High-affinity targeting of biotin-labeled liposomes to streptavidin-conjugated ligands

Abstract

Cell-specific delivery of drug-loaded liposomal carrier systems can be achieved through the use of liposomes with covalently attached proteins. For such targeting strategies to be successful a number of potential difficulties, related to the preparation of the liposomes as well as optimization of properties that maximize in vivo access and binding to a defined target cell population, must be overcome. The studies summarized here have attempted to identify specific factors that will promote binding of targeted liposomes to defined target surfaces. Liposomes containing biotinylated phospha-tidylethanolamine were used to demonstrate that the avidity of a targeted liposome for streptavidin-coated ELISA plates and cells is influenced by liposome lipid composition, the amount of targeting molecule present per liposome, the nature of the targeting ligand, and the target surface. Specifically, it is demonstrated that the three most important factors (in order of importance) controlling the apparent affinity of targeted liposomes are (1) target ligand concentration in the liposomal membrane; (2) the presence of a spacer grout between the biotin and the phospholipid headgroup; and (3) the addition of cholesterol. Other less important factors that influence target liposome binding include whether the target ligand is attached to a saturated phospholipid compared to an unsaturated lipid and whether the bulk phospholipid species in the liposome is unsaturated versus saturated. These studies suggest that targeted liposomes exhibiting a broad range of binding avidities, as estimated by the concentration of liposomes required to achieve saturation of a target surface, can be prepared by selective design of the liposomal carrier. Advantages of the biotinylated liposome for targeting include the relative ease of preparation the possibility of preparation of large-scale batches suitable for clinical development), the ease of incorporation of the targeting ligand, and, importantly, the ability to alter the apparent affinity of the liposome for the target cell through choice of the biotin-labeled lipid and targeting molecule concentration. The potential for developing a two-step targeting strategy based on the use of biotinylated liposomes is discussed.     

Landscape phage fusion protein-mediated targeting of nanomedicines enhances their prostate tumor cell association and cytotoxic efficiency

Tumor-specific cytotoxicity of drugs can be enhanced by targeting them to tumor receptors using tumor-specific ligands. Phage display offers a high-throughput approach to screen for the targeting ligands. We have successfully isolated phage fusion peptides selective and specific for PC3 prostate cancer cells. Also, we have demonstrated a novel approach of targeting liposomes through tumor-specific phage fusion coat proteins, exploiting the intrinsic properties of the phage coat protein as an integral membrane protein. Here we describe the production of Rhodamine-labeled liposomes as well as doxorubicin-loaded long-circulating liposomes targeted to PC3 prostate tumor cells via PC-specific phage peptides, as an extension of our previous studies. Targeting of labeled liposomes was demonstrated using fluorescence microscopy as well as flow cytometry. Targeting of doxorubicin-loaded liposomes enhanced their cytotoxic effect against PC3 cells in vitro, indicating a possible therapeutic advantage. The simplicity of the approach for generating targeted liposomes coupled with the ability to rapidly obtain tumor-specific phage fusion proteins via phage display may contribute to a combinatorial system for the production of targeted liposomal therapeutics for advanced stages of prostate tumor.From the Clinical EditorThis paper demonstrates targeting cytotoxic agents to tumor receptors using tumor-specific ligands. The authors describe the production of Rhodamine-labeled liposomes as well as doxorubicin loaded long circulating liposomes targeted to PC3 prostate tumor cells via PC-specific phage peptides. This approach may be especially relevant for advanced prostate tumors.     

High-affinity targeting of biotin-labeled liposomes to streptavidin-conjugated ligands

Cell-specific delivery of drug-loaded liposomal carrier systems can be achieved through the use of liposomes with covalently attached proteins. For such targeting strategies to be successful a number of potential difficulties, related to the preparation of the liposomes as well as optimization of properties that maximize in vivo access and binding to a defined target cell population, must be overcome. The studies summarized here have attempted to identify specific factors that will promote binding of targeted liposomes to defined target surfaces. Liposomes containing biotinylated phospha-tidylethanolamine were used to demonstrate that the avidity of a targeted liposome for streptavidin-coated ELISA plates and cells is influenced by liposome lipid composition, the amount of targeting molecule present per liposome, the nature of the targeting ligand, and the target surface. Specifically, it is demonstrated that the three most important factors (in order of importance) controlling the apparent affinity of targeted liposomes are (1) target ligand concentration in the liposomal membrane; (2) the presence of a spacer grout between the biotin and the phospholipid headgroup; and (3) the addition of cholesterol. Other less important factors that influence target liposome binding include whether the target ligand is attached to a saturated phospholipid compared to an unsaturated lipid and whether the bulk phospholipid species in the liposome is unsaturated versus saturated. These studies suggest that targeted liposomes exhibiting a broad range of binding avidities, as estimated by the concentration of liposomes required to achieve saturation of a target surface, can be prepared by selective design of the liposomal carrier. Advantages of the biotinylated liposome for targeting include the relative ease of preparation the possibility of preparation of large-scale batches suitable for clinical development), the ease of incorporation of the targeting ligand, and, importantly, the ability to alter the apparent affinity of the liposome for the target cell through choice of the biotin-labeled lipid and targeting molecule concentration. The potential for developing a two-step targeting strategy based on the use of biotinylated liposomes is discussed.     

Liposomal drug delivery systems: an update review

The discovery of liposome or lipid vesicle emerged from self forming enclosed lipid bi-layer upon hydration; liposome drug delivery systems have played a significant role in formulation of potent drug to improve therapeutics. Recently the liposome formulations are targeted to reduce toxicity and increase accumulation at the target site. There are several new methods of liposome preparation based on lipid drug interaction and liposome disposition mechanism including the inhibition of rapid clearance of liposome by controlling particle size, charge and surface hydration. Most clinical applications of liposomal drug delivery are targeting to tissue with or without expression of target recognition molecules on lipid membrane. The liposomes are characterized with respect to physical, chemical and biological parameters. The sizing of liposome is also critical parameter which helps characterize the liposome which is usually performed by sequential extrusion at relatively low pressure through polycarbonate membrane (PCM). This mode of drug delivery lends more safety and efficacy to administration of several classes of drugs like antiviral, antifungal, antimicrobial, vaccines, anti-tubercular drugs and gene therapeutics. Present applications of the liposomes are in the immunology, dermatology, vaccine adjuvant, eye disorders, brain targeting, infective disease and in tumour therapy. The new developments in this field are the specific binding properties of a drug-carrying liposome to a target cell such as a tumor cell and specific molecules in the body (antibodies, proteins, peptides etc.); stealth liposomes which are especially being used as carriers for hydrophilic (water soluble) anticancer drugs like doxorubicin, mitoxantrone; and bisphosphonate-liposome mediated depletion of macrophages. This review would be a help to the researchers working in the area of liposomal drug delivery.     

Increasing the antitumor efficacy of doxorubicin-loaded liposomes with peptides anchored via a chelator lipid

The therapeutic efficacy of anticancer drugs like doxorubicin can be significantly increased by their incorporation into liposomes, but an ability to actively target the drug-containing liposomes to tumors could well provide an even greater curative effect. In this work, a commercial preparation of doxorubicin-loaded liposomes (Caelyx) was modified by incorporation of the metal chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA₃-DTDA) to enable engraftment of histidine-tagged targeting molecules. Our results show that when engrafted with p15-RGR, a His-tagged peptide containing a sequence purported to bind platelet-derived growth factor receptor β (PDGFRβ), NTA₃-DTDA-containing Caelyx (3NTA-Caelyx) can be targeted to NIH-3T3 cells in vitro, leading to increased cytotoxicity compared with non-targeted 3NTA-Caelyx. PDGFRβ is known to be expressed on pericytes in the tumor vasculature; however, when radiolabeled p15-RGR liposomes were administered to mice bearing subcutaneous B16-F1 tumors, minimal accumulation into tumors was observed. In contrast, an alternative targeting peptide, p46-RGD, was found to actively direct liposomes to tumors (4.7 %ID/g). Importantly, when injected into tumor-bearing mice, p46-RGD-engrafted 3NTA-Caelyx significantly decreased the tumor growth rate compared with controls. These results indicate that the incorporation of NTA₃-DTDA into liposomal drugs could represent a simple modification to the drug to allow engraftment of targeting molecules and to increase its efficacy.     

Predicting the influence of liposomal lipid composition on liposome size,zeta potential and liposome-induced dendritic cell maturation using a design of experiments approach

In this study, the effect of liposomal lipid composition on the physicochemical characteristics and adjuvanticity of liposomes was investigated. Using a design of experiments (DoE) approach, peptide-containing liposomes containing various lipids (EPC, DOPE, DOTAP and DC-Chol) and peptide concentrations were formulated. Liposome size and zeta potential were determined for each formulation. Moreover, the adjuvanticity of the liposomes was assessed in an in vitro dendritic cell (DC) model, by quantifying the expression of DC maturation markers CD40, CD80, CD83 and CD86. The acquired data of these liposome characteristics were successfully fitted with regression models, and response contour plots were generated for each response factor. These models were applied to predict a lipid composition that resulted in a liposome with a target zeta potential. Subsequently, the expression of the DC maturation factors for this lipid composition was predicted and tested in vitro; the acquired maturation responses corresponded well with the predicted ones. These results show that a DoE approach can be used to screen various lipids and lipid compositions, and to predict their impact on liposome size, charge and adjuvanticity. Using such an approach may accelerate the formulation development of liposomal vaccine adjuvants.     

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.     

A folate receptor-targeted liposomal formulation for paclitaxel

A novel liposomal formulation of paclitaxel targeting the folate receptor (FR) was synthesized and characterized. This formulation was designed to overcome vehicle toxicity associated with the traditional Cremophor EL-based formulation and to provide the added advantages of prolonged systemic circulation time and selective targeting of the FR, which is frequently overexpressed on epithelial cancer cells. The formulation had the composition of dipalmitoyl phosphatidylcholine/dimyristoyl phosphatidylglycerol/monomethoxy-polyethylene glycol (PEG)2000-distearoyl phosphatidylethanolamine/folate-PEG3350-distearoyl phosphatidylethanolamine (DPPC/DMPG/mPEG-DSPE/folate-PEG-DSPE) at molar ratios of (85.5:9.5:4.5:0.5) and a drug-to-lipid molar ratio of 1:33. The liposomes were prepared by polycarbonate membrane extrusion. The mean particle size of the liposomes was 97.1 nm and remained stable for at least 72 h at 4 degrees C. FR-targeted liposomes of the same lipid composition entrapping calcein were shown to be efficiently taken up by KB oral carcinoma cells, which are highly FR+. FR-targeted liposomes containing paclitaxel showed 3.8-fold greater cytotoxicity compared to non-targeted control liposomes in KB cells. Plasma clearance profiles of paclitaxel in the liposomal formulations were then compared to paclitaxel in Cremophor EL formulation. The liposomal formulations showed much longer terminal half-lives (12.33 and 14.23 h for FR-targeted and non-targeted liposomes, respectively) than paclitaxel in Cremophor EL (1.78 h). In conclusion, the paclitaxel formulation described in this study has substantial stability and favorable pharmacokinetic properties. The FR-targeted paclitaxel formulation is potentially useful for treatment of FR+ tumors and warrants further investigation.     

Synthesis and Evaluation of High Functionality and Quality Cell-penetrating Peptide Conjugated Lipid for Octaarginine Modified PEGylated Liposomes In U251 and U87 Glioma Cells

The use of peptide ligand modified PEGylated liposomes has been widely investigated for tumor targeting. Peptides are mainly inserted in the liposomal lipid bilayer using PEG_2K-lipid spacer (Peptide-PEG_2K-DSPE). However, a lower cellular uptake from longer nonlinear PEG_2K spacer was reported, we here synthesized a high functionality and quality (HFQ) lipid with a short, linear serine-glycine repeated peptide [(SG)₅] spacer. The objective of the current study is to develop novel octaarginine (R₈) peptide-HFQ lipid grafted PEGylated liposomes for glioma cells targeting. In vitro liposomes characterization showed that the mean particle size of all liposomal formulations was in the nano-scale range < 120 nm, with a small PDI value (i.e. ～0.2) and had a spherical shape under Transmission Electron Microscope, indicating a homogenous particle size distribution. The flow cytometry in vitro cellular association data with U251 MG and U87 cells revealed that 1.5% R₈-(SG)₅-lipid-PEGylated liposomes exhibited significantly higher cellular association of ～15.87 and 7.59-fold than the conventional R₈-PEG_2K-lipid-PEGylated liposomes (10.4 and 6.19-fold), respectively, relative to the unmodified PEGylated liposomes. Moreover, intracellular distribution studies using confocal laser scanning microscopy (CLSM) corroborated the results of the in vitro cell association. The use of ligand-HFQ-lipid liposomes could be a potential alternative to ligand-PEG_2K-lipid-modified liposomes as a drug delivery system for tumor targeting.     

Receptor-Specific Targeting with Liposomes <Emphasis Type="BoldItalic">In Vitro</Emphasis> Based on Sterol-PEG<Subscript>1300</Subscript> Anchors

Purpose  The challenge in developing liposomes to be used in active drug targeting is to design a method that can be used for modifying liposomal membranes that is applicable for a number of different specific ligands. In this study, the post insertion technique was used with activated sterol-PEG₁₃₀₀ anchors and was evaluated with regard to its effectiveness in active targeting in vitro. The key advantage of these anchors is that the insertion step into the liposomal membrane takes place at room temperature and is very fast. Materials and Methods  For in vitro experiments, neuroblastoma cell lines overexpressing GD2 antigen on their surface as a target structure were chosen. This allowed the use of anti-GD2 antibodies coupled to the liposomal surface for testing of specific binding. These modified liposomes were labelled with rhodamine-PE and their cellular association was analyzed by flow cytometry. Results  It was shown that the activated sterol-PEG₁₃₀₀ anchors allow specific and significant interactions of the modified liposomes with GD2 positive cells. Conclusion  Coupling using sterol-PEG₁₃₀₀ anchors is both simple and rapid. It is reproducible and applicable for all ligands bearing amino groups. This method demonstrates the advantage of a ready-to-use system for the modification of pre-formed liposomes with different ligands. M. Gantert and F. Lewrick contributed equally to this publication.     

Plasma membrane targeting by short chain sphingolipids inserted in liposomes improves anti-tumor activity of mitoxantrone in an orthotopic breast carcinoma xenograft model

Mitoxantrone (MTO) is clinically used for treatment of various types of cancers providing an alternative for similarly active, but more toxic chemotherapeutic drugs such as anthracyclines. To further decrease its toxicity MTO was encapsulated into liposomes. Although liposomal drugs can accumulate in target tumor tissue, they still face the plasma membrane barrier for effective intracellular delivery. Aiming to improve MTO tumor cell availability, we used short chain lipids to target and modulate the tumor cell membrane, promoting MTO plasma membrane traversal. MTO was encapsulated in liposomes containing the short chain sphingolipid (SCS), C₈-Glucosylceramide (C₈-GluCer) or C₈-Galactosylceramide (C₈-GalCer) in their bilayer. These new SCS-liposomes containing MTO (SCS-MTOL) were tested in vivo for tolerability, pharmacokinetics, biodistribution, tumor drug delivery by intravital microscopy and efficacy, and compared to standard MTO liposomes (MTOL) and free MTO.Liposomal encapsulation decreased MTO toxicity and allowed administration of higher drug doses. SCS-MTOL displayed increased clearance and lower skin accumulation compared to standard MTOL. Intratumoral liposomal drug delivery was heterogeneous and rather limited in hypoxic tumor areas, yet SCS-MTOL improved intracellular drug uptake in comparison with MTOL. The increased MTO availability correlated well with the improved antitumor activity of SCS-MTOL in a MDAMB-231 breast carcinoma model. Multiple dosing of liposomal MTO strongly delayed tumor growth compared to free MTO and prolonged mouse survival, whereas among the liposomal MTO treatments, C8-GluCer-MTOL was most effective. Targeting plasma membranes with SCS improved MTO tumor availability and thereby therapeutic activity and represents a promising approach to improve MTO-based chemotherapy.     

Cell Culture and Animal Studies for Intracerebral Delivery of Borocaptate in Liposomal Formulation

The efficacy of a liposomal formulation for intracerebral delivery of borocaptate (BSH) to brain tumor cells has been investigated using cell culture to study BSH uptake and persistence and using tumor-bearing rats to determine BSH distribution in the brain. During a 16-hr incubation, cellular uptake of BSH solution or BSH liposomal formulation was similar. However, the cellular persistence of BSH greatly increased when BSH was present in liposome. The differences in cellular persistence for BSH solution and BSH-loaded liposomes were significant both in 12-hr and 24-hr incubation experiments (p &lt; 0.05 and p &lt; 0.01, respectively). For the studies involving tumor-bearing rats, BSH level in tumor tissue was significantly higher than that in normal brain tissue at 2 hr and 6 hr after intracerebral injection of BSH-loaded liposomes (p &lt; 0.01). Our study indicated that the liposomal formulation enhanced cellular persistence of BSH in tumor cells and therefore favored the boron accumulation in the cells. With the prolonged physical retention of liposomes at the local injection site and the cellular retention of BSH enhanced by the liposomes, the intracerebral delivery of BSH using liposomal formulation may provide an effective boron delivery approach for boron neutron capture therapy of brain tumors.     

Targeted Delivery of a Heme Oxygenase Inhibitor with a Lyophilized Liposomal Tin Mesoporphyrin Formulation

Tin mesoporphyrin (SnMP) is a competitive inhibitor of heme oxygenase being examined clinically for the treatment of hyperbilirubinemia. Since liposomes have been shown to target SnMP to the spleen and increase its efficacy (S. A. Landaw, G. S. Drummond, and A. Kappas, Pediatrics 84, 1091–1096, 1989), we began investigating the feasibility of the preparation and scaleup of a liposomal SnMP formulation for clinical use. SnMP liposomes were prepared by high-pressure homogenization of a suspension of SnMP and egg phosphatidylcholine (1:20, w/w) in lactose-phosphate buffer, resulting in SnMP liposomes that were less than 200 nm in diameter and had encapsulation efficiencies of up to 90% at pH 5. The SnMP liposomes could be sterile filtered and lyophilized in a 1-day cycle with retention of the encapsulation efficiency and particle size. Following injection into rats, the distribution of liposomal SnMP to spleen at 2 and 6 hr after dosing was 5–20 times higher than for aqueous SnMP. Lyophilized SnMP liposomes were also more effective than aqueous SnMP in decreasing bilirubin production in bile-cannulated rats. The results suggest the potential for producing a safe, sterile, and effective lyophilized formulation of SnMP liposomes for targeting of heme oxygenase inhibitors to the spleen.     

Studies for the osmotic parameter of liposomes

By using the former equation (8), we modified the equation which can show the dissimilar osmotic behavior of liposome with composition change. The slope of the new equation was presented as the ratio of osmotically active volume (V _act ) to the total volume (V _total =V _act +V _dead ; V _dead is osmotically inactive volume) of liposomes. We defined it as a Z-value, which can elucidate the dissimilarity of the osmotic activity of multilamellar liposomes with the change of phospholipid composition and the differences of physicochemical properties of liposomes. Z-value was applied for studying the physico-chemical properties of liposomal membrane. The factor that affects on the Z-value was not the lipid concentration of liposome stock dispersion but the lipid composition of liposomal membrane. As the content of dicetylphosphate, the negative charged phospholipid, was increased, the osmotic activity, represented by Z-value, of multilamellar liposome was decreased. Under the hypertonic conditions (shrinking region), Z-value steadily increased and reached a maximum at 10 mole percent cholesterol with increasing the cholesterol content.     

Tumor cell targeting of liposome-entrapped drugs with phospholipid-anchored folic acid-PEG conjugates

Targeting of liposomes with phospholipid-anchored folate conjugates is an attractive approach to deliver chemotherapeutic agents to folate receptor (FR) expressing tumors. The use of polyethylene glycol (PEG)-coated liposomes with folate attached to the outer end of a small fraction of phospholipid-anchored PEG molecules appears to be the most appropriate way to combine long-circulating properties critical for liposome deposition in tumors and binding of liposomes to FR on tumor cells. Although a number of important formulation parameters remain to be optimized, there are indications, at least in one ascitic tumor model, that folate targeting shifts intra-tumor distribution of liposomes to the cellular compartment. In vitro, folate targeting enhances the cytotoxicity of liposomal drugs against FR-expressing tumor cells. In vivo, the therapeutic data are still fragmentary and appear to be formulation- and tumor model-dependent. Further studies are required to determine whether folate targeting can confer a clear advantage in efficacy and/or toxicity to liposomal drugs.     

On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters

The integration of pharmaceutical nanocarriers with phage display techniques is emerging as a new paradigm for targeted cancer nanomedicines. We explored the direct use of landscape phage fusion proteins for the self-assembly of phage-derived binding peptides to liposomes for cancer cell targeting. The primary purpose of this study was to elucidate the targeting mechanism with a particular emphasis on the relative contributions of the two motifs that make up the landscape phage fusion protein (a binding peptide and the phage pVIII coat protein) to the targeting efficiency. Using transmission electron microscopy and dynamic light scattering, we confirmed the formation of phage-liposomes. Using FACS analysis, fluorescence microscopy, and fluorescence photospectrometry, we found that liposomes modified with MCF-7-specific phage fusion proteins (MCF-7 binding peptide, DMPGTVLP, fused to the phage PVIII coat protein) provided a strong and specific association with target MCF-7 cancer cells but not with cocultured, nontarget cells including C166-GFP and NIH3T3. The substitution for the binding peptide fused to phage pVIII coat protein abolished the targeting specificity. The addition of free binding peptide, DMPGTVLP, competitively inhibited the interaction of MCF-7-specific phage-liposomes with target MCF-7 cells but showed no reduction of MCF-7-associated plain liposomes. The proteolysis of the binding peptide reduced MCF-7 cell-associated phage-liposomes in a proteinase K (PK) concentration-dependent manner with no effect on the binding of plain liposomes to MCF-7 cells. Overall, only the binding peptide motif was involved in the targeting specificity of phage-liposomes. The presence of phage pVIII coat protein did not interfere with the targeting efficiency.     

Targeting Anticancer Drugs to Tumor Vasculature Using Cationic Liposomes

Liposomal drug delivery systems improve the therapeutic index of chemotherapeutic agents, and the use of cationic liposomes to deliver anticancer drugs to solid tumors has recently been recognized as a promising therapeutic strategy to improve the effectiveness of conventional chemotherapeutics. This review summarizes the selective targeting of cationic liposomes to tumor vasculature, the merits of incorporating the polymer polyethylene-glycol (PEG), and the impact of the molar percent of the cationic lipid included in cationic liposomes on liposomal targeting efficacy. In addition, the discussion herein includes the therapeutic benefit of a dual targeting approach, using PEG-coated cationic liposomes in vascular targeting (of tumor endothelial cells), and tumor targeting (of tumor cells) of anticancer drugs. Cationic liposomes have shown considerable promise in preclinical xenograft models and are poised for clinical development.     

Vascular targeting of doxorubicin using cationic liposomes

Tumor vessel has been recognized as an important target for anticancer therapy. Cationic liposomes have been shown to selectively target tumor endothelial cells, thus can potentially be used as a carrier for chemotherapy agents. In this study, cationic liposomes containing 20 mol% cationic lipid dimethyl dioctadecyl ammonium bromide (DDAB) and loaded with doxorubicin (DOX) were prepared and characterized. The cationic liposomal DOX showed 10.8 and 9.1 times greater cytotoxicity than control PEGylated liposomal DOX in KB oral carcinoma and L1210 murine lymphocytic leukemia cells, and 7.7- and 6.8-fold greater cytotoxicity compared to control neutral non-PEGylated liposomal DOX, repectively, in these two cell lines. Although cationic liposomal DOX had higher tumor accumulation at 30 min after intravenous administration compared to control liposomes (p<0.05), DOX uptake of these liposomes at 24h post-injection was similar to that of PEGylated liposomal DOX (p>0.05) and approximately twice the levels of the free drug and non-PEGylated liposomes. In a murine tumor model generated using L1210 cells, increased survival rate was obtained with cationic liposomal DOX treatment compared to free DOX (p<0.01), neutral liposome control (p<0.01), as well as PEGylated liposomes (p<0.05). In conclusion, the cationic liposomal DOX formulation produced superior in vitro cytotoxicity and in vivo antitumor activity, and warrants further investigation.     

Characterization of Cationic Liposome Formulations Designed to Exhibit Extended Plasma Residence Times and Tumor Vasculature Targeting Properties

Cationic liposomes exhibit a propensity to selectively target tumor-associated blood vessels demonstrating potential value as anti-cancer drug delivery vehicles. Their utility however, is hampered by their biological instability and rapid elimination following i.v. administration. Efforts to circumvent rapid plasma elimination have, to date, focused on decreasing cationic lipid content and incorporating polyethylene glycol (PEG)-modified lipids. In this study we wanted to determine whether highly charged cationic liposomes with surface-associated PEG could be designed to exhibit extended circulation lifetimes, while retaining tumor vascular targeting properties in an HT29 colorectal cancer xenograft model. Cationic liposomes prepared of DSPC, cationic lipids (DODAC, DOTAP, or DC-CHOL), and DSPE-PEG₂₀₀₀ were studied. Our results demonstrate that formulations prepared with 50 mol% DODAC or DC-CHOL, and 20 mol% DSPE-PEG₂₀₀₀ exhibited circulation half-lives ranging from 6.5 to 12.5 h. Biodistribution studies demonstrated that DC-CHOL formulations prepared with DSPE-PEG₂₀₀₀ accumulated threefold higher in s.c. HT29 tumors than its PEG-free counterpart. Fluorescence microscopy studies suggested that the presence of DSPE-PEG₂₀₀₀ did not adversely affect liposomal tumor vasculature targeting. We show for the first time that it is achievable to design highly charged, highly pegylated (20 mol% DSPE-PEG₂₀₀₀) cationic liposomes which exhibit both extended circulation lifetimes and tumor vascular targeting properties     

Liposomes as drug carrier systems. Preparation, classification and therapeutic advantages of liposomes]

Bangham et al. (1965) created first the concept of the liposome as a microparticulate lipoidal vesicle separated from its aqueous environment by one or more lipid bilayers. Later Gregoriadis and Ryman (1972) suggested to use liposomes as drug carrier systems. Nowadays liposomes are under extensive investigation for improving the delivery of therapeutic agents, enzymes, vaccines and genetic materials. Liposomes offer an excellent opportunity to selective targeting of drugs which is expected to optimize the pharmacokinetical parameters, the pharmacological effect and to reduce the toxicity of the encapsulated drugs. To understand the system it is important to know the basic properties of these lipoidal vesicles. Our aim was to focus on the lipid composition and the method of liposome preparation what determine the liposomal membrane fluidity, permeability, vesicle size, charge density, steric hindrance and stability of the liposomes as principle factors those influence the fate of liposomes, their interactions with the blood components and other tissues after systemic administration or local use.