The Effect of Shear on the Desorption of Liposomes Adsorbed to Bacterial Biofilms

Abstract

With the aid of a flow cell assembly the desorption of cationic liposomes prepared from mixtures of dipalmitoylphoshatidylcholine (DDPC), cholesterol, and either dimethyldioctadecylammonium bromide (DDAB) or 3,β[N-(N¹,N-dimethylethylenediamine)-carbamoyl]cholesterol (DC-chol) from immoblized biofilms of Staphylococcus aureus has been studied as a function of shear stress by confocal microscopy. A shear stress theory has been adapted from fluid mechanics of laminar flow between parallel plates and used to determine the critical shear stress for liposome desorption. The critical shear stress for both DDAB and DC-chol liposomes has been determined as a function of cationic lipid content and hence surface charge as reflected in their zeta potentials. The critical shear stress has been used to obtain the potential energy of liposome–biofilm interaction which together with the electrostatic interaction energy has enabled estimates of the London-Hamaker constants to be made. The values of the London-Hamaker constants at small liposome-bacterial cell separation were found to be independent of liposome composition.     

Induction of alloreactive cytotoxic T lymphocytes by intra-splenic immunization with allogeneic class I Major Histocompatibility Complex DNA and DC-chol cationic liposomes

Abstract

A simple strategy for designing a cancer immunotherapeutic system involves modification of tumor cells from tumor-bearing animals in vivo in such a way that the host can evoke a specific immune response against them. We have expressed allogeneic class I major histocompatibility complex (MHC) molecules on tumor cells, through ex vivo DNA-mediated gene transfer. These molecules are potent immuno-modulators for the stimulation of strong immune reactions against certain malignancies. In order to achieve efficient gene delivery to tumor cells in vivo we have compared the efficiencies of gene transfer into mammalian tumor cells by the biolistic particle delivery system and cationic liposomes. In this report, we have demonstrated that cationic liposomes prepared by DC-chol and DOPE gives the best efficiency of transfection for tumor cells in vivo. We also showed that a strong anti-H-2K^b allo-reactive cytotoxic T lymphocyte (CTL) response could be generated following in vivo immunization of AKR/J mouse spleens with the H-2K^b gene and DC-chol cationic liposomes. The direct immunization of mouse spleens to induce cell-mediated immunity against exogenous antigens may allow alternative treatment strategies for cancer immunotherapy.     

Intranasal Immunization with DOTAP Cationic Liposomes Combined with DC-Cholesterol Induces Potent Antigen-Specific Mucosal and Systemic Immune Responses in Mice

Despite the progress made by modern medicine, infectious diseases remain one of the most important threats to human health. Vaccination against pathogens is one of the primary methods used to prevent and treat infectious diseases that cause illness and death. Vaccines administered by the mucosal route are potentially a promising strategy to combat infectious diseases since mucosal surfaces are a major route of entry for most pathogens. However, this route of vaccination is not widely used in the clinic due to the lack of a safe and effective mucosal adjuvant. Therefore, the development of safe and effective mucosal adjuvants is key to preventing infectious diseases by enabling the use of mucosal vaccines in the clinic. In this study, we show that intranasal administration of a cationic liposome composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N'',N''-dimethylaminoethane)-carbamoyl] (DC-chol) (DOTAP/DC-chol liposome) has a potent mucosal adjuvant effect in mice. Intranasal vaccination with ovalbumin (OVA) in combination with DOTAP/DC-chol liposomes induced the production of OVA-specific IgA in nasal tissues and increased serum IgG1 levels, suggesting that the cationic DOTAP/DC-chol liposome leads to the induction of a Th2 immune response. Additionally, nasal-associated lymphoid tissue and splenocytes from mice treated with OVA plus DOTAP/DC-chol liposome showed high levels of IL–4 expression. DOTAP/DC-chol liposomes also enhanced OVA uptake by CD11c⁺ dendritic cells in nasal-associated lymphoid tissue. These data demonstrate that DOTAP/DC-chol liposomes elicit immune responses via an antigen-specific Th2 reaction. These results suggest that cationic liposomes merit further development as a mucosal adjuvant for vaccination against infectious diseases.     

Targeted Delivery of Drugs and DNA with Liposomes

Abstract

This presentation is divided into three parts: long-circulating liposomes, immunoliposomes and gene transfer with liposomes. The mechanism of action for the poly(ethylene glycol)-phospholipid conjugates to prolong the circulation time of liposomes can be understood on the basis of steric barrier activity imposed by the flexible PEG chains on the liposome surface. The action of ganglioside GM₁, on the other hand, probably involves specific interactions with serum protein(s). Immunoliposomes can efficiently bind with the target only if the target is readily accessible and the liposomes stay in the circulation for a relatively long period of time. Coating the liposome surface with PEG chains or GM₁ enhances the target binding of immunoliposomes, except when PEG of greater than 5000 dalton is used. In this case, immunoliposome binding to the target is sterically hindered by the long PEG chains. To overcome the problem, antibody molecule is conjugated to the distal end of the PEG chain. This approach works well except that the liver uptake of immunoliposomes is somewhat enhanced. For the delivery of DNA into cells, a novel cationic amphiphile (DC-chol) is synthesized and is now used in clinical trials of gene therapy for melanoma. Current effort is concentrated on the means to enhance the level and duration of transgene expression.     

Enhanced Gene Delivery and Expression in Human Hepatocellular Carcinoma Cells by Cationic Immunoliposomes

Abstract

A targeted vector allowing enhanced gene transfer to human hepatocellular carcinoma (HCC¹) cells in vitro was developed using cationic liposomes covalently conjugated with the mAb AF-20. This high affinity antibody recognizes a rapidly internalized 180 kDa cell surface glycoprotein which is abundantly expressed on the surface of human HCC and other cancer cells. Quantitative binding analysis of liposomes with target cells by flow cytometry showed specific association of mAb-targeted liposomes with human HCC cells. Using mAb-targeted cationic liposomes containing 20% DOTAP, in the presence or absence of serum, gene expression in HuH-7 cells was enhanced up to 40-fold as compared to liposomes conjugated with an isotype-matched non-relevant control antibody. Transfection specificity was not observed in a control cell line that does not express the antigen recognized by mAb AF-20. This study demonstrates that cationic liposome formulations can be targeted with monoclonal antibodies (mAbs) to enhance specific in vitro gene delivery and expression in the presence or absence of serum.     

Liposome-mediated therapy of human immunodeficiency virus type-1 and mycobacterium infections

Abstract

We review our recent work on the use of liposomes for the delivery of antiviral agents to human immunodeficiency virus type-1 (HIV-1) infected cells, and antimycobactcrial drugs to cells harboring Mycobacterium avium complex or Mycobacterium tuberculosis. Soluble CD4 has been used to target liposomes to HIV-1-infected cells. Antisense oligodeoxynucleotides have been effectively delivered into HIV-1-infected macrophages using pH-sensitive liposomes. pH-sensitive liposomes with serum stability are being developed as in vivo delivery vehicles. Liposomes encapsulating an HIV-1 protease inhibitor were more effective in inhibiting virus production in infected macrophages than the free drug. Anionic liposomes were found to inhibit HIV-1 infectivity, while cationic liposomes had a differential toxicity for HIV-1-infected macrophages. Lipophilic sulfated cyclodextrins have been synthesized as novel antiviral agents. Liposome-encapsulated ciprofloxacin treatment reduced the number of viable M. avium in macrophages more than the free antibiotic. Liposome-encapsulated paromomycin and sparfloxacin were effective against M. tuberculosis inside macrophages, including multi-drug-resistant strains. Streptomycin encapsulated in liposomes and delivered intravenously or subcutaneously reduced the number of viable M. tuberculosis in infected mice and prevented mortality.     

Targeting of Liposomes Within Cardiovascular System

Abstract

Our studies on the targeting of liposomes and liposome-associated pharmaceuticals within the cardiovascular system are reviewed. The delivery of diagnostic and therapeutic agents in plain liposomes, immunoliposomes, long-circulating liposomes and long-circulating immunoliposomes into the sites of vascular injuries and myocardial infarction is discussed. In vitro, ex vivo, and in vivo experiments present a general view on the advantages and limitations of using liposome-mediated targeting. Liposomes capable of targeting pathological areas of the blood vessel wall both, in vitro and ex vivo are described, as well as liposome able to be internalized by normal endothelial cells. Liposome-mediated drug targeting to compromised myocardium is reviewed with a primary impact on liposomes with anti-cardiac myosin antibodies. Targeted visualization of myocardial infarction with diagnostic liposomes is discussed. Efficient accumulation of long-circulating immunoliposomes in the infarct zone is demonstrated, and a relative importance of different variables, such as liposome size, targetability, and prolonged circulation time, for target accumulation is analyzed. The use of immunoliposomes for targeted sealing of hypoxia-caused damages in plasmic membranes of cardiocytes is considered as a new approach in the therapeutic use of liposomes.     

Cationic Liposome-Mediated Gene Delivery Via Systemic Administration

Abstract

Cationic liposomes have been studied as a potential carrier for delivering genes to cells for the purpose of gene therapy. This report summarizes our efforts to characterize the in vivo expression of transgene delivered by cationic liposomes via intravenous administrtion. Using a CMV driven gene expression system containing cDNA of luciferase or green fluorescence protein gene as a reporter and two commonly used cationic lipids, 2, 3-dioleoyloxypropyl-1-trimethyl ammonium chloride (DOTMA) and 2, 3-dioleoyloxyl-1-trimethylammonium propanyl chloride (DOTAP), we demonstrate that a significant level of gene expression can be obtained in different organs including the lung, heart, spleen, liver and kidneys following intravenous administration in the mouse. Our finding show that the transfection efficiency of cationic liposomes is determined by the structure of the cationic lipids, the lipid composition of liposomes and cationic lipid to DNA ratio. Furthermore, gene expression was short in duration, peaked between 4-24 hours post injection, and dropped to less than 1% of the peak level within a 4 day period. Experiments with repeated injections revealed that cells initially transfected by the first transfection were not fully responsive to the subsequent second transfection for approximately 14 days.     

Liposomes loaded with a dirhenium compound and cisplatin: preparation,properties and improved in vivo anticancer activity

Abstract

Liposomes loaded with the rhenium compound (bis-dimethylsulfoxido-cis-tetrachlorodi-μ-pivalatodirhenium(III) (cis–Re₂((CH₃)₃CCOO)₂Cl₄?2DMSO, I) and cisplatin in the molar ratio of 4:1 as well as those loaded only with I were synthesized and characterized by scanning electron microscopy, transmission electron microscopy, dynamic light scattering and electronic absorption spectroscopy. The relative stability of liposomes loaded with I is reflected by a minimal change in the electronic absorption spectra over a period of 8 days whereas the stability of those loaded with both drugs is lower, which we ascribe to the formation of new Re-Pt species inside the liposomes. Furthermore, the investigations of the co-encapsulation effects on the anticancer activity of the Re-Pt system were undertaken. Importantly, the co-encapsulated liposomes exhibit synergistic or additive anticancer activities in vivo, e.g. introduction of these liposomes into tumor-bearing rats demonstrated their antianemic, nephro- and hepato-protecting effects. These liposomes, which are active in cancer treatment, protect the dirhenium compounds from hydrolysis and preserve the biological properties of the Re-Pt hybrid. This study reveals the importance of combined therapy in nanotechnology and medicine.     

Ganglioside GM1 and Hydrophilic Polymers Increase Liposome Circulation Times by Inhibiting the Association of Blood Proteins

Abstract

Several agents have been shown to prolong the circulation lifetime of liposomes. These agents, such as ganglioside G_M1 or phosphatidylethanolamine-derivatives of monomethoxypolyethyleneglycols, provide insight into the mechanism(s) by which liposomes are cleared from the circulation. It is suggested here that the primary mechanism by which these molecules alter the biodistribution of liposomes in vivo involves an inhibition of the association of blood proteins to liposomes, resulting in a diminished rate of clearance of liposomes from the circulation.     

Impact of Mycobacterium Avium Infection on Macrophage-Liposome Interaction

Abstract

Mycobacterium avium (M. avium) can survive within macrophages, and alters various functions of its cellular host. Liposomes and other particulate drug carriers have been investigated as a means for enhancing drug delivery to the intracellular site of infection via the endocytic pathway. We have investigated the impact of M. avium infection on the endocytosis, retention, and intracellular disposition of liposomes, as these are essential macrophage functions that may determine the activity of liposome-encapsulated antibiotics. J774, a macrophage-like cell line, was infected with M. avium at various multiplicities. Infected cells accumulated and retained liposomes in a manner similar to uninfected cells, regardless of the time after infection. Moreover, the intracellular trafficking and disposition of liposomes was unaltered in J774 cells that contained live or heat-killed M. avium, as evidenced by the vesicular pH which liposomes encountered within cells. These results suggest that specific essential cellular functions involved in processing particulate drug carriers remain intact in macrophages infected with M. avium.     

Cationic liposome and plasmid DNA complexes formed in serum-free medium under optimum transfection condition are negatively charged

In medium where in vitro transfection is routinely performed, DC-chol liposomes alone were nearly neutral, whereas the DC-chol liposome/DNA complexes were largely negatively charged which changed only slightly at all [liposome]/[DNA] ratios (zeta=-27.1 to -21.8 mV). Three other commercial transfection reagents, Lipofectin(R), LipofectAMINE 2000, and SuperFect, were also largely negatively charged when complexed with DNA. The aggregation of liposomes in medium was prevented by the addition of DNA. Incubation of the complexes in medium did not change their size, charge or lipofection activity for 30 min. These results suggest that, in medium, the liposome/DNA complexes were formed at the time of mixing with negative charges.     

The effect of shear on the desorption of liposomes adsorbed to bacterial biofilms

With the aid of a flow cell assembly the desorption of cationic liposomes prepared from mixtures of dipalmitoylphoshatidylcholine (DDPC), cholesterol, and either dimethyldioctadecylammonium bromide (DDAB) or 3,beta[N-(N1,N-dimethylethylenediamine)-carbamoyl]cholesterol (DC-chol) from immobilized biofilms of Staphylococcus aureus has been studied as a function of shear stress by confocal microscopy. A shear stress theory has been adapted from fluid mechanics of laminar flow between parallel plates and used to determine the critical shear stress for liposome desorption. The critical shear stress for both DDAB and DC-chol liposomes has been determined as a function of cationic lipid content and hence surface charge as reflected in their zeta potentials. The critical shear stress has been used to obtain the potential energy of liposome-biofilm interaction which together with the electrostatic interaction energy has enabled estimates of the London-Hamaker constants to be made. The values of the London-Hamaker constants at small liposome-bacterial cell separation were found to be independent of liposome composition.     

Novel liposome-based formulations for prolonged delivery of proteins and vaccines

Abstract

Two strategies for increasing liposome stability in vivo are described in this review. The first strategy involves the encapsulation of liposomes within polymeric microcapsules of alginate-poly(L-lysine) that retained the liposomes inside but allowed the outward diffusion of proteins of 100 kDa or less, once they were released from the encapsulated liposomes. In vivo studies revealed that the microencapsulated liposome systems (MELs) extended the delivery of a model antigen, bovine serum albumin (BSA), for more that 80 days, resulting in the prolonged production of high levels of antigen-specific antibodies. The antibody levels were higher that those obtained with rats injected with BSA in complete Freund's adjuvant, or in liposomes. The unique construction of MELs enabled also the enzymatically-triggered pulsatile delivery of proteins from encapsulated liposomes, which was not possible before with liposomes.     

Cationic Liposomes Containing Disulfide Bonds in Delivery of Plasmid DNA

Abstract

Cationic liposomes are non-viral gene transfer vectors for in vitro and in vivo experiments. In the present studies, we investigated whether a disulfide linkage in a cationic lipid was reducible by cell lysate resulting in the release of plasmid DNA and enhanced gene transfection. We also investigated if the differences in transgene production were from differences in total amount of cellular associated plasmid DNA. We systematically compared the gene transfection of disulfide bond containing-cationic lipid, 1', 2'-dioleoyl-sn-glycero-3'-succinyl-2-hydroxyethyl disulfide ornithine conjugate (DOGSDSO), its non-disulfide-containing analog, 1', 2'-dioleyl-sn-glycero-3'-succinyl-1, 6-hexanediol ornithine conjugate (DOGSHDO), 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP). Two transgene reporter systems (i.e., luciferase and green fluorescent protein (GFP)) were used to address transgene transgene expression and transgene efficiency. Experiments with the luciferase expression plasmid resulted in transgene activity up to 11 times greater transgene production for the disulfide containing lipid in at least two different cell lines, COS 1 and CHO cells. When transgene expression was determined by GFP activity, DOGSDSO liposomes were four times greater than the non-disulfide lipid or positive control (DOTAP) liposomes. By quantifying nucleic acid uptake by flow cytometry it was also demonstrated that increase expression was not solely from an increase in cellular plasmid DNA accumulation. These results demonstrate that cationic lipids containing a disulfide linkage are a promising method for gene transfer.     

Lipid reducing potential of liposomes loaded with ethanolic extract of purple pitanga (Eugenia uniflora) administered to Caenorhabditis elegans

Abstract

The ethanolic extract obtained from purple pitanga fruit (Eugenia uniflora – PPE) has been previously described by its potential to reduce lipid accumulation in vitro. In this study, we aimed to study this potential in vivo using Caenorhabditis elegans as animal model. Considering the low pH of the extract, its hydrophilic characteristic, its absorption by the medium where the worms are cultivated and the need of a chronic exposure in the worms solid medium, we have loaded liposomes with PPE and investigated its potential for oral administration. Following 48?h exposure to the PPE-loaded liposomes on worms nematode growth medium, we did not observe any toxic effects of the formulation. Under high cholesterol diet, which increased worms total lipid and also triacylglycerides levels, liposomes containing PPE were able to significantly attenuate these alterations, which could not be observed when worms were treated with free PPE. Furthermore, we could evidence that liposomes were ingested by worms through their labelling to uranin fluorescence dye. Through total phenolic compounds quantification, we estimated an entrapment efficacy of PPE into liposomes of 87.7%. The high levels of phenolic compounds present in PPE, as previously described by our group, indicate that these antioxidants may interfere in worms lipid metabolism, which may occur through many and intricated mechanisms. Although the use of conventional liposomes for human consumption may not be pragmatic, its application for oral delivery of a hydrophilic substance in C. elegans was absolutely critical for our experimental design and has proven to be efficient.     

Pore size and properties of channels from mitochondria isolated fromNeurospora crassa

Summary A triton X100 extract of mitochondria, isolated from a wall-less mutant ofNeurospora crassa, can be used to insert channels into planar lipid bilayers. These channels have the same properties as the VDAC channels previously reported (Colombini, 1979,Nature (London) 279:643) in the outer membrane of rat liver mitochondria. When large multiwalled liposomes are produced from mixtures of phospholipids andN. crassa mitochondrial membrane material, these liposomes are now permeable to nonelectrolytes up to the size of polyethylene glycol of 3400 mol wt. This yields an estimated radius for the channels inserted into the liposomes of 20 Å.It is proposed that VDAC is the channel which allows the outer mitochondrial membrane to be permeable to small molecules and that this channel has a pore size of 20 Å in radius.     

Phospholipases. II. Enzymatic hydrolysis of lecithin: Effects of structure,cholesterol content,and sonication

Summary Hydrolysis of unsonicated liposomes of egg lecithin catalyzed by several phospholipases is markedly activated by addition ofn-alkanols [Jain & Cordes,J. Membrane Biol. 14:101 (1973)]. Further pursuit of these systems has established that several factors, including higher temperatures, increasing unsaturation of fatty acyl chains of the substrate, incorporation of cholesterol into the liposomes, and sonication, reduce the concentration ofn-hexanol required to elicit maximal activation for enzymatic hydrolysis. Moreover, sonication or incorporation of cholesterol into lecithin liposomes reduces from C₈ to C₇ and C₆, respectively, the chain length of that alcohol eliciting maximal activation. These results are consistent with the hypothesis that sonication and increasing cholesterol content lead to liposomes which have a diminished thickness of the hydrocarbon region compared to that for unmodified liposomes derived from the same lecithin.     

Improved Encapsulation of DNA in pH-Sensitive Liposomes for Transfection

Abstract

A simple method has been developed to prepare liposomes containing large amounts of DNA. The procedure consisted of three cycles of freeze-thawing a mixture of sonicated liposomes and DNA. The encapsulation efficiency depended on the size of DNA. For a small plasmid (2.7 kb), approximately 40% of input DNA was entrapped with an efficiency of 16 μgDNA/μmol lipid. For larger plasmids, the encapsulation efficiency decreased considerably. Transfection of cultured mouse L929 cells mediated by the DNA-containing liposomes was assayed with a plasmid containing the E. coli chloramphenicol acetyl transferase gene. The transfection activity of the liposome was primarily determined by its pH sensitivity. Acid-sensitive liposomes transfected cells efficiently, whereas pH-insensitive liposomes were much less active. The level of the expression of the exogenous gene in the treated cells could be further modulated by protein kinase C (PKC) activators that were incorporated into the liposomal membrane as a minor lipid component. Transfection conditions were optimized with respect to DNA, lipid, and PKC activator concentrations. The results of the current study may help the use of liposomal delivery system for applications in gene therapy.     

Gene Transfer to Lentil Protoplasts by Lipofection and Electroporation

Abstract

Cationic liposomes made of dipalmitoylphosphatidylcholine and stearylamine (9:1) were prepared by reverse-phase evaporation and were able to interact spontaneously with plasmid DNA. The loaded vesicles delivered a β-glucuronidase (GUS)-carrying plasmid to lentil Lens culinaris) protoplasts, leading to transient expression of the GUS reporter gene. The transfection efficiency achieved by using stearylamine-containing liposomes (lipofection) was comparable to the one obtained by electroporating the protoplasts at 500 μF and different field strengths. Furthermore, the combination of electroporation and lipofection, though reducing cell survival, increased the activity of the reporter enzyme detected in the cell lysates, yielding transient expression levels higher than those recorded after lipofection or electroporation alone.