fluid liposomal-encapsulated tobramycin was prepared, and the in vitro bactericidal effects were also investigated.

Synthesis and physicochemical property of the hydrophobic bisphosphate amidated derivatives

Hydrophobic bisphosphate amidated derivatives (1a–1d) anchored long alkyl chains were designed and obtained through the reaction of -NH2 in BPs and -COOH in fat acid under anhydrous condition. The structure of derivatives were investigated by fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and mass spectrometer (MS) and the results showed that the final products were amidated compounds with the long alkyl anchored in alendroantes lateral chain R2. The physicochemical parameters, such as the solubility and partition coefficient in n-octanol/water, were also determined through calculated by performing reversed phase high performance liquid chromatography (RP-HPLC). Compared with alendronate, the derivatives had a very low aqueous solubility and had little or even no measurable solubility values in PBS. While it had different solubility in organic solvents with different polarities. The partition coefficient Po/w of derivatives in n-octacol/water mixture was calculated and the results showed that the derivatives had the high Po/w data than that of alendronate and the longer the alkyl chain was introduced, the higher the partition coefficient was. All of the above results showed the improved lipophilicity of the derivatives through long alkyl chain introduced into its R2 chain.

Preparation and properties of a new artemisinin lipid emulsion for parenteral administration

Artemisinin is difficult to formulate for parenteral administration because of its low aqueous solubility and Cremophor EL, the excipient used for its formulation, has been shown to cause serious side effects. This study reports the preparation method and properties of artemisinin lipid emulsion for parenteral administration. High-pressure homogenization was used to prepare artemisinin lipid emulsion. The factors influencing the stability of artemisinin lipid emulsion were monitored, such as drug loading methods and sterilization methods. It has been found that the optimum formulation of artemisinin lipid emulsion were composed of artemisinin 0.4%(w/v), soybeen oil 10%(w/v), egg lecithin 1.2%(w/v), F68 0.4%(w/v), oleate 0.02%(w/v) and glycerol 2.5%(w/v). Artemisinin lipid emulsion was stable enough to withstand rotating steam sterilization at 121°C for 15 min.