寡核苷酸聚赖氨酸修饰物的脂质体制备及其对HepG2细胞的细胞毒活性研究

目的研究反义寡核苷酸的聚赖氨酸修饰物对脂质体包封率的影响及对HepG2细胞活性的初步测定。方法利用反义药物的聚赖氨酸修饰物,采用薄膜分散法制备反义寡核苷酸及其聚赖氨酸修饰物的脂质体;紫外分光光度法测定包封率的差异并考察对HepG2细胞细胞毒活性的影响。结果高密度正电荷的聚赖氨酸与带负电荷的反义寡核苷酸偶联后,脂质体的载药量大大增加,并且细胞的摄入量增加,对HepG2细胞生长具有抑制作用。结论聚赖氨酸修饰物增加了反义寡核苷酸的脂质体包封率,有效诱导人肝癌细胞的凋亡作用。     

Mitochondrial delivery of mastoparan with transferrin liposomes equipped with a pH-sensitive fusogenic peptide for selective cancer therapy

Mastoparan (MP), a potent facilitator of mitochondrial permeability transition (PT), could be used as an antitumor agent, if it were encapsulated in a tumor selective delivery system. We recently developed transferrin-modified liposomes (Tf-L) with a pH-sensitive fusogenic peptide (GALA), which delivers an encapsulated fluorescent marker into cytosol efficiently. In this study, we encapsulated MP into Tf-L with GALA for the selective delivery to mitochondria of tumor cells. The MP showed potent PT activity at concentrations above 25 microM in a homogenate of K 562 cells as well as in isolated mitochondria in the presence of phosphate. Tf-L equipped with cholesteryl GALA can release encapsulated sulforhodamine B, while Tf-L failed, as evidenced by confocal laser scanning microscopy. The MP, which was delivered with Tf-L with GALA, released cytochrome c (cyt c) from mitochondria to the cytosol, while free MP released cyt c not only to the cytosol but also extracellulary. These results demonstrate the utility of MP in Tf-L with GALA for cancer therapy.     

Studies on uptake, sub-cellular trafficking and efflux of antisense oligodeoxynucleotides in glioma cells using self-assembling cationic lipoplexes as delivery systems

The cellular uptake of antisense oligodeoxynucleotides (ODNs) may be enhanced by the use of carriers such as cationic liposomes or lipoplexes, but little is known about the intracellular fate and subcellular trafficking of these systems in target cells. In this study, we report on the cellular uptake and biodistribution of ODNs in the presence and absence of optimised self-assembled cationic lipoplexes using the C6 glioma cell line as an in vitro model. Biotin or radiolabelled 15-mer phosphorothioate (PS) ODNs were synthesised and their cellular uptake and subcellular biodistribution characterised in the presence and absence of an optimised cationic lipoplex delivery system using studies ranging from cellular association, cellular efflux and transmission electron microscopy (TEM). Ultrastructural studies clearly showed PS ODNs in the absence of liposomal delivery to be sequestered within endosomal and lysosomal vesicular bodies indicative of endocytic uptake. ODNs were also visible, to a lesser extent, in the nucleus and cytoplasm. By employing DOSPA (2'-(1",2"-dioleoyloxypropyldimethyl-ammonium bromide)-N-ethyl-6-amidospermine tetra trifluoroacetic acid) and DOPE (dioleoylphosphatidylethanolamine) complex in a 3 : 1 ratio, as a delivery system for ODNs at a optimal lipid/DNA charge ratio of 1 : 1, the level of ODN cellular association was significantly increased by approximately 10-12 fold with a concomitant change in subcellular distribution of PS ODN. TEM studies indicated enhanced penetration of ODN within the cytosol and the cell nucleus with reduced presence in vesicular compartments. Efflux studies confirmed that cationic lipoplexes promoted entry of ODNs into 'deeper' cellular compartments, consistent with endosomal release. Optimised cationic lipoplexes improved cellular delivery of ODNs by enhancing cell association, uptake and by favourably modulating the intracellular trafficking and distribution of ODNs into non-vesicular compartments including the cytosol and nucleus.     

Liposomal vaccine delivery systems

Introduction: Liposomes remain at the forefront of drug and vaccine design owing to their well-documented abilities to act as delivery vehicles. Nevertheless, the concept of liposomes as delivery vehicles is not a new one, with most works focusing on their use for the delivery of genes and drugs. However, in the last 10 years a significant amount of research has focused on using liposomes as vaccine adjuvants, not only as an antigen delivery vehicle but also as a tool to increase the immunogenicity of peptide and protein antigens.

Areas covered: This paper reviews liposomal adjuvants now in vaccine development, with particular emphasis on their adjuvant mechanism and how specific physicochemical characteristics of liposomes affect the immune response. The inclusion of immunomodulators is also discussed, with prominence given to Toll-like receptor ligands.

Expert opinion: The use of liposomes as vaccine delivery systems is evolving rapidly owing to the combined increase in technological advances and understanding of the immune system. Liposomes that contain and deliver immunostimulators and antigens are now being developed to target diseases that require stimulation of both humoral and cell-mediated immune responses. The CAF liposomal system, described in detail in this review, is one liposomal model that shows such flexibility.     

Site-Specific Administration of Antisense Oligonucleotides using Biodegradable Polymer Microspheres Provides Sustained Delivery and Improved Subcellular Biodistribution in the Neostriatum of the Rat Brain

Abstract

Antisense oligonucleotides (ODNs) are being increasingly used in the central nervous system as biological tools, as drug-target validation agents and as potential therapeutic agents. Although the local delivery of naked ODNs to the brain can result in the desired biological effects, the duration of efficacy is relatively short lived due to the combined effects of rapid ODN degradation and elimination half-lives in vivo. In this study, we have examined the use of biodegradable polymer microspheres as a site-specific delivery system for targeting ODNs to the neostriatum of the rat brain. Model phosphorothioate backbone-modified ODNs were entrapped within poly(D,L-lactide-co-glycolide) (PLAGA) microspheres using a double emulsion-deposition method and the formulations characterised in terms of particle size, surface morphology, percent encapsulation efficiency, ODN loading and in vitro release profiles. For in vivo evaluation, PLAGA microspheres containing fluorescently-labelled ODNs were stereotaxically administered to the neostriatum of the rat brain and biodistribution of ODNs monitored after 48 h. Administration of free fluorescently-labelled ODNs to the neostriatum resulted in a punctate cellular distribution of ODNs after 24 h with little or no ODN remaining in the neostriatum after 48 h. In comparison, fluorescently-labelled ODNs delivered using polymer microspheres were intensely visible in cells after 48 h post-administration and the fluorescence appeared to be diffuse covering both cytosolic and nuclear regions. Dual-label immunohistochemical analyses suggested that ODNs were mainly distributed to neuronal cells. These data indicate that site-specific administration of ODNs using biodegradable polymer microspheres will not only provide sustained delivery of nucleic acids but can also improve the cellular distribution of ODNs to brain cells. Sustained or controlled-release biodegradable polymer formulations, therefore, represent an attractive strategy for improved local delivery of ODNs to the CNS.     

Design and characterization of liposomes containing long-chain N-acylPEs for brain delivery: penetration of liposomes incorporating GM1 into the rat brain

Purpose. To develop a suitable liposomal carrier to encapsulate neu- roactive compounds that are stable enough to carry them to the brain across the blood-brain barrier with the appropriate surface characteristics for an effective targeting and for an active membrane transport. Methods. Liposomes containing glycosides and a fusogenic lipid were prepared by extrusion. Photon correlation spectroscopy, fluorescence spectroscopy, and differential scanning calorimetry were used to characterize liposomal preparations. Tissue distribution was determined by using ³H-cholesterylhexadecylether as a marker. Results. The incorporation of glycoside determinants and N-palmitoylphosphatidylethanolamine gives liposomes with similar initial size, trapped volume, negative surface charge, bilayer fluidity, and melting temperature, except for monosialoganglioside-containing liposomes, which showed less negative surface charge and the highest size, trapped volume and melting temperature. All glycosilated formulations gave liposomes able to retain up to the 95% of encapsulated carboxyfluorescein after 90 min at physiologic temperature even in the presence of serum. Monosialoganglioside liposomes were recovered in the cortex, basal ganglia, and mesencephalon of both brain hemispheres. The liver uptake was higher for sulfatide- and glucose-liposomes, whereas the higher blood levels were observed for glucose- and mannose-liposomes. Conclusions. These results show the suitability of such liposomal formulations to hold encapsulated drugs. Moreover, the brain uptake of monosialoganglioside liposomes makes them good candidates as drug delivery systems to the brain.     

Liposomal adjuvants for human vaccines

ABSTRACT

Introduction: Liposomes are well-known as drug carriers, and are now critical components of two of six types of adjuvants present in licensed vaccines. The liposomal vaccine adjuvant field has long been dynamic and innovative, and research in this area is further examined as new commercial products appear in parallel with new vaccines. In an arena where successful products exist the potential for new types of vaccines with liposomal adjuvants, and alternative liposomal adjuvants that could emerge for new types of vaccines, are discussed.

Areas covered: Major areas include: virosomes, constructed from phospholipids and proteins from influenza virus particles; liposomes containing natural and synthetic neutral or anionic phospholipids, cholesterol, natural or synthetic monophosphoryl lipid A, and QS21 saponin; non-phospholipid cationic liposomes; and combinations and mixtures of liposomes and immunostimulating ingredients as adjuvants for experimental vaccines.

Expert opinion: Liposomes containing monophosphoryl lipid A and QS21 have considerable momentum that will result soon in emergence of prophylactic vaccines to malaria and shingles, and possible novel cancer vaccines. The licensed virosome vaccines to influenza and hepatitis A will be replaced with virosome vaccines to other infectious diseases. Alternative liposomal formulations are likely to emerge for difficult diseases such as tuberculosis or HIV-1 infection.     

Quantitation of nanoparticle accumulation in flow using optimized microfluidic chambers

Abstract

Background: The vascular cell adhesion molecule-1 (VCAM-1) targeting peptide sequence, VHPKQHR, is a promising moiety for targeting atherosclerosis through incorporation into nanoparticles such as dendrimers and liposomes.

Purpose: We aim to develop VCAM-1-targeted nanoparticles that effectively accumulate on the endothelium under shear conditions and to develop robust microfluidic chambers able to house sufficient cells for flow cytometric measurements.

Methods: Carboxyfluorescein-labeled monomeric VHP-peptide, tetrameric VHP-dendrimers (bisbidentate or radial architecture, with or without N-terminal acetylation) and VHP-peptide liposomes were prepared. Human umbilical vein endothelial cells were treated with nanoparticles under 0 or 2.9 dyne/cm² shear, and particle binding was quantified. Flow chambers cured at various temperatures, with or without glass backings were fabricated, characterized for deformation and applied in experiments.

Results: Although liposomes accumulated with highest efficiency, dendrimers also demonstrated specific binding. N-terminal acetylation significantly reduced dendrimer binding, and despite shorter movement range, bisbidentate dendrimers outperformed radial dendrimers, suggesting multiple epitope presence within its estimated arm-span of 57?Å. Under shear, while liposome binding increased 300%, dendrimer binding to cells decreased 65%. Through higher temperature curing and glass backing insertion, polydimethylsiloxane flow chambers maintaining rectangular cross-section with aspect-ratio as low as 1:111 were achieved.

Conclusion: Optimized dendrimers and liposomal nanocarriers specifically accumulated onto cells within microfluidic chambers.     

Evaluation of Generation 2 and 3 Poly(Propylenimine) Dendrimers for the Potential Cellular Delivery of Antisense Oligonucleotides Targeting the Epidermal Growth Factor Receptor

PURPOSE: To evaluate low generation, G2 and G3, poly(propylenimine) dendrimers for the potential cellular delivery of antisense oligonucleotides (ODNs) targeting the epidermal growth factor receptor (EGFR) in A431 epidermoid carcinoma cells. METHODS: Cell cytotoxicity of the dendrimers was evaluated using trypan blue exclusion assays. Cellular uptake studies of fluorescently labeled ODNs were performed using fluorescence-activated cell sorting analysis. Intracellular fate of dendrimer-delivered ODNs was assessed in both fixed and live cells using fluorescent microscopy. Antisense ODN activity was assessed in terms of cancer cell growth, inhibition of target EGFR protein, and reduction in mRNA levels. RESULTS: G2 dendrimer (DAB-8) was less toxic than G3 (DAB-16) dendrimer in A431 cells, with IC50 of >175 and approximately 30 microg/ml, respectively. Uptake of fluorescently labeled ODN:dendrimer complexes was increased by up to 100-fold compared to a marker of fluid-phase endocytosis and up to 9-fold over free ODN at the optimal dendrimer:ODN (w/w) ratio of 5:1. Uptake of dendrimer:ODN complexes was significantly reduced at 4 degrees C (p < 0.05). Live cell fluorescent microscopy resulted in an intracellular distribution of dendrimer:ODN complexes that was suggestive of endocytic uptake; in contrast, cell fixation resulted in an artefactual nuclear localization. Treatment of A431 cells with anti-EGFR antisense ODN:dendrimer complexes inhibited cell growth, protein, and mRNA expression to levels comparable to Oligofectamine-mediated delivery. CONCLUSIONS: G2 and G3 poly(propylenimine) dendrimers markedly improved the delivery and activity of ODNs and thus may represent general reagents for the delivery of ODNs to cells in culture.     

Targeted thermosensitive liposomes: an attractive novel approach for increased drug delivery to solid tumors

Introduction: Currently available chemotherapy is hampered by a lack in tumor specificity and resulting toxicity. Small and long-circulating liposomes can preferentially deliver chemotherapeutic drugs to tumors upon extravasation from tumor vasculature. Although clinically used liposomal formulations demonstrated significant reduction in toxicity, enhancement of therapeutic activity has not fully met expectations.

Areas covered: Low drug bioavailability from liposomal formulations and limited tumor accumulation remain major challenges to further improve therapeutic activity of liposomal chemotherapy. The aim of this review is to highlight strategies addressing these challenges. A first strategy uses hyperthermia and thermosensitive liposomes to improve tumor accumulation and trigger liposomal drug bioavailability. Image-guidance can aid online monitoring of heat and drug delivery and further personalize the treatment. A second strategy involves tumor-specific targeting to enhance drug delivery specificity and drug internalization. In addition, we review the potential of combinations of the two in one targeted thermosensitive-triggered drug delivery system.

Expert opinion: Heat-triggered drug delivery using thermosensitive liposomes as well as the use of tumor vasculature or tumor cell-targeted liposomes are both promising strategies to improve liposomal chemotherapy. Preclinical evidence has been encouraging and both strategies are currently undergoing clinical evaluation. A combination of both strategies rendering targeted thermosensitive liposomes (TTSL) may appear as a new and attractive approach promoting tumor drug delivery.     

Synthesis of cholesterol modified cationic lipids for liposomal drug delivery of antisense oligonucleotides.

The paper describes a novel synthesis of cholest-5-en-3 beta-yl-6-aminohexyl ether (AH-Chol). AH-Chol was used to prepare positively charged liposomes. The liposomes consisted of phospholipon 90H and the cationic cholesterol derivative in an equimolar ratio. Liposome preparation was achieved by membrane homogenization after rehydration of a dry lipid film. Oligonucleotides (ODN) were adsorbed to the cationic liposomes very efficiently. At an ODN/liposome ratio of 1:5 (10:50 micrograms/ml) 84.2 +/- 5.4% of the ODNs were bound to the liposomal membrane. Within the range of 1:40 and 1:100 charge neutralization occurred and the liposome dispersion showed an increase in particle size due to aggregation. Below or above this range of charge neutralization the ODN loaded liposome preparation was physically stable, no sedimentation, increase of vesicle size or vesicle aggregation occurred.     

Physicochemical Properties of Liposomes Affecting Apoptosis Induced by Cationic Liposomes in Macrophages

Purpose. Cationic liposomes are expected to be useful as nonviral vectors for gene delivery. Cationic liposomes showed cytotoxicity, and we proposed that the cytotoxicity is through apoptosis. In this study, we examined the effects of liposomal properties, such as liposomal charge, size, membrane fluidity, and PEG coating, on the induction of apoptosis in the macrophage-like cell line RAW264.7. Methods. RAW264.7 cells were treated with liposomes, and the induction of apoptosis was evaluated by monitoring the changes in DNA content by flow cytometry. The association of liposomes with cells and the generation of reactive oxygen species (ROS) were also measured by flow cytometry. Results. The induction of apoptosis of RAW264.7 cells was dependent on the concentrations of stearylamine or cholesterol, a component of cationic liposomes. A significant correlation was observed between the degree of apoptosis and association of cationic liposomes with the cells. Coating the liposomal surface with polyethylene glycol (PEG) decreased the association of cationic liposomes with RAW264.7 cells and reduced the induction of apoptosis. Liposomal size also affected the induction of apoptosis, and larger liposomes showed a higher degree of apoptosis induction. Furthermore, ROS, which were required for the induction of apoptosis by cationic liposomes, were generated in a cholesterol content-dependent manner, and ROS generation was also decreased by PEG coating as the association and the induction of apoptosis were reduced. Conclusions. The degree of apoptosis is related to the extent of association of cationic liposomes with cells and is related to the generation of ROS.     

The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment

Importance of the field: Targeted liposomal drugs represent the next evolution of liposomal drug delivery in cancer treatment. In various preclinical cancer models, antibody-targeted PEGylated liposomal drugs have demonstrated superior therapeutic effects over their non-targeted counterparts. Single chain Fv (scFv) has gained popularity in recent years as the targeting agent of choice over traditional targeting agents such as monoclonal antibodies (mAb) and antibody fragments (e.g., Fab′).

Areas covered in this review: This review is focused mainly on advances in scFv-targeted liposomal drug delivery for the treatment of cancers, based on a survey of the recent literature, and on experiments done in a murine model of human B-lymphoma, using anti-CD19 targeted liposomes targeted with whole mAb, Fab′ fragments and scFv fragments.

What the reader will gain: This review examines the recent advances in PEGylated immunoliposomal drug delivery, focusing on scFv fragments as targeting agents, in comparison with Fab′ and mAb.

Take home message: For clinical development, scFv are potentially preferred targeting agents for PEGylated liposomes over mAb and Fab′, owing to factors such as decreased immunogenicity, and pharmacokinetics/biodistribution profiles that are similar to non-targeted PEGylated (Stealth^®) liposomes.     

Efficient Introduction of Macromolecules and Oligonucleotides into Brain Capillary Endothelial Cells Using HVJ-Liposomes

Abstract

In this study, we examined the feasibility of introducing macromolecules into cultured mouse brain capillary endothelial cells (MBEC4 cells) by utilizing the hemagglutating virus of Japan (HVJ)-liposomes with fusogenic activity. We used fluorescein isothiocyanate dextran (FITC-Dextran) and FITC-labeled oligodeoxynucleotide (FITC-ODN) as models of a macromolecule and an ODN, respectively. Intracellular fluorescence appeared rapidly after the exposure of MBEC4 cells to FITC-Dextran-containing HVJ-liposomes, and remained detectable for at least 3 days. Only a control level of intracellular fluorescence was seen after treatment with FITC-Dextran alone, FITC-Dextran with empty HVJ-liposomes or FITC-Dextran-containing liposomes without fusogenic activity. In the early phase after administration (0–30 min), the introduction of FITC-Dextran into MBEC4 cells by the HVJ-liposome method resulted in a rapid and time-dependent increase of intracellular fluorescence intensity. Moreover, FITC-ODN was also introduced into MBEC4 cells by the HVJ-liposome method, although FITC-ODN alone was not introduced. These results indicate that the HVJ-liposome method is useful for the efficient introduction of macromolecules, including ODN, into brain capillary endothelial cells.     

Radionuclide imaging of liposomal drug delivery

ABSTRACT

Introduction: Ever since their discovery, liposomes have been radiolabeled to monitor their fate in vivo. Despite extensive preclinical studies, only a limited number of radiolabeled liposomal formulations have been examined in patients. Since they can play a crucial role in patient management, it is of importance to enable translation of radiolabeled liposomes into the clinic.

Areas covered: Liposomes have demonstrated substantial advantages as drug delivery systems and can be efficiently radiolabeled. Potentially, radiolabeled drug-loaded liposomes form an elegant theranostic system, which can be tracked in vivo using single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. In this review, we discuss important aspects of liposomal research with a focus on the use of radiolabeled liposomes and their potential role in drug delivery and monitoring therapeutic effects.

Expert opinion: Radiolabeled drug-loaded liposomes have been poorly investigated in patients and no radiolabeled liposomes have been approved for use in clinical practice. Evaluation of the risks, pharmacokinetics, pharmacodynamics and toxicity is necessary to meet pharmaceutical and commercial requirements. It remains to be demonstrated whether the results found in animal studies translate to humans before radiolabeled liposomes can be implemented into clinical practice.     

In Vitro-In Vivo Myotoxicity of Intramuscular Liposomal Formulations

Purpose. The first objective was to study the in vitro myotoxicity of empty liposomes and to examine whether liposome size, charge and fluidity affect liposome myotoxicity. The second objective was to investigate the effect of liposomal encapsulation on the in vitro and in vivo myotoxicity of loxapine compared to the loxapine commercial preparation (Loxitane^®). Methods. The in vitro myotoxicity of empty liposomes and loxapine liposomes was evaluated by the cumulative efflux of the cytosolic enzyme creatine kinase (CK) from the isolated rat extensor digitorum longus (EDL) muscle over a 2 hour period. In the in vivo studies, the area under plasma CK curve over 12 hours was used to evaluate muscle damage. Results. The in vitro myotoxicity for all empty liposomal formulations was not statistically different from negative controls (untreated control muscles and normal saline injected muscles). However, these empty liposomal formulations were significantly less myotoxic than the positive controls (muscles injected with phenytoin and muscle sliced in half). In vitro-in vivo studies showed that the liposomal encapsulation of loxapine resulted in significant (P < 0.05) reduction in myotoxicity (80% in vitro and 60% in vivo) compared to the commercially available formulation which contains propylene glycol (70% V/V) and polysor-bate 80 (5% W/V) prepared at equal concentration. Conclusions. Results indicate that empty liposomes do not induce myotoxicity. Furthermore, liposomal size, charge and fluidity do not affect myotoxicity. In addition, in vitro and in vivo studies have demonstrated that liposomal encapsulation of loxapine can reduce myotoxicity compared to a formulation containing organic cosolvents.     

Fusogenic pH sensitive liposomal formulation for rapamycin: Improvement of antiproliferative effect

Context: Liposomes are increasingly employed to deliver chemotherapeutic agents, antisense oligonucleotides, and genes to various therapeutic targets.

Objective: The present investigation evaluates the ability of fusogenic pH-sensitive liposomes of rapamycin in increasing its antiproliferative effect on human breast adenocarcinoma (MCF-7) cell line.

Materials and methods: Cholesterol (Chol) and dipalmitoylphosphatidylcholine (DPPC) (DPPC:Chol, 7:3) were used to prepare conventional rapamycin liposomes by a modified ethanol injection method. Dioleoylphosphatidylethanolamine (DOPE) was used to produce fusogenic and pH-sensitive properties in liposomes simultaneously (DPPC:Chol:DOPE, 7:3:4.2). The prepared liposomes were characterized by their size, zeta potential, encapsulation efficiency percent (EE%), and chemical stability during 6 months. The antiproliferative effects of both types of rapamycin liposomes (10, 25, and 50?nmol/L) with optimized formulations were assessed on MCF-7 cells, as cancerous cells, and human umbilical vein endothelial cells (HUVEC), as healthy cells, employing the diphenyltetrazolium bromide (MTT) assay for 72?h.

Results and discussion: The particle size, zeta potential, and EE% of the liposomes were 165?±?12.3 and 178?±?15.4?nm, ?39.6?±?1.3, and ?41.2?±?2.1?mV as well as 76.9?±?2.6 and 76.9?±?2.6% in conventional and fusogenic pH-sensitive liposomes, respectively. Physicochemical stability results indicated that both liposome types were relatively stable at 4?°C than 25?°C. In vitro antiproliferative evaluation showed that fusogenic pH-sensitive liposomes had better antiproliferative effects on MCF-7 cells compared to the conventional liposomes. Conversely, fusogenic pH-sensitive liposomes had less cytotoxicity on HUVEC cell line.     

Stabilization of liposomes during drying

Introduction: During the past 40 years, liposomes have been investigated intensively as drug carriers for anticancer drugs and as the adjuvant components of vaccines, for example. In this context, the development of dry formulations of liposomes is important to ensure a more stable drug product and to avoid the use of the ‘cold chain’ during distribution.

Areas covered: This review provides an overview of the technologies commonly used for the drying of liposomal formulations and the significance of formulation and processing parameters for the drying process. In addition, a review is provided of the protective mechanisms proposed to be responsible for stabilization during processing and in the dry state, with special emphasis on the techniques used for the characterization of the mechanisms. Parameters are discussed that critically influence the liposomal stability during drying and the underlying stabilization mechanisms, including the water replacement theory, vitrification and kosmotropic effects.

Expert opinion: Drying of liposomal formulations has contributed to the development of more stable products because liposomes can be dehydrated in the presence of appropriate stabilizing excipients, without affecting the size or the drug encapsulation efficiency. The key to the successful design and preparation of optimal liposomal dry powder formulations is an understanding of the significance of the drying process parameters, and the mechanisms responsible for the stabilization of liposomes during drying and in the dry state.     

Delivery of different length poly(L-lysine)-conjugated ODN to HepG2 cells using N-stearyllactobionamide-modified liposomes and their enhanced cellular biological effects

Short (14-20-mer range) synthetic oligodeoxynucleotides (ODNs) allow specific modulation of cellular gene expression at various stages, thus providing a versatile tool for fundamental studies and a rational approach to anticancer chemotherapy. However, several problems, such as metabolic stability, efficient cell internalization of ODNs and their efficient entrapment into liposomes continue to markedly limit this approach. To improve the target specificity and biological activity of ODN, three different length of poly(L-lysine) (PLL) were conjugated to ODN and these conjugates were encapsulated in N-stearyllactobionamide (N-SLBA)-modified liposomes, N-SLBA is a ligand for the asialoglycoprotein receptor. Then, we investigated their effects on cell cycle and survivin protein levels of HepG2 cells. The results showed that the encapsulation efficiency was improved because the polycationic charges of PLL neutralized the polyanionic charges of ODN. Among them, PLL (M(W) 2000 and 10,000)-conjugated ODN encapsulated in N-SLBA liposomes induced apoptosis of HepG2 cells and highly inhibited survivin gene expression.