Search

New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.

Several plasminogen activators (PAs) have been found effective in treating different thromboembolic diseases. However, administration of conventional thrombolytic therapy is limited by a low efficacy of present formulations of PAs. Conventional treatments using these therapeutic proteins are associated with several limitations including rapid inactivation and clearance, short half-life, bleeding complications or non-specific tissue targeting. Liposome-based formulations of PAs such as streptokinase, tissue-plasminogen activator and urokinase have been developed to improve the therapeutic efficacy of these proteins. Resulting liposomal formulations were found to preserve the original activity of PAs, promote their selective delivery and improve thrombus targeting. Therapeutic potential of these liposome-based PAs has been demonstrated successfully in various pre-clinical models in vivo. Reductions in unwanted side effects (e.g., hemorrhage or immunogenicity) as well as enhancements of efficacy and safety were achieved in comparison to currently existing treatment options based on conventional formulations of PAs. This review summarizes present achievements in: (i) preparation of liposome-based formulations of various PAs, (ii) development of PEGylated and targeted liposomal PAs, (iii) physico-chemical characterization of these developed systems, and (iv) testing of their thrombolytic efficacy. We also look to the future and the imminent arrival of theranostic liposomal formulations to move this field forward., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Liposomal nanoparticles have proven to be versatile systems for drug delivery. However, the progress in clinic has been slower and less efficient than expected. This suggests a need for further development using carefully designed chemical components to improve usefulness under clinical conditions and maximize therapeutic effect. For cancer chemotherapy, PEGylated liposomes were the first nanomedicine to reach the market and have been used clinically for several years. Approaches toward targeted drug delivery using next generation "thermally triggered" nanoparticles are now in clinical trials. However, clinically tested thermosensitive liposomes (TSLs) lack the markers that allow tumor labeling and improved imaging for tissue specific applied hyperthermia. Here we describe the development of optically labeled TSLs for image guidance drug delivery and proof-of-concept results for their application in the treatment of murine xenograft tumors using the anticancer drug topotecan. These labeled TSLs also allow the simultaneous, real-time diagnostic imaging of nanoparticle biodistribution using a near-infrared (NIR; 750-950 nm) fluorophore coupled to a lipidic component of the lipid bilayer. When combined with multispectral fluorescence analysis, this allows for specific and high sensitivity tracking of the nanoparticles in vivo. The application of NIR fluorescence-labeled TSLs could have a transformative effect on future cancer chemotherapy.

Background: Nonviral lipid-based gene delivery vectors have been shown to possess better stability and a longer circulation time after surface poly(ethylene glycol) PEG modification. However, surface PEGylation may decrease the transfection efficiency dramatically. In the present study, we addressed the hypothesis that down-regulating lysosomal processing with a clinical available proton pump inhibitor omeprazole might decrease the sequestration of PEGylated Lipid-Mu-DNA (LMD) in intracellular organelles, thereby increasing their transfection efficiencies., Methods: LMD nanoparticles were prepared by the self-assembling of cationic liposomes, peptide Mu and plasmid DNA. The characteristics of LMD lipopolyplexes were detected by scanning electron microscopy, photon correlation spectroscopy and DNA gel electrophoresis. They were added to cultured cells with or without omeprazole pretreatment. The detailed cellular uptake and subcellular distribution were followed by flow cytometry and confocal microscopy. Gene transfection efficiencies were evaluated in four cell lines, as well as in xenograft tumor models., Results: Lysosome staining revealed that 0.1 mg/ml (290 μM) omeprazole raised the pH value of intracellular acidic organelles and induced alterations in the lysosomal compartments. Confocal microscopy showed that more gene materials distributed from intracellular organelles to cytoplasms with omeprazole treatment. A luciferase gene transfection assay showed that omeprazole (probably at a nontoxic concentration) increased PEGylated LMD transfection efficiency significantly in human (NCI-H1299, HT-1080 and A375) and mouse (4T1) cell lines, as well as in tumors of H1299 xenograft tumor models (p < 0.05)., Conclusions: Omeprazole might be used as a helper to improve gene transfection efficiencies of some PEGylated gene delivery vectors both in vitro and in vivo., (Copyright © 2013 John Wiley & Sons, Ltd.)

Liposomes represent a biocompatible platform for the construction of self-assembling proteoliposomes using nickel or zinc metallochelation. Potential applications of such structures consist in the development of new biocompatible vaccination nanoparticles and drug delivery nanoparticle systems. Here, we describe the design and construction of a flow-through ultrafiltration cell suitable for the preparation of monodisperse liposomes enabled for metallochelation and, hence, the formation of proteoliposomes. The linkage of the cell with a fast protein liquid chromatography system facilitates automation of the procedure, which fits the criteria for upscaling. Proof-of-concept experiments are performed using a mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (1,2-dioleoyl-sn-glycero-3-{[N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl}(nickel salt)) to formulate proteoliposomes with proteins attached by metallochelation, including histidine (His)-tagged recombinant green fluorescent protein and rgp120 (derived from HIV-1 Env). These model proteoliposomes are characterized by gel permeation chromatography and by dynamic light scattering. Transmission electron microscopy and immunogold staining are used to characterize surface-bound proteins, revealing the tendency of rgp120 to form microdomains on liposome surfaces. These microdomains possess a two-dimensional crystal-like structure that is seen more precisely by atomic force microscopy., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In this review we discuss the development of paramagnetic liposomes incorporating MRI contrast agents and show how these are utilized in cellular imaging in vitro. Bi-functional, bi-modal imaging paramagnetic liposome systems are also described. Next we discuss the upgrading of paramagnetic liposomes into bi-modal imaging neutral nanoparticles for in vivo imaging applications. We discuss the development of such systems and show how paramagnetic liposomes and imaging nanoparticles could be developed as platforms for future multi-functional, multi-modal imaging theranostic nanodevices tailor-made for the combined imaging of early stage disease pathology and functional drug delivery.

Epidermal growth factor receptor (EGF-R) is an important target in anticancer therapy. Here we report how a novel EGF-R peptide ligand (D4: Leu-Ala-Arg-Leu-Leu-Thr) is identified using a computer-aided design approach from a virtual peptide library of putative EGF-R binding peptides by screening against the EGF-R X-ray crystal structure in silico and in vitro. The selected peptide is conjugated with a polyethylene glycol (PEG) lipid, and the lipid moiety of the peptide-PEG-lipid conjugate is inserted into liposome membranes by a postmodification process. D4 peptide-conjugated liposomes are found to bind to and enter cells by endocytosis specifically and efficiently in vitro in a process apparently mediated by EGF-R high-expressing cancer cells (H1299). In vivo, the D4 peptide-conjugated liposomes are found to accumulate in EGF-R-expressing xenograft tumor tissues up to 80 h after intravenous delivery, in marked contrast to controls. These results demonstrate how structure-based peptide design can be an efficient approach to identify highly novel binding ligands against important receptors. These data could have important consequences for the development of peptide-directed drug delivery systems with engineered specificities and prolonged times of action.

Folate-targeted bimodal paramagnetic and fluorescent liposomes were developed and showed enhanced accumulation in a folate receptor expressing tumor model. These bimodal liposomes were composed of both a paramagnetic and a fluorescent lipid, and utilized a PEG-lipid amphiphile for prolonged in vivo circulation. The particles were formulated to ensure a size distribution of approximately 100 nm with a low polydispersity index. IGROV-1 cells were used to induce tumors in nude Balb/c mice, and the folate-targeted liposomes were injected intravenously. Rapid accumulation of the folate-targeted liposomes within the tumor tissue compared to nontargeted liposomes was observed. Furthermore, folate-labeled liposomes showed a 4-fold increase in tumor T(1) signal intensity at just 2 h postinjection with similar results being obtained for the nontargeted liposomes only 24 h postinjection. In addition, the folate-targeted liposomes were injected at half the nontargeted liposome dose, further demonstrating their effectiveness. Histological analysis of sectioned tumor slices revealed distinct fluorescence patterns between the targeted and nontargeted systems, with a more localized and hyperintense fluorescence signal observed from tumor sections post-folate-targeted liposome injections. These results demonstrate the effectiveness of folate targeting for dynamic real-time solid tumor MRI and provide insight into kinetics of targeted and nontargeted nanoparticles to solid tumors.

Targeted transfection of the CNS with synthetic, nonviral vectors represents a huge technical challenge. The approach explored here attempts to combine self-assembly ABCD nanoparticles (Kostarelos and Miller, Chem. Soc. Rev. 2005, 34, 970), with the potential of Clostridium neurotoxin fragments to effect receptor-mediated transfection of neuronal cells. Cationic liposome-plasmid DNA complexes were first modified with a PEG stealth layer, before the addition of C-terminal fragments of tetanus toxin (TH(C)), botulinum toxin (BH(C)) or the truncated C-terminal domain of TH(C) as biological "targeting" ligands. First-generation nanoparticles were identified for the transfection of two neuronal cell lines (human SH-5YSY and rat/mouse hybrid N18-RE105); control studies were also performed with HeLa cells. ABCD nanoparticle transfections of the neuronal cell lines were up to 30-fold higher than corresponding control transfections with nanoparticles that lacked the protein ligand. We also demonstrate apparent receptor-mediated uptake by means of competition-binding and real-time confocal experiments. By contrast, nanoparticle transfection of HeLa cells appeared to involve alternative nonspecific enhanced cellular uptake mechanism(s). Receptor-mediated and nonspecific mechanisms appear to be in competition, potentially harming the capacity of receptor-mediated delivery to effect proper targeted delivery of nucleic acids to cells ex vivo and in vivo.

A novel bimodal fluorescent and paramagnetic liposome is described for cellular labeling. In this study, we show the synthesis of a novel gadolinium lipid, Gd.DOTA.DSA, designed for liposomal cell labeling and tumor imaging. Liposome formulations consisting of this lipid were optimized in order to allow for maximum cellular entry, and the optimized formulation was used to label HeLa cells in vitro. The efficiency of this novel bimodal Gd-liposome formulation for cell labeling was demonstrated using both fluorescence microscopy and magnetic resonance imaging (MRI). The uptake of Gd-liposomes into cells induced a marked reduction in their MRI T 1 relaxation times. Fluorescence microscopy provided concomitant proof of uptake and revealed liposome internalization into the cell cytosol. The optimized formulation was also found to exhibit minimal cytotoxicity and was shown to have capacity for plasmid DNA (pDNA) transfection. A further second novel neutral bimodal Gd-liposome is described for the labeling of xenograft tumors in vivo utilizing the enhanced permeation and retention effect (EPR). Balb/c nude mice were inoculated with IGROV-1 cells, and the resulting tumor was imaged by MRI using these in vivo Gd-liposomes formulated with low charge and a poly(ethylene glycol) (PEG) calyx for long systemic circulation. These Gd-liposomes which were less than 100 nm in size were shown to accumulate in tumor tissue by MRI, and this was also verified by fluorescence microscopy of histology samples. Our in vivo tumor imaging results demonstrate the effectiveness of MRI to observe passive targeting of long-term circulating liposomes to tumors in real time, and allow for MRI directed therapy, wherein the delivery of therapeutic genes and drugs to tumor sites can be monitored while therapeutic effects on tumor mass and/or size may be simultaneously observed, quantitated, and correlated.

Cellular entry of imaging probes, such as contrast agents for magnetic resonance imaging (MRI), is a key requirement for many molecular imaging studies, particularly imaging intracellular events and cell tracking. Here, we describe the successful development and in vitro analysis of MAGfect, a novel liposome formulation containing a lipidic gadolinium contrast agent for MRI, Gd-DOTA-Chol , designed to enter and label cells. Liposome formulation and cell incubation time were optimised for maximum cellular uptake of the imaging probe in a variety of cell lines. MRI analysis of cells incubated with MAGfect showed them to be highly MRI active. This formulation was examined further for cytotoxicity, cell viability and mechanism of cell labelling. One of the key advantages of using MAGfect as a labelling vehicle arises from its potential for additional functions, such as concomitant drug or gene delivery and fluorescent labelling. The gadolinium liposome was found to be an effective vehicle for transport of plasmid DNA (pDNA) into cells and expression levels were comparable to the commercial transfection agent Trojene.

The need for safe and structurally defined immunomodulators and adjuvants is increasing in connection with the recently observed marked increase in the prevalence of pathological conditions characterized by immunodeficiency. Important groups of such compounds are muramyl glycopeptides, analogs of muramyl dipeptide (MDP), glucosaminyl-muramyl dipeptide (GMDP), and desmuramylpeptides. We have designed and synthesized new types of analogs with changes in both the sugar and the peptide parts of the molecule that show a high immunostimulating and adjuvant activity and suppressed adverse side effects. The introduction of lipophilic residues has also improved their incorporation into liposomes, which represent a suitable drug carrier. The proliposome-liposome method is based on the conversion of the initial proliposome preparation into liposome dispersion by dilution with the aqueous phase. The description of a home-made stirred thermostated cell and its link-up with a liquid delivery system for a rapid and automated preparation of multilamellar liposomes at strictly controlled conditions (sterility, temperature, dilution rate and schedule) is presented. The cell has been designed for laboratory-scale preparation of liposomes (300-1000 mg of phospholipid per run) in a procedure taking less than 90 min. The method can be readily scaled up. Examples of adjuvant and immunostimulatory effect of liposomal preparation in mice model will be presented.

Positively-charged gene delivery agents, such as cationic liposomes, typically prepared by mixing a cationic lipid and a neutral lipid in a 1 : 1 molar ratio, exhibit a fundamental flaw: on the one hand, the charge encourages cell uptake; on the other hand, the charge leads to aggregation in vivo with anionic serum components. We herein report a more phase-stable analogue of the zwitterionic and fusogenic lipid DOPE that allows for the reduction of the cationic lipid component of the liposome from 50 to 9 mol% with almost no apparent loss in transfection activity. This reduction in charge may induce important in vivo stability whilst still imparting high cell uptake and transgene expression.

Gene therapy research is still in trouble owing to a paucity of acceptable vector systems to deliver nucleic acids to patients for therapy. Viral vectors are efficient but may be too dangerous for routine clinical use. Synthetic non-viral vectors are inherently much safer but are currently not efficient enough to be clinically viable. The solution for gene therapy lies with improved synthetic non-viral vectors based upon well-found platform technologies and a thorough understanding of the barriers to efficient gene delivery and expression (transfection) relevant to clinical applications of interest. Here we introduce and interpret synthetic non-viral vector systems through the ABCD nanoparticle structural paradigm that represents, in our view, an appropriate lens through which to view all synthetic, non-viral vector systems applicable to in vitro use or in vivo applications and gene therapy. Our intention in introducing this paradigm is to shift the focus of organic and physical chemists away from the design of yet another cytofectin, and instead encourage them to appreciate the wider challenges presented by the need to produce tool kits of meaningful chemical components from which to assemble viable, tailor-made nanoparticles for in vivo applications and gene therapy, both now and in the future.

Novel carbohydrate-based agents for the stabilization of ternary liposome:mu:DNA (LMD) nonviral vector systems are described. LMD vector systems comprise plasmid DNA (pDNA; D,7.5 kb) expressing a reporter gene (in this instance beta-galactosidase expressing gene) that is precondensed with the adenoviral core peptide mu (mu, M; MRRAHHRRRRASHRRMRGG) and then further packaged by means of DC-Chol:DOPE (3:2; m/m) cationic liposomes. Final optimized lipid:mu:pDNA ratio is typically 12:0.6:1 (w/w/w). We report the synthesis of a series of nine neoglycolipids prepared by coupling completely unprotected sugar monomers or oligomers (mannose, glucose, galactose, glucuronic acid, maltose, lactose, maltotriose, maltotetraose, and maltoheptaose) through their reducing-residue termini to an aminoxy-functionalized cholesterol-based lipid. Characterization of these novel neoglycolipids by (1)H NMR reveals that the coupling reaction has a major configurational preference for the beta-anomer. Unusually, even mannose coupling results in a neoglycolipid product with a predominantly beta-anomeric conformation (>85%). Formulation of neoglycolipids into LMD vector systems by incubation of LMD particles with neoglycolipid micelles results in the formation of a range of potential stabilized-LMD (sLMD) vector systems. Those potential sLMD systems prepared with longer chain neoglycolipids are found to have enhanced stabilities, with respect to aggregation in high ionic strength buffers, and enhanced transfection efficacies in comparison to the transfection properties of the naked first generation LMD vector system (i.e., gene delivery and expression). By contrast, when LMD vector systems are incubated with poly(ethylene glycol) DSPE-PEG micelles, resulting PEG-LMD vector systems are very stable with respect to colloidal instablility and aggregation in high ionic strength buffers and in serum, but are completely refractory to transfection. These data suggest that oligosaccharides could represent an alternative to PEG as a stealth polymer able to stabilize synthetic nonviral vector systems in some fluids but without impairing transfection efficiency. Furthermore, sLMD systems prepared with longer chain neoglycolipids appear to have sufficient useful characteristics to form the basis of viable second-generation LMD vector systems after further development.

Previously we have described the development and applications of an important new platform system for gene delivery known as liposome-mu-DNA (LMD), prepared from cationic liposomes (L), plasmid DNA (D) and the mu(M) peptide derived from the adenovirus core. In an attempt to improve upon mu, an alternative peptide (pepV) derived from the adenovirus peptide/protein-DNA core complex was identified, synthesised and studied alongside mu using a number of biophysical techniques including gel retardation, ethidium bromide exclusion, CD binding titration, DNA melting, and plasmid protection assays. PepV binds to pDNA less efficiently than mu but is able to charge neutralise and condense pDNA into negatively charged pepVD particles comparable in dimension to MD particles. The results of CD studies and plasmid protection assays suggest that peptide-DNA interactions are likely to cause pDNA condensation by a combination of charge neutralisation, base pair tilting, double helix destabilisation and the induction of pDNA superfolding. Data suggest the pepVD particles may be formulated with cationic liposomes to give defined LpepVD particles that appear to transfect HeLa cells with marginally more efficiency than LMD particles suggesting that pepV may have some effect on the pDNA transcription process. Although pepV harbours a nuclear-nucleolar localisation sequence (NLS), transfection data show that this capacity is not being appropriately harnessed by the current LpepVD formulation. Further improvements may be required in terms of optimising LpepVD formulations--for instance, to ensure the integrity of the peptide-DNA complexes following cell entry--in order to fully exploit the full NLS capacity of the peptide, thereby facilitating the transfection of slowly dividing or quiescent cells.

Gene therapy research is in crisis owing to the lack of acceptable vector systems to deliver nucleic acids to patients for therapy. Viral vectors are efficient but currently appear to be too dangerous for routine clinical use. Synthetic non-viral vectors are inherently much safer but are currently not efficient enough to be clinically viable. The solution for gene therapy lies with improved synthetic non-viral vectors based upon well-found platform technologies and a thorough understanding of the barriers to efficient gene delivery and expression (transfection) relevant to clinical applications of interest. In this review, the current status and prospects for cationic liposome/micelle-based synthetic non-viral vector systems are discussed including a description of the barriers to efficient transfection, a summary of the main structure/activity studies and mention of ternary cationic liposome/micelle-nucleic acid (LD) systems. The review culminates with a description of two promising cationic liposome/micelle-based non-viral vector platform systems known as liposome:mu:DNA (LMD) and stabilised plasmid-lipid particles (SPLP) that should create a real opportunity for the development of clinically viable synthetic vector systems within the next few years.

The DNA complexation and condensation properties of two established cationic liposome formulations, CDAN/DOPE (50:50, m/m; Trojene) and DC-Chol/DOPE (60:40, m/m), were investigated by using a combination of isothermal titration calorimetry (ITC), circular dichroism (CD), photon correlation spectroscopy (PCS), and turbidity assays. Plasmid DNA (7528 bp) was titrated with extruded liposomes (90 +/- 15 nm) and a thermodynamic profile established. ITC data revealed that the two liposome formulations differ substantially in their DNA complexation characteristics. Equilibrium dissociation constants for CDAN/DOPE (K(d) = 19 +/- 3 microM) and DC-Chol/DOPE liposomes (K(d) = 2 +/- 0.5 microM) were obtained by fitting the experimental data in a one-site binding model. Both CDAN/DOPE and DC-Chol/DOPE binding events take place with a negative binding enthalpy (DeltaH degrees = -0.5 and -1.7 kcal/mol, respectively) and increasing system entropy (TDeltaS = 6 +/- 0.3 and 6.2 +/- 0.3 kcal/mol, respectively). Interestingly, CDAN/DOPE liposomes undergo substantial rehydration and protonation prior to complexation with pDNA, which is observed as two discrete exothermic signals during titration. No such biphasic effects are seen with respect to the binding between DC-Chol/DOPE and pDNA that appears to be otherwise instantaneous with no rehydration effects. The rehydration and protonation characteristics of CDAN/DOPE liposomes in comparison with those of DC-Chol/DOPE cationic liposomes are confirmed by ITC; CDAN/DOPE liposomes have strongly exothermic dilution characteristics and DC-Chol/DOPE liposomes only mildly endothermic characteristics. Furthermore, analysis of cationic liposome-pDNA binding by CD spectroscopy reveals that CDAN/DOPE-pDNA lipoplexes are more structurally fluid than DC-Chol/DOPE-pDNA lipoplexes. CDAN/DOPE liposomes induced considerable fluctuation in the DNA structure for at least 60 min, whereas liposomes obtained from DC-Chol/DOPE lack the same effect on the DNA structure. Turbidity studies show that DC-Chol/DOPE lipoplexes exhibit greater resistance to serum than CDAN/DOPE lipoplexes, which showed substantial precipitation after incubation for 100 min with serum. Transfection studies on HeLa and Panc-1 cells reveal that CDAN/DOPE lipoplexes are superior in efficacy to DC-Chol/DOPE lipoplexes. CDAN/DOPE liposomes tend to transfect best in normal growth medium (including 10% serum and antibiotics), whereas DC-Chol/DOPE lipoplexes transfect best under serum free transfection conditions.

The decoration of nanoparticle surfaces with receptor-specific ligands can improve the delivery of their therapeutic load to cancer cells. Using as the ligand, U11, a peptide specific for the cancer-associated urokinase plasminogen activator receptor, we examined the effect of manipulation of nanoparticle properties on the targeting specificity of U11-nanoparticles. The nanoparticle surface charge was controlled by varying the amount of the cationic lipid within the nanoparticle formulation. A reduction in surface charge led to an increase in the targeting effect of U11-nanoparticles, a result of limiting its non-specific interactions with negatively charged cellular membranes. Furthermore, fluorescence and circular dichroism spectroscopy were used to probe changes in the U11 presentation on the surfaces of nanoparticles. Optimized ligand conformations were when ligands were separated into monomers and protruded from the nanoparticle surface. It is concluded that both the ligand organization as well as the nanoparticle platform require control to give optimal cell uptake efficiencies. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (EGF-R) is an important target in anticancer therapy. Here we report how a novel EGF-R peptide ligand (D4: Leu-Ala-Arg-Leu-Leu-Thr) is identified using a computer-aided design approach from a virtual peptide library of putative EGF-R binding peptides by screening against the EGF-R X-ray crystal structure in silico and in vitro. The selected peptide is conjugated with a polyethylene glycol (PEG) lipid, and the lipid moiety of the peptide-PEG-lipid conjugate is inserted into liposome membranes by a postmodification process. D4 peptide-conjugated liposomes are found to bind to and enter cells by endocytosis specifically and efficiently in vitro in a process apparently mediated by EGF-R high-expressing cancer cells (H1299). In vivo, the D4 peptide-conjugated liposomes are found to accumulate in EGF-R-expressing xenograft tumor tissues up to 80 h after intravenous delivery, in marked contrast to controls. These results demonstrate how structure-based peptide design can be an efficient approach to identify highly novel binding ligands against important receptors. These data could have important consequences for the development of peptide-directed drug delivery systems with engineered specificities and prolonged times of action. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) has become a popular tool for downregulating specific gene expression in many species, including mammalian cells [Novina, C. D., and Sharp, P. A. (2004) The RNAi revolution, Nature 430, 161-164]. Synthetic double-stranded RNA sequences (siRNA) of 21-23 nucleotides have been shown in particular to have the potential to silence specifically gene function in cultured mammalian cells. As a result, there has been a significant surge of interest in the application of siRNA in functional genomics programs as a means of deciphering specific gene function. However, for siRNA functional genomics studies to be valuable and effective, specific silencing of any given target gene is essential, devoid of nonspecific knockdown and toxic side effects. For this reason, we became interested in investigating cationic liposome/lipid-mediated siRNA delivery (siFection) as a meaningful and potentially potent way to facilitate effective functional genomics studies. Accordingly, a number of cationic liposome/lipid-based systems were selected, and their formulation with siRNA was studied, with particular emphasis on formulation parameters most beneficial for siRNA use in functional genomics studies. Cationic liposome/lipid-based systems were selected from a number of commercially available products, including lipofectAMINE2000 and a range of CDANIDOPE systems formulated from different molar ratios of the cationic cholesterol-based polyamine lipid N1-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN) and the neutral helper lipid dioleoyl-L-α-phosphatidylethanolamine (DOPE). Parameters that were been investigated included the lipid:nucleic acid ratio of mixing, the extent of cationic liposome/lipid-nucleic acid complex (lipoplex) formation plus medium used, the lipoplex particle size, the mode of delivery, and dose-response effects. Results suggest that concentrations during siRNA lipoplex (LsiR) formation are crucial for maximum knockdown, but the efficacy of gene silencing is not influenced by the size of LsiR particles. Most significantly, results show that most commercially available cationic liposome/lipid-based systems investigated here mediate a significant nonspecific downregulation of the total cellular protein content at optimal doses for maximal specific gene silencing and knockdown. Furthermore, one pivotal aspect of using siRNA for functional genomics studies is the need for at least minimal cellular toxicity. Results demonstrate that CDAN and DOPE with and without siRNA confer low toxicity to mammalian cells, whereas lipofectAMINE2000 is clearly toxic both as a reagent and after formulation into LsiR particles. Interestingly, LsiR particles formulated from CDAN and DOPE (45:55, rn/rn; siFECTamine) seem to exhibit a slower cellular uptake than LsiR particles formulated from lipofectAMINE2000. Intracellularly, LsiR particles formulated from CDAN and DOPE systems also appear to behave differently, amassing in distinct but diffuse small nonlysosomal compartments for at least 5 h after siFection. By contrast, LsiR particles formulated from lipofectAMINE2000 accumulate in fewer larger intracellular vesicles. [ABSTRACT FROM AUTHOR]

Image guided drug delivery using imageable thermosensitive liposomes (iTSLs) and high intensity focused ultrasound (FUS or HIFU) has attracted interest as a novel and non-invasive route to targeted delivery of anti-cancer therapeutics. FUS-induced hyperthermia is used as an externally applied “trigger” for the release of a drug cargo from within thermosensitive drug carriers. It is suggested that sub-ablative hyperthermia significantly modifies the permeability of tumour vasculature and enhances nanoparticle uptake. Here we describe the preparation and use of magnetic resonance imaging (MRI) and near infrared fluorescence (NIRF) labelled thermosensitive liposomes for imaging and tracking of biodistribution and drug release in a murine cancer model. We prepared iTSLs to encapsulate topotecan (Hycamtin®), a chemotherapeutic agent which when released in tumours can be monitored by an increase in its intrinsic drug fluorescence. FUS was applied using feedback via subcutaneously placed fine-wire thermocouples to maintain and monitor hyperthermic temperatures. iTSL accumulation was detected within tumours using NIRF imaging immediately after liposome administration. Mild FUS-induced hyperthermia (3 min at 42 °C, 30 min post i.v. administration) greatly enhanced iTSLs uptake. A co-localised enhancement of topotecan fluorescence emission was also observed immediately after application of FUS indicating rapid triggered drug release. The phenomena of increased iTSL accumulation and concomitant topotecan release appeared to be amplified by a second mild hyperthermia treatment applied one hour after the first. MRI in vivo also confirmed enhanced iTSLs uptake due to the FUS treatments. Our imaging results indicate the effects of hyperthermia on the uptake of carriers and drug. FUS-induced hyperthermia combined with real time imaging could be used as a tool for tumour targeted drug delivery. [ABSTRACT FROM AUTHOR]

Nanofibre-based mucoadhesive films were invented for oromucosal administration of nanocarriers used for delivery of drugs and vaccines. The mucoadhesive film consists of an electrospun nanofibrous reservoir layer, a mucoadhesive film layer and a protective backing layer. The mucoadhesive layer is responsible for tight adhesion of the whole system to the oral mucosa after application. The electrospun nanofibrous reservoir layer is intended to act as a reservoir for polymeric and lipid-based nanoparticles, liposomes, virosomes, virus-like particles, dendrimers and the like, plus macromolecular drugs, antigens and/or allergens. The extremely large surface area of nanofibrous reservoir layers allows high levels of nanoparticle loading. Nanoparticles can either be reversibly adsorbed to the surface of nanofibres or they can be deposited in the pores between the nanofibres. After mucosal application, nanofibrous reservoir layers are intended to promote prolonged release of nanoparticles into the submucosal tissue. Reversible adsorption of model nanoparticles as well as sufficient mucoadhesive properties were demonstrated. This novel system appears appropriate for the use in oral mucosa, especially for sublingual and buccal tissues. To prove this concept, trans-/intramucosal and lymph-node delivery of PLGA-PEG nanoparticles was demonstrated in a porcine model. This system can mainly be used for sublingual immunization and the development of “printed vaccine technology”. [ABSTRACT FROM AUTHOR]

Pro-apoptotic analogues of vitamin E (VE) exert selective anti-cancer effect on various animal cancer models. Neither suitable formulation of α-tocopheryl succinate (α-TOS), representative semi-synthetic VE analogue ester, nor suitable formulations of the other VE analogues for clinical application have been reported yet. The major factor limiting the use of VE analogues is their low solubility in aqueous solvents. Due to the hydrophobic character of VE analogues, liposomes are predetermined as suitable delivery system. Liposomal formulation prevents undesirable side effects of the drug, enhances the drug biocompatibility, and improves the drug therapeutic index. Liposomal formulations of VE analogues especially of α-TOS and α-tocopheryl ether linked acetic acid (α-TEA) have been developed. The anti-cancer effect of these liposomal VE analogues has been successfully demonstrated in pre-clinical models in vivo. Present achievements in: (i) preparation of liposomal formulations of VE analogues, (ii) physico-chemical characterization of these developed systems and (iii) testing of their biological activity such as induction of apoptosis and evaluation of anti-cancer effect are discussed in this review. [ABSTRACT FROM AUTHOR]

Abstract: Lyme disease caused by spirochete Borrelia burgdorferi sensu lato, is a tick-born illness. If the infection is not eliminated by the host immune system and/or antibiotics, it may further disseminate and cause severe chronic complications. The immune response to Borrelia is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies associated with Th1 cell activation. A new experimental vaccine was constructed using non-lipidized form of recombinant B. burgdorferi s.s. OspC protein was anchored by metallochelating bond onto the surface of nanoliposomes containing novel nonpyrogenic lipophilized norAbuMDP analogues denoted MT05 and MT06. After i.d. immunization, the experimental vaccines surpassed Alum with respect to OspC-specific titers of IgG2a, IgG2b isotypes when MT06 was used and IgG3, IgM isotypes when MT05 was used. Both adjuvants exerted a high adjuvant effect comparable or better than MDP and proved themselves as nonpyrogenic. [Copyright &y& Elsevier]

Abstract: We designed and synthesised a series of new cationic lipids based on spermine linked to various hydrophobic anchors. These lipids could be potentially useful for the preparation of stable cationic liposomes intended for the construction of drug targeting systems applicable in the field of anticancer/antiviral therapy, vaccine carriers, and vectors for the gene therapy. Low in vitro toxicity was found for these compounds, especially for LD1, in several cell lines. The delivery of both a fluorescence marker (calcein) and antiviral drugs into cells has been achieved owing to a large extent of internalization of cationic liposomes (labelled by Lyssamine-Rhodamine PE or fluorescein-PE) as demonstrated by fluorescent microscopy and quantified by flow cytometry. The bovine herpes virus type 1 (BHV-1) virus infection in vitro model using MDBK cells was employed to study the effect of the established antiviral drug HPMPC (Cidofovir®) developed by Prof. A. Holý. Inhibition of BHV-1 virus replication was studied by quantitative RT-PCR and confirmed by both Hoffman modulation contrast microscopy and transmission electron microscopy. We found that in vitro antiviral activity of HPMPC was significantly improved by formulation in cationic liposomes, which decreased the viral replication by about 2 orders of magnitude. [Copyright &y& Elsevier]

Abstract: Non-viral vectors are considered safer than viral vectors and show clinical potential, but remain less efficient in terms of DNA delivery. Here we report how cationic liposomes, prepared from new cationic lipid, N′,N′,-dioctadecyl-N-4,8-diaza-10-aminodecanoylglycine amide (DODAG) and neutral lipid dioleoyl-l-α-phos-phatidylethanolamine (DOPE), can be formulated with plasmid DNA (pDNA) in the presence of stabilizer cholesteryl-oxycarbonylpolyethlylene glycol4600 (PEG4600-Chol) giving PEGylated pDNA nanoparticles (pDNA-ABC nanoparticles) that are proposed to be half-life triggered nanoparticles. In particular, the PEGylated pDNA nanoparticle formulation DODAG/DOPE/PEG4600-Chol (43:43:14, m/m/m)−pDNA (total lipid/pDNA ratio 4:1 w/w) (pTRANSplus nanoparticles) is shown to mediate efficient transfection of murine lung tissue in vivo. Levels of transfection compare well with the results of polyethylenimine (PEI) mediated pDNA transfection in vivo and even of adenovirus mediated transduction. Cryo-EM imaging indicates that pTRANSplus formulations are somewhat heterogeneous but do consist primarily of bilammellar lipoplex nanoparticles with a few multilammellar nanoparticle aggregates. Lung histology confirms that pTRANSplus mediated transfection in vivo targets substantially the epithelial cells of bronchii and bronchioli airway passages. The pTRANSplus nanoparticle system is a useful new starting point for nucleic acid therapeutic strategies to counter lung disorders such as viral infection and possibly cystic fibrosis. [Copyright &y& Elsevier]

Abstract: Hsp90-CA is present in cell wall of Candida pseudohyphae or hyphae — typical pathogenic morphotype for both systemic and mucosal Candida infections. Heat shock protein from Candida albicans (hsp90-CA) is an important target for protective antibodies during disseminated candidiasis of experimental mice and human. His-tagged protein rHsp90 was prepared and used as the antigen for preparation of experimental recombinant liposomal vaccine. Nickel-chelating liposomes (the size around 100nm, PDI≤0.1) were prepared from the mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (molar ratio 95:5%) by hydration of lipid film and extrusion methods. New non-pyrogenic hydrophobised derivative of MDP (C18-O-6-norAbuMDP) was incorporated into liposomes as adjuvans. rHsp90 was attached onto the surface of metallochelating liposomes by metallochelating bond and the structure of these proteoliposomes was studied by dynamic light scattering, AF microscopy, TEM and GPC. The liposomes with surface-exposed C18-O-6-norAbuMDP were well recognised and phagocyted by human dendritic cells in vitro. In vivo the immune response towards this experimental vaccine applied in mice (i.d.) demonstrated both TH1 and TH2 response comparable to FCA, but without any side effects. Metallochelating liposomes with lipophilic derivatives of muramyl dipeptide represent a new biocompatible platform for construction of experimental recombinant vaccines and drug-targeting systems. [Copyright &y& Elsevier]

Abstract: RNA interference (RNAi) is being widely explored as a means of tumour therapy due to the specific and potent silencing of targeted genes. However, in vivo delivery of RNAi effectors, such as small interfering RNA (siRNA) and detection of delivery is fraught with problems. Here, we describe novel theranostic PEGylated siRNA nanoparticles termed liposome-entrapped siRNA (LEsiRNA) nanoparticles. Our LEsiRNA nanoparticles are MR sensitive, contain labels for fluorescence microscopy/histology and promote functional siRNA delivery to tumours in mice leading to a significant reduction in both Survivin expression and tumour growth. LEsiRNA nanoparticles, administered by intravenous injection, were shown to accumulate in xenograft tumours by MR contrast image enhancements 24h post-administration. Fluorescence microscopy was used to corroborate the MR results and simultaneously demonstrate co-localisation of nanoparticles and siRNA within the tumours. The LEsiRNA nanoparticle-mediated delivery of the anti-cancer Survivin siRNA causes significant reduction in tumour growth when compared to controls. Our results suggest that LEsiRNA nanoparticles can be valuable as an in vivo delivery agent for siRNA therapy to tumours. [ABSTRACT FROM AUTHOR]

Liposomal vectors have been widely studied for targeted gene and drug delivery to tumour cells. In vivo these systems encounter problems with serum stability, tumour localisation, cell internalisation and endosomal escape. Modification or shielding of the liposome surface helps to inhibit non-specific interaction with blood components and enables an extended circulation of the vectors after systemic administration. This is achieved with a hydrophilic polymer of which polyethylene glycol (PEG) has been found to be best. Targeting cell-surface receptors is an attractive concept to achieve specific binding and internalisation of the liposome. To this end, cell-binding ligands are displayed from the surface of the liposome. The common denominator for these ligands is that their cell receptor targets are over-expressed in tumours.We are developing a post-modification methodology for coupling cell or tissue targeting moieties onto liposomes via chemioselective oxime bond formation. Thus, liposomes comprising a highly nucleophilic aminoxy-functionalised lipid are formulated. The aminoxy lipid readily reacts with aldehyde functionalised targeting ligands in aqueous conditions forming a stable oxime bond conjugated targeted liposome Fig 1.A peptide displaying the RGD sequence was selected as the targeting ligands to validate our post-coupling methodology. A cyclic RGD peptide conjugate has been successfully synthesised using a combination of solution- and solid-phase synthesis. Additionally, control RGE peptide and PEG linker conjugates have been prepared. All the ligands terminate in an aldehyde functional group separated from the targeting moiety by a PEG linker.The couplings between the RGD conjugate and aminoxy liposomes in water at pH 4 were shown to be efficient, simple and reproducible. In vitro HUVEC cell uptake studies show a definite targeted uptake of the RGD-targeted liposomes relative to appropriate control liposomes.Molecular Therapy (2006) 13, S74–S75; doi: 10.1016/j.ymthe.2006.08.216 [ABSTRACT FROM AUTHOR]

Abstract: We report the syntheses of novel cationic lipids comprised of cholesteryl-moieties linked to guanidinium functional groups, and also cationic lipids comprising a dialkylglycylamide moiety conjugated with a polyamine or a guanidinium functional group. In plasmid DNA (pDNA) transfection studies, these cationic lipids were formulated into cationic liposomes with the neutral co-lipid dioleoyl-L-α-phosphatidylethanolamine (DOPE) or with a recently reported neutral lipophosphoramidate derivative of histamine (MM27). We observe that cationic liposomes prepared from the cationic lipid N′,N′-dioctadecyl-N-4,8-diaza-10-aminodecanoylglycine amide (DODAG) and DOPE frequently mediate the highest levels of transfection in vitro in all three different cell lines studied (OVCAR-3, IGROV-1 and HeLa) both in the presence or absence of serum. In addition, in vitro cellular toxicity was found to be minimal. Alternatively, we observe that DODAG alone forms lipoplex nanoparticles with small interfering RNA (siRNA) that are able to mediate the functional delivery of two previously validated anti-hepatitis B virus (HBV) — siRNAs to murine liver in vivo with minimal observable liver toxicity and immune stimulation. Specific knock-down of HBV infection parameters (virion and hepatic mRNA levels) is observed that is at least equivalent to the impact of extensive treatment with lamivudine (a licensed antiviral drug). [Copyright &y& Elsevier]