Physico-chemical and physiological determinants of lipo-nanoparticle stability.

Liposome-based nanoparticles (NPs) comprised mostly of phospholipids (PLs) have been developed to deliver diagnostic and therapeutic agents. Whereas reassembled plasma lipoproteins have been tested as NP carriers of hydrophobic molecules, they are unstable because the components can spontaneously transfer to other PL surfaces-cell membranes and lipoproteins-and can be degraded by plasma lipases. Here we review two strategies for NP stabilization. One is to use PLs that contain long acyl-chains: according to a quantitative thermodynamic model and in vivo tests, increasing the chain length of a PL reduces the spontaneous transfer rate and increases plasma lifetime. A second strategy is to substitute ether for ester bonds which makes the PLs lipase resistant. We conclude with recommendations of simple ex vivo and in vitro tests of NP stability that should be conducted before in vivo tests are begun.

Pharmacokinetics of 10-hydroxycamptothecin-tetrandrine liposome complexes in rat by a simple and sensitive ultra-high performance liquid chromatography with tandem mass spectrometry.

10-Hydroxycamptothecin is a drug to cure various cancers. However, the 10-hydroxycamptothecin cannot be widely applied in clinics due to fast elimination and resistance of various cancers to the drug. Nevertheless, co-treatment with tetrandine is known to reverse the resistance of multi-drug resistant cancers, and may present an effective strategy to improve the efficacy of 10-hydroxycamptothecin. In order to improve the bioavailability and prolong the treatment time of the 10-hydroxycamptothecin in vivo, we prepared 10-hydroxycamptothecin-tetrandrine liposome complexes with 10-hydroxycamptothecin as the basic anticancer drug, tetrandrine and liposomes as carriers. In this article, an ultra-high performance liquid chromatography tandem mass spectrometry method for the analysis of 10-hydroxycamptothecin and tetrandrine in plasma has been developed, validated, and utilized to compare the pharmacokinetics of both drugs in the original dosage form and administered as liposome complexes. According to the pharmacokinetic parameters of mean residence time, half-life period and clearance rate, the 10-hydroxycamptothecin-tetrandrine liposome complexes prolongs the retention and circulation time of 10-hydroxycamptothecin in vivo, achieving a good sustained release effect. To the best of our current knowledge, the pharmacokinetic properties of 10-hydroxycamptothecin-tetrandrine liposome complexes in rats have not been reported yet. Our study can provide a helpful reference for further related study.

Improving the accuracy of pancreatic cancer clinical staging by exploitation of nanoparticle-blood interactions: A pilot study.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) early diagnosis is  crucial  and new, cheap and user-friendly techniques for biomarker identification  are  needed. "Protein corona" (PC) is emerging a new bio-interface potentially useful in tumor early diagnosis. In a previous investigation, we showed that relevant differences between the  protein patterns of  PCs formed on lipid NPs after exposure to PDAC and non-cancer plasma  samples exist. To extend that research, We performed this pilot study to investigate the effect of PDAC tumor size and distant metastases on PC composition. METHODS: Twenty PDACs were clinically staged according to the UICC TNM staging system 8 t h Edition. Collected plasma samples were let to interact with lipid NPs; resulting PCs were characterized by SDS-PAGE. To properly evaluate changes in the PC, the protein intensity profiles were reduced to four regions of molecular weight: < 25 kDa, 25-50 kDa, 50-120 kDa, > 120 kDa.  RESULTS: Data analysis allowed to distinguish T1-T2 cases from T3 and above all from metastatic ones (p < 0.05). Discrimination power was particularly due to a subset of plasma proteins with molecular  weight comprised between 25-50 kDa  and 50-120 kDa. CONCLUSIONS: PC composition is critically influenced by tumor size and presence of distant metastases in PDAC. If our findings will be further confirmed, we envision that future developments of cheap and user-friendly PC-based tools will allow to improve the accuracy of PDAC clinical staging, identifying among resectable  PDACs with potentially better prognosis (i.e. T1 and T2) those at higher risk of occult distant metastases.

Dual-targeting for brain-specific liposomes drug delivery system: Synthesis and preliminary evaluation.

The treatment of glioma has become a great challenge because of the existence of brain barrier (BB). In order to develop an efficient brain targeting drug delivery system to greatly improve the brain permeability of anti-cancer drugs, a novel brain-targeted glucose-vitamin C (Glu-Vc) derivative was designed and synthesized as liposome ligand for preparing liposome to effectively deliver paclitaxel (PTX). The liposome was prepared and its particle size, zeta potential, encapsulation efficiency, release profile, stability, hemolysis and cytotoxicity were also characterized. What's more, the cellular uptake of CFPE-labeled Glu-Vc-Lip on GLUT1- and SVCT2-overexpressed C6 cells was 4.79-, 1.95-, 4.00- and 1.53-fold higher than that of Lip, Glu-Lip, Vc-Lip and Glu + Vc-Lip. Also, the Glu-Vc modified liposomes showed superior targeting ability in vivo evaluation compared with naked paclitaxel, non-coated, singly-modified and co-modified by physical blending liposomes. The relative uptake efficiency was enhanced by 7.53 fold to that of naked paclitaxel, while the concentration efficiency was up to 7.89 times. What's more, the Glu-Vc modified liposomes also displayed the maximum accumulation of DiD-loaded liposomes at tumor sites with the strongest fluorescence in the brain in vivo imaging. Our results suggest that chemical modification of liposomes with warheads of glucose and vitamin C represents a promising and efficient strategy for the development of brain-specific liposomes drug delivery system by utilizing the endogenous transportation mechanism of the warheads.

Computer simulations of drug release from a liposome into the bloodstream.

I propose two-dimensional simulations of drug release from a liposome into the bloodstream. I perform the fluid-structure coupling, between the particles deformation (the liposome and the red blood cells) and the plasma flow, using the immersed boundary method. I compute both the flow and the drug mass transport using the lattice Boltzmann method. The simulations allow computing the instantaneous amount of the released drug, its distribution and its accumulation in the blood vessel wall. These quantities are sensitive to multiple factors and parameters. Here, I briefly explore the impact of having surrounding red blood cells, which are found to enhance slightly the drug release at large Schmidt numbers. In the limit of extremely large permeability of the particles, the drug transport is mainly affected by the complex flow induced by the interplay between the applied flow and the collective motion of the particles.

Gas chromatography/mass spectrometry measurement of xenon in gas-loaded liposomes for neuroprotective applications.

RATIONALE: We have produced a liposomal formulation of xenon (Xe-ELIP) as a neuroprotectant for inhibition of brain damage in stroke patients. This mandates development of a reliable assay to measure the amount of dissolved xenon released from Xe-ELIP in water and blood samples. METHODS: Gas chromatography/mass spectrometry (GC/MS) was used to quantify xenon gas released into the headspace of vials containing Xe-ELIP samples in water or blood. In order to determine blood concentration of xenon in vivo after Xe-ELIP administration, 6 mg of Xe-ELIP lipid was infused intravenously into rats. Blood samples were drawn directly from a catheterized right carotid artery. After introduction of the samples, each vial was allowed to equilibrate to 37°C in a water bath, followed by 20 minutes of sonication prior to headspace sampling. Xenon concentrations were calculated from a gas dose-response curve and normalized using the published xenon water-gas solubility coefficient. RESULTS: The mean corrected percent of xenon from Xe-ELIP released into water was 3.87 ± 0.56% (SD, n = 8), corresponding to 19.3 ± 2.8 µL/mg lipid, which is consistent with previous independent Xe-ELIP measurements. The corresponding xenon content of Xe-ELIP in rat blood was 23.38 ± 7.36 µL/mg lipid (n = 8). Mean rat blood xenon concentration after intravenous administration of Xe-ELIP was 14 ± 10 µM, which is approximately 15% of the estimated neuroprotective level. CONCLUSIONS: Using this approach, we have established a reproducible method for measuring dissolved xenon in fluids. These measurements have established that neuroprotective effects can be elicited by less than 20% of the calculated neuroprotective xenon blood concentration. More work will have to be done to establish the protective xenon pharmacokinetic range. Copyright © 2016 John Wiley & Sons, Ltd.

Surprising lack of liposome-induced complement activation by artificial 1,3-diamidophospholipids in vitro.

Cardio-vascular diseases are the main cause of death, emphasizing the need to improve patient treatment and survival. One therapeutic approach is a liposome-based drug carrier system specifically targeting constricted arteries. The recently discovered mechano-sensitive liposomes use hemodynamic shear-stress differences between healthy and constricted blood vessels as trigger for drug release. Liposomes are promising delivery containers but are being recognized as foreign by the immune system. Complement activation as essential factor of the recognition leads to adverse effects. Here, we tested complement activation by liposomes formulated from the artificial phospholipid Pad-PC-Pad in vitro. Surprisingly no complement activation was detected in human sera and porcine plasma. In in vivo experiments with three pigs, neither anaphylactic reactions nor other significant hemodynamic changes were observed even at comparably high liposome doses. The pilot study holds promise for an absence of complement-mediated adverse effects of Pad-PC-Pad liposomes in human. FROM THE CLINICAL EDITOR: A lot of research has been done on new treatment for cardiovascular diseases. Liposome-based carrier systems have also shown promises. In this article, the authors studied the potential risks of complement activation by liposomes in in-vivo experiments. The absence of complement activation by Pad-PC-Pad liposomes may indicate its use in humans.

Production of limit size nanoliposomal systems with potential utility as ultra-small drug delivery agents.

Previous studies from this group have shown that limit size lipid-based systems--defined as the smallest achievable aggregates compatible with the packing properties of their molecular constituents--can be efficiently produced using rapid microfluidic mixing technique. In this work, it is shown that similar procedures can be employed for the production of homogeneously sized unilamellar vesicular systems of 30-40 nm size range. These vesicles can be remotely loaded with the protonable drug doxorubicin and exhibit adequate drug retention properties in vitro and in vivo. In particular, it is demonstrated that whereas sub-40 nm lipid nanoparticle (LNP) systems consisting entirely of long-chain saturated phosphatidylcholines cannot be produced, the presence of such lipids may have a beneficial effect on the retention properties of limit size systems consisting of mixed lipid components. Specifically, a 33-nm diameter doxorubicin-loaded LNP system composed of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1,2-dipalmitoyl phosphatidylcholine (DPPC), cholesterol, and PEGylated lipid (DSPE-PEG2000) demonstrated adequate, stable drug retention in the circulation, with a half-life for drug release of ∼ 12 h. These results indicate that microfluidic mixing is the technique of choice for the production of bilayer LNP systems with sizes less than 50 nm that could lead to development of a novel class of ultra-small drug delivery vehicles.

Evaluation of multifunctional liposomes in human blood serum by light scattering.

To overcome the limited functionality of "stealth" lipids based on linear poly(ethylene glycol) (PEG) chains, hyperbranched polyether-based lipids that bear multiple hydroxyl groups for further chemical modification may be a suitable replacement. This study focuses on the development and characterization of "stealth" liposomes modified with a novel hyperbranched polyglycerol lipid (cholesterol-PEG30-hbPG23). An emphasis was placed on the stability of these liposomes in comparison to those containing a linear PEG derivative (cholesterol-PEG44) directly in human blood serum, characterized via dynamic light scattering (DLS). Polymer lipid contents were varied between 0 and 30 mol %, resulting in liposomes with sizes between 150 and 80 nm in radius, depending on the composition. DLS analysis showed no aggregation inducing interactions between serum components and liposomes containing 10-30 mol % of the hyperbranched lipid. In contrast, liposomes functionalized with comparable amounts of linear PEG exhibited aggregate formation in the size range of 170-330 nm under similar conditions. In addition to DLS, cryo-transmission electron microscopy (TEM) was employed for all liposome samples to prove the formation of unilamellar vesicles. These results demonstrate the outstanding potential of the introduction of hyperbranched polyglycerol into liposomes to stabilize the assemblies against aggregation while providing additional functionalization sites.

Ursolic acid incorporation does not prevent the formation of a non-lamellar phase in pH-sensitive and long-circulating liposomes.

Ursolic acid (UA) is a triterpene found in different plant species that has been shown to possess significant antitumor activity. However, UA presents a low water solubility, which limits its biological applications. In this context, our research group has proposed the incorporation of UA in long-circulating and pH-sensitive liposomes (SpHL-UA).These liposomes, composed of dioleylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), and distearoylphosphatidylethanolamine-polyethylene glycol2000 (DSPE-PEG2000), were shown to be very promising carriers for UA. Considering that the release of UA from SpHL-UA and its antitumor activity depend upon the occurrence of the lamellar to non-lamellar phase transition of DOPE, in the present work, the interactions of UA with the components of the liposomes were evaluated, aiming to clarify their role in the structural organization of DOPE. The study was carried out by differential scanning calorimetry (DSC) and small-angle X-ray scattering (SAXS) under low hydration conditions. DSC studies revealed that DOPE phase transition temperatures did not shift significantly upon UA addition. On the other hand, in SAXS studies, a different pattern of DOPE phase organization was observed in the presence of UA, with the occurrence of the cubic phase Im3m at 20 °C and the cubic phase Pn3m at 60 °C. These findings suggest that UA interacts with the lipids and changes their self-assembly. However, these interactions between the lipids and UA were unable to eliminate the lamellar to non-lamellar phase transition, which is essential for the cytoplasmic delivery of UA molecules from SpHL-UA.

Meta-analysis of inter-patient pharmacokinetic variability of liposomal and non-liposomal anticancer agents.

A meta-analysis was conducted to evaluate the inter-patient pharmacokinetic (PK) variability of liposomal and small molecule (SM) anticancer agents. Inter-patient PK variability of 9 liposomal and SM formulations of the same drug was evaluated. PK variability was measured as coefficient of variance (CV%) of area under the plasma concentration versus time curve (AUC) and the fold-difference between AUCmax and AUCmin (AUC range). CV% of AUC and AUC ranges were 2.7-fold (P<0.001) and 16.7-fold (P=0.13) greater, respectively, for liposomal compared with SM drugs. There was an inverse linear relationship between the clearance (CL) of liposomal agents and PK variability with a lower CL associated with greater PK variability (R(2)=0.39). PK variability of liposomal agents was greater when evaluated from 0-336 h compared with 0-24h. PK variability of liposomes is significantly greater than SM. The factors associated with the PK variability of liposomal agents need to be evaluated. FROM THE CLINICAL EDITOR: In this meta-analysis, the inter-patient pharmacokinetic variability of 9 liposomal and small molecule anti-cancer agents was studied. The authors determined that several parameters are in favor of the liposomal formulation; however, the PK variability of the formulation was higher compared with small molecule agents, the reason for which remains to be determined in future studies.

Pharmacokinetic and tissue distribution of paclitaxel in rabbits assayed by LC-UV after intravenous administration of its novel liposomal formulation.

A simple, rapid and sensitive LC-UV method was developed and validated for the determination of paclitaxel (PTX) in rabbit plasma and tissues. A 2 mL aliquot of acetonitrile and 10 µL ammonium acetate (pH 5.0, 6 m) as extraction agents were used to markedly increase the extraction recoveries and greatly reduce the endogenous substances. The separation was achieved on a C18 column at 30 °C using an acetonitrile-ammonium acetate buffer (pH 5.0, 0.02 m; 55:45, v/v) at a flow rate of 1.0 mL/min; UV detection was used at 227 nm. Good linearity was obtained between 0.025 and 10,000 µg/mL for plasma and between 0.025-200,000 µg/g for tissue samples (r > 0.999). The limit of detection was 6 ng/mL in plasma, 8 ng/g in heart and 12.5 ng/g in other tissues. The limit of quantitation was 25 ng/mL in plasma and heart, 125 ng/g in other tissues. The intra- and inter-day assays of precision and accuracy for all bio-samples ranged from 1.38 to 9.60% and from 83.6 to 114.5%, respectively. The extraction recoveries ranged from 70.1 to 109.5%. Samples were stable during three freeze-thaw cycles or stored in a freezer at -20 °C for 30 days. The assay method was successfully applied to a study of the pharmacokinetics and tissue distribution of novel PTX lung targeting liposomes.

[Repeated injection of mitoxantrone containing thermosensitive liposomes in rat induced ABC phenomenon].

To investigate whether accelerated blood clearance (ABC) phenomenon could be induced after repeated injection of mitoxantrone thermosensitive liposomes, LC-MS/MS and enzyme linked immunosorbent assay (ELISA) were used to measure the concentration of mitoxantrone and the anti-polyethylene glycol (PEG) IgM levels in rat plasma, separately. The drug was rapidly cleared away after the second administration. The anti-PEG IgM was detected after the first dose which was neutralized quickly after the second dose. It is proved that repeated administration of mitoxantrone thermosensitive liposomes in rat caused the ABC phenomenon.

[Preparation of novel gypenosides long-circulating liposomes consisted by sphingomyelin and beta-sitosterol modified by PEG].

OBJECTIVE: To explore the feasibility of preparing novel gypenosides long-circulating liposomes with PEG grafted on beta-sitosterol (PEG-Sito). METHOD: Succinicanhydride was adopted to connect beta-sitosterol and PEG 2000. Sphingomyelin and PEG-Sito was used as material to prepare gypenosides long-circulating liposomes by using ethanol injection method. Encapsulation efficiency was determined by using protamine precipitation method. H-NMR was used to verify the synthesis of PEG-Sito, the novel gypenosides long-circulating liposomes were characterized by particle size, zeta potential and atomic force microscope. RESULT: The synthesis of PEG-Sito was verified by 1H-NMR. Encapsulation efficiency of long-circulating liposomes prepared by ethanol injection method was 74.3%, particle size was 288.1 nm, zeta potential was -20.25 mV, the morphology were round observed by AFM. CONCLUSION: The novel gypenosides long-circulating liposomes prepared with PEG-Sito was feasible, it had a high encapsulation efficiency and good morphology.

Quantitative determination of liposomal irinotecan and SN-38 concentrations in plasma samples from children with solid tumors: Use of a cryoprotectant solution to enhance liposome stability.

Preclinical studies have demonstrated that liposomal irinotecan (CPT-11), a topoisomerase I inhibitor, has broad activity against adult cancers, including pancreatic, gastric, colon, lung, glioma, ovarian, and breast cancer. Encapsulation of irinotecan into liposomes can modify its pharmacokinetic properties dramatically. Also, the pharmacokinetic profiles of liposomal drug formulations are not fully understood; thus, bioanalytical methods are needed to separate and quantify nonencapsulated vs. encapsulated concentrations. In this study, two robust, specific, and sensitive LC-MS/MS methods were developed and validated to separate and quantify the nonencapsulated CPT-11 (NE-CPT-11) from the sum-total CPT-11 (T-CPT-11) and its major metabolite, SN-38, in human plasma after intravenous administration of liposomal irinotecan. NE-CPT-11 and SN-38 were separated from plasma samples by using solid-phase extraction, and T-CPT-11 was measured by protein precipitation. The liposomal CPT-11 formulation was unstable during sample storage and handling, resulting in elevated NE-CPT-11 concentration. To improve the stability of liposomal CPT-11, a cryoprotectant solution was added to human plasma samples prior to storage and processing. CPT-11, SN-38, and their respective internal standards, CPT-11-d10 and SN-38-d3, were chromatographically separated on a reversed-phase C18 analytical column. The drugs were detected on a triple quadrupole mass spectrometer in the positive MRM ion mode by monitoring the transitions 587.3 > 124.1 (CPT-11) and 393.0 > 349.1 (SN-38). The calibration curves demonstrated a good fit across the concentration ranges of 10-5000 ng/mL for T-CPT-11, 2.5-250 ng/mL for NE-CPT-11, and 1-500 ng/mL for SN-38. The accuracy and precision were within the acceptable limits, matrix effects were nonsignificant, recoveries were consistent and reproducible, and the analytes were stable under all tested storage conditions. Finally, the LC-MS/MS methods were successfully applied in a phase I clinical pharmacokinetic study of nanoliposomal irinotecan (Onivyde®) in pediatric patients with recurrent solid malignancies or Ewing sarcoma.

Metallomics in drug development: characterization of a liposomal cisplatin drug formulation in human plasma by CE-ICP-MS.

A capillary electrophoresis inductively coupled plasma mass spectrometry method for separation of free cisplatin from liposome-encapsulated cisplatin and protein-bound cisplatin was developed. A liposomal formulation of cisplatin based on PEGylated liposomes was used as model drug formulation. The effect of human plasma matrix on the analysis of liposome-encapsulated cisplatin and intact cisplatin was studied. The presence of 1 % of dextran and 4 mM of sodium dodecyl sulfate in HEPES buffer was demonstrated to be effective in improving the separation of liposomes and cisplatin bound to proteins in plasma. A detection limit of 41 ng/mL of platinum and a precision of 2.1 % (for 10 µg/mL of cisplatin standard) were obtained. Simultaneous measurements of phosphorous and platinum allows the simultaneous monitoring of the liposomes, liposome-encapsulated cisplatin, free cisplatin and cisplatin bound to plasma constituents in plasma samples. It was demonstrated that this approach is suitable for studies of the stability of liposome formulations as leakage of active drug from the liposomes and subsequent binding to biomolecules in plasma can be monitored. This methodology has not been reported before and will improve characterization of liposomal drugs during drug development and in studies on kinetics.

Hydrogel-embeded vesicles, as a novel approach for prolonged release and delivery of liposome, in vitro and in vivo.

A novel delivery concept based on the integration of liposomes in hydrogel for the controlled release of liposomes was developed. As an in situ forming hydrogel, chitosan-glycerophosphate was used and gelation time at different temperatures was studied. Liposomes (DSPC/chol/DOPE) were labelled with (99m)Tc-hexamethylpropyleneamineoxime ((99m)Tc-HMPAO). (99m)Tc-HMPAO solution, hydrogel/(99m)Tc-HMPAO, (99m)Tc-HMPAO liposomes and hydrogel/(99m)Tc-HMPAO liposomes were injected into mouse peritoneum. The percentages of radioactive injected dose per gram of tissue (%ID/g) and %ID of peritoneum lavage were obtained. Results showed that free label left the peritoneal cavity rapidly in both solution and hydrogel forms, such that the activity was 2.5 and 3.8 (%ID) after one hour, respectively. The values for liposomes and liposomal hydrogel were 25.8 and 51.2 (%ID) and decreased to 1.9 and 19.2 after 24 h, respectively. The blood profile of liposomal hydrogel showed a two-phase profile including a descending trend in early hours regarding gel formation followed by an ascending trend due to gel disappearance by time. Free label had high activity in reticuloendothelial system (RES) and the gastrointestinal tract during the early hours and then dropped. In contrast, the accumulation of liposomes increased in RES during 24 h in the range of 1-34.5 and 1.1-35.1 (%ID/g) for plain liposomes and liposomal hydrogel, respectively. Overall, incorporation of liposomes in hydrogel could be a useful strategy to prolong the release of liposomes.

Effectiveness of submicronized chitosan-coated liposomes in oral absorption of indomethacin.

The plasma profile of indomethacin (IMC) after oral administration of IMC-loaded submicronized chitosan-coated liposomes (ssCS-Lip) was evaluated to reveal the effectiveness of the mucoadhesive function for improving the absorption of this poorly absorbable drug. The stomach and small intestine were removed from rats after 1, 2, and 4 hours of oral administration of submicron-sized liposomes (ssLip) or ssCS-Lip containing fluorescent dye, and the retentive properties were confirmed by measuring the amount of dye in each part of the gastrointestinal (GI) tract. Results showed that ssCS-Lip tended to be better retained in the upper part of the GI tract, compared with ssLip, at 1, 2, and 4 hours after administration, and was significantly better retained in the small intestine at 4 hours. The plasma profile and bioavailability of IMC after oral administration of both types of liposomes were improved, compared with oral administration of IMC solution. The maximum residence time of ssCS-Lip was significantly longer than those of ssLip. The extended plasma profile of ssCS-Lip was attributed to its prolonged retention in the upper region of the GI tract, and its delayed migration to the lower part of the intestine, the neutral pH of which is more soluble for IMC, an acidic drug. Therefore, the chitosan-coated ssLip, with its higher retention in the GI tract, is a promising drug carrier for the oral administration of poorly absorbed compounds.

Study of quercetin-loaded liposomes as potential drug carriers: in vitro evaluation of human complement activation.

Liposomes have been employed as potential drug carriers. However, after their in vivo administration, they can be destabilized by proteins of complement system, contributing to the clearance of vesicles from blood circulation. Antioxidant flavonoids such as quercetin have been reported to be beneficial to human health, but their low water solubility and bioavailability limit their enteric administration. Therefore, the development of appropriate flavonoid-carriers could be of great importance to drug therapy. The aim of the present study was to evaluate the activation of human complement system proteins by liposomes composed of soya phosphatidylcholine (SPC) and cholesterol (CHOL) or cholesteryl ethyl ether (CHOL-OET) loaded with quercetin or not. The consumption of complement, via classical (CP) and alternative (AP) pathways, by different vesicles was evaluated using a hemolytic assay and quantitative determination of iC3b and natural antibodies deposited on empty liposomal surfaces by ELISA. The main results showed that empty liposomes composed of large amounts of CHOL consumed more complement components than the others for both CP and AP. Furthermore, replacement of CHOL with CHOL-OET reduced complement consumption via both CP and AP. Incorporation of quercetin did not change CP and AP consumption. Deposition of iC3b, IgG and IgM in vesicles composed of SPC:CHOL-OET at a molar ratio of 1.5:1 was lower compared to the others. Taken together, these observations suggest that liposomes composed of SPC:CHOL-OET at a molar ratio of 1.5:1 are the most appropriate among the vesicles studied herein to be used as a drug carrier system in further investigations.

A novel cationic liposome formulation for efficient gene delivery via a pulmonary route.

The clinical success of gene therapy for lung cancer is not only dependent on efficient gene carriers but also on a suitable delivery route. A pulmonary delivery route can directly deliver gene vectors to the lung which is more efficient than a systemic delivery route. For gene carriers, cationic liposomes have recently emerged as leading non-viral vectors in worldwide gene therapy clinical trials. However, cytotoxic effects or apoptosis are often observed which is mostly dependent on the cationic lipid used. Therefore, an efficient and safe cationic lipid, 6-lauroxyhexyl lysinate (LHLN), previously synthesized by our group was first used to prepare cationic liposomes. Physicochemical and biological properties of LHLN-liposomes were investigated. LHLN-liposome/DNA complexes showed positive surface charge, spherical morphology, a relatively narrow particle size distribution and strong DNA binding capability. Compared with Lipofectamine2000, the new cationic liposome formulation using LHLN exhibited not only lower cytotoxicity (P < 0.05) but also similar transfection efficiency in A549 and HepG2 lung cancer cells for in vitro tests. When administered by intratracheal instillation into rat lungs for in vivo evaluation, LHLN-liposome/DNA complexes exhibited higher pulmonary gene transfection efficiency than Lipofectamine2000/DNA complexes (P < 0.05). These results suggested that LHLN-liposomes may have great potential for efficient pulmonary gene delivery.