ORAL TREATMENT OF HEMOPHILIA

Disclosed herein is a simple method for the treatment of antigen-deficiency diseases, by orally administering to a subject a therapeutically effective amount of the deficient antigen, wherein the antigen is not present in a liposome. In one embodiment, the method increases hemostasis in a subject having hemophilia A or B, by orally administering to the hemophiliac a therapeutically effective amount of the appropriate clotting factor other than in a liposome, sufficient to induce oral tolerance and supply exogenous clotting factor to the subject

INDUCTION OF TOLERANCE BY ORAL ADMINISTRATION OF FACTOR VIII AND TREATMENT OF HEMOPHILA

Disclosed herein is a simple method for the treatment of antigen-deficiency diseases, by orally administering to a Subject a therapeutically effective amount of the deficient anti gen, wherein the antigen is not present in a liposome. In one embodiment, the method increases hemostasis in a subject having hemophilia A or B, by orally administering to the hemophiliac atherapeutically effective amount of the appropriate clotting factor other than in a liposome, Sufficient to induce oral tolerance and Supply exogenous clotting factor to the subject

A pH-sensitive stearoyl-PEG-poly(methacryloyl sulfadimethoxine)-decorated liposome system for protein delivery: an application for bladder cancer treatment

Stealth pH-responsive liposomes for the delivery of therapeutic proteins to the bladder epithelium were prepared using methoxy-poly(ethylene glycol)5kDa-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG5kDa-DSPE) and stearoyl-poly(ethylene glycol)-poly(methacryloyl sulfadimethoxine) copolymer (stearoyl-PEG-polySDM), which possesses an apparent pKa of 7.2. Liposomes of 0.2:0.6:100, 0.5:1.5:100 and 1:3:100 mPEG5kDa-DSPE/stearoyl-PEG-polySDM/(soybean phosphatidylcholine + cholesterol) molar ratios were loaded with bovine serum albumin (BSA) as a protein model. The loading capacity was 1.3% w/w BSA/lipid. At pH 7.4, all liposome formulations displayed a negative zeta-potential and were stable for several days. By pH decrease or addition to mouse urine, the zeta potential strongly decreased, and the liposomes underwent a rapid size increase and aggregation. Photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) analyses showed that the extent of the aggregation depended on the stearoyl-PEG-polySDM/lipid molar ratio. Cytofluorimetric analysis and confocal microscopy showed that at pH 6.5, the incubation of MB49 mouse bladder cancer cells and macrophages with fluorescein isothiocyanate-labelled-BSA (FITC-BSA) loaded and N-(Lissamine Rhodamine B sulfonyl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (rhodamine-DHPE) labelled 1:3:100 mPEG5kDa-DSPE/stearoyl-PEG-polySDM/lipid molar ratio liposomes resulted in a time-dependent liposome association with the cells. At pH 7.4, the association of BSA-loaded liposomes with the MB49 cells and macrophages was remarkably lower than at pH 6.5. Confocal images of bladder sections revealed that 2 h after the instillation, liposomes at pH 7.4 and control non-responsive liposomes at pH 7.4 or 6.5 did not associate nor delivered FITC-BSA to the bladder epithelium. On the contrary, the pH-responsive liposome formulation set at pH 6.5 and soon administered to mice by bladder instillation showed that, 2 h after administration, the pH-responsive liposomes efficiently delivered the loaded FITC-BSA to the bladder epitheliu

Layer-By-Layer Assembly of Graphene Oxide on Thermosensitive Liposomes for Photo-Chemotherapy

Stimuli responsive polyelectrolyte nanoparticles have been developed for chemo-photothermal destruction of breast cancer cells. This novel system, called layer by layer Lipo-graph (LBL Lipo-graph), is composed of alternate layers of graphene oxide (GO) and graphene oxide conjugated poly (l-lysine) (GO-PLL) deposited on cationic liposomesencapsulating doxorubicin. Various concentrations of GO and GO-PLL were examined and the optimal LBL Lipo-graph was found to have a particle size of 267.9 ± 13 nm, zeta potentialof +43.9 ± 6.9 mV and encapsulation efficiency of 86.4 ± 4.7%. The morphology of LBL Lipo-graph was examined by cryogenic-transmission electron microscopy (Cryo-TEM), atomic force microcopy (AFM) and scanning electron microscopy (SEM). The buildup of LBL Lipo-graph was confirmed via ultraviolet-visible (UV–Vis) spectrophotometry, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) analysis. Infra-red (IR) response suggests that four layers are sufficient to induce a gel-to-liquid phase transition in response to near infra-red (NIR) laser irradiation. Light-matter interaction of LBL Lipo-graph was studied by calculating the absorption cross section in the frequency domain by utilizing Fourier analysis. Drug release assay indicates that the LBL Lipo-graph releases much faster in an acidic environment than a liposome control. A cytotoxicity assay was conducted to prove the efficacy of LBL Lipo-graph to destroy MD-MB-231 cells in response to NIR laser emission. Also, image stream flow cytometry and two photon microcopy provide supportive data for the potential application of LBL Lipo-graph for photothermal therapy. Study results suggest the novel dual-sensitive nanoparticles allow intracellular doxorubin delivery and respond to either acidic environments or NIR excitation. Statement of Significance Stimuli sensitive hybrid nanoparticles have been synthesized using a layer-by-layer technique and demonstrated for dual chemo-photothermal destruction of breast cancer cells. The hybrid nanoparticles are composed of alternating layers of graphene oxide and graphene oxide conjugated poly-l-lysine coating the surface of a thermosensitive cationic liposome containing doxorubicin as a core. Data suggests that the hybrid nanoparticles may offer many advantages for chemo-photothermal therapy. Advantages include a decrease of the initial burst release which may result in the reduction in systemic toxicity, increase in pH responsivity around the tumor environment and improved NIR light absorption

In vivo stability of ester- and ether-linked phospholipid-containing liposomes as measured by perturbed angular correlation spectroscopy

To evaluate liposome formulations for use as intracellular sustained-release drug depots, we have compared the uptake and degradation in rat liver and spleen of liposomes of various compositions, containing as their bulk phospholipid an ether-linked phospholipid or one of several ester-linked phospholipids, by perturbed angular correlation spectroscopy. Multilamellar and small unilamellar vesicles (MLVs and SUVs), composed of egg phosphatidylcholine, sphingomyelin, distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC) or its analog dihexadecylglycerophosphorylcholine (DHPC), and cholesterol plus phosphatidylserine, and containing (111)In complexed to nitrilotriacetic acid, were injected intravenously in rats. Recovery of (111)In-labeled liposomes in blood, liver, and spleen was assessed at specific time points after injection and the percentage of liposomes still intact in liver and spleen was determined by measurement of the time-integrated angular perturbation factor ([G22(∞)] of the (111)In label. We found that MLVs but not SUVs, having DHPC as their bulk phospholipid, showed an increased resistance against lysosomal degradation as compared to other phospholipid-containing liposomes. The use of diacyl phospholipids with a high gel/liquid-crystalline phase-transition temperature, such as DPPC and DSPC, also retarded degradation of MLV, but not of SUV in the dose range tested, while the rate of uptake of these liposomes by the liver was lower

Influence of Micro-mixing on the Size of Liposomes Self-Assembled from Miscible Liquid Phases

Ethanol injection and variations of it are a class of methods where two miscible phases---one of which contains dissolved lipids---are mixed together leading to the self-assembly of lipid molecules to form liposomes. This method has been suggested, among other applications, for in-situ synthesis of liposomes as drug delivery capsules. However, the mechanism that leads to a specific size selection of the liposomes in solution based self-assembly in general, and in flow-focussing microfluidic devices in particular, has so far not been established. Here we report two aspects of this problem. A simple and easily fabricated device for synthesis of monodisperse unilamellar liposomes in a co-axial flow-focussing microfluidic geometry is presented. We also show that the size of liposomes is dependent on the extent of micro-convective mixing of the two miscible phases. Here, a viscosity stratification induced hydrodynamic instability leads to a gentle micro-mixing which results in larger liposome size than when the streams are mixed turbulently. The results are in sharp contrast to a purely diffusive mixing in macroscopic laminar flow that was believed to occur under these conditions. Further precise quantification of the mixing characteristics should provide the insights to develop a general theory for size selection for the class of ethanol injection methods. This will also lay grounds for obtaining empirical evidence that will enable better control of liposome sizes and for designing drug encapsulation and delivery devices.Comment: 11 pages, 14 Figure

Study of macroscopic and microscopic properties of liposomes produced using microfluidic methods

For the last decades, lipid vesicles or liposomes, vesicles formed by a bilayer of amphiphilic lipids, have been used as a toy model for studying the cell membrane and for applications in cosmetics and drug delivery. Traditional methods for producing liposomes face some problems such as the heterogeneity in size and composition of the liposomes produced. A few years ago, a novel method that produces liposomes with homogeneous size and composition was developed. This novel method is based on the use of water in oil in water ultra-thin double emulsions, with lipids dissolved in the oil phase, as templates for the liposome production. These ultra-thin double emulsions are produced using glass capillary microfluidic devices. This new method for producing liposomes seems very promising, but since the liposomes are formed by the oil phase evaporation of the double emulsions, the doubt that some residual oil in the bilayer may alter the properties of the liposomes appears. In this work different phenomena and properties of liposomes that have been studied for the ones produced using conventional methods are studied for liposomes produced using microfluidic methods. The microfluidic apprOutgoin

An intelligent liposome that may deliver drug molecules in a well controlled fashion

The passage of molecules, especially large ones, through the cellular membrane is a very important problem for some biotechnological applications, such as drug delivery. The appearance of pores in the lipid bilayer following some controlled mechanisms may be an adequate and interesting way. Some pores, named stochastic pores, can appear due to structural and dynamic properties of lipid bilayer, but others may be favored by mechanical tension induced by different ways. Recently, a sequence of 30-40 pores was observed in the same vesicle, a pore at a time, which can appear in vesicles stretched by optical induced mechanical tension. There are two very interesting biotechnological applications that require the increase of membrane permeability: gene therapy and targeted drug delivery. In the first one, the transport of DNA fragments through cellular and nuclear membranes is required. The second application uses drug molecules encapsulated in vesicles, which have to be transported to a target place. Having reached that point, one supposes that the liposome discharges its content by its breakdown. In this paper, we will write about how a lipid vesicle has to release the drug molecules in a well-controlled fashion. Such liposomes are named pulsatory liposomes and they induce cyclic activity. We will demonstrate that this liposome may be programmed to work a certain number of cycles, settled in advance. Also, we will calculate the amount of drug delivered during each cycle. In fact, a pulsatory liposome may be conceived as a drug dose micro device, which works according to a medical prescription established _a priori_