A mechanistic study of the effect of transferrin conjugation on cytotoxicity of targeted liposomes

Abstract

This study was performed to prepare 5-fluorouracil (5FU) containing targeted liposomes for the safety and efficacy enhancement. Liposomes were prepared using thin layer method and transferrin (Tf) was employed as the targeting ligand. Morphology of 5FU-loaded liposomes was assessed by transmission electron microscopy (TEM). The in vitro cytotoxicity was investigated via MTT assay on HT-29, CT26 and fibroblast cells. Mitochondrial membrane and cell death evaluations were also investigated. Resulted showed that the encapsulation efficiency (EE%) and particle size of the liposomes were 40.12% and 130?nm, respectively. TEM image implied that liposomes were spherical in shape. In cancer cells, targeted liposomes triggered the mitochondrial apoptotic pathway by lower production of reactive oxygen species (ROS) (63.58 vs 84.95 fluorescence intensity), reduced mitochondrial membrane potential and releasing of cytochrome c (68.66 vs 51.13?ng/mL). The results of this study indicated that Tf-targeted 5FU liposomes can be employed as promising nanocarrier for the delivery of drugs to cancer cells.     

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.     

Integrin Targeting and Toxicological Assessment of Peptide‐Conjugated Liposome Delivery Systems to Activated Endothelial Cells

Utilization of functionalized liposomes as the means of targeted delivery of therapeutics may enhance specific transport of biologically active drugs to target tissues, while avoiding or reducing undesired side effects. In the present investigation, peptide‐conjugated cationic liposomes were constructed with the aim of targeting integrins (i.e. vitronectin and/or fibronectin receptors) on activated endothelial cells. The peptide‐conjugated liposomes induced only cytotoxicity at the highest concentration in non‐activated or activated endothelial cells, as well as in co‐culture of endothelial cells and macrophages. There was unaltered secretion of cytokines after exposure of peptide‐conjugated liposomes to endothelial cells, indicating that the materials were not inflammogenic. Liposomes with a peptide targeting the fibronectin receptor (integrin α5β1) were more effective in targeting of activated endothelial cells, as compared to a liposome with a peptide that targeted both the fibronectin and vitronectin receptors, as well as liposomes with a control peptide. The liposome targeted to the fibronectin receptor also displayed uptake in endothelial cells in co‐culture with activated macrophages. Therefore, this study demonstrates the feasibility of constructing a peptide‐conjugated cationic liposome, which displays targeting to activated endothelial cells at concentrations that are not cytotoxic or inflammogenic to the cells.     

双配体修饰的阿霉素脂质体靶向于脑胶质瘤的体外研究

目的筛选和优化转铁蛋白、叶酸共同修饰的阿霉素脂质体的处方及制备工艺,以期得到具有良好的脑胶质瘤靶向治疗作用的给药系统。方法采用薄膜分散和硫酸铵梯度法制备阿霉素脂质体。将叶酸连接至二硬脂酸磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000-NH2)得到DSPE-PEG2000-Folic,考察不同磷脂种类、药脂比、水化介质和载药时间,对脂质体粒径、包封率和稳定性的影响,确定脂质体的处方工艺。以大鼠的脑毛细血管内皮细胞(bEnd3)和星形胶质细胞组成体外血脑屏障(blood-brain barrier,BBB),并结合大鼠胶质瘤C6细胞,构建体外模拟胶质瘤靶向治疗的复合BBB模型。考察阿霉素脂质体在bEnd3细胞中的摄取机制和透过BBB的转运速率及对C6细胞的毒性。结果确定了DSPC作为主要磷脂组分,并以120 mmol.L 1的硫酸铵作为水化介质,药脂比为1∶1 5,载药时间选择60 min,成功制备了高包封率和稳定性的双配体脂质体。其在bEnd3细胞中摄取远大于普通脂质体(P<0.05),摄取过程受网格蛋白和小窝内陷介导的细胞内吞作用,并受转铁蛋白和叶酸的影响;同时其在BBB模型中的药物透过速率、及其进一步透过BBB后对下层C6细胞的毒性,均显著高于其他脂质体组。结论转铁蛋白和叶酸共同修饰的阿霉素脂质体具有较好的体外脑胶质瘤靶向治疗作用。     

Folate receptor–targeted quantum dot liposomes as fluorescence probes

In this study, the preparation of the novel imaging agents, folate receptor (FR)–targeted liposomes encapsulating hydrophilic CdTe quantum dots (QDs), and their use as luminescence probes for live cell imaging are reported. Hydrophilic CdTe QDs were directly synthesized in the water phase, and FR-targeted QD liposomes were prepared by hydrating the lipid thin film with CdTe suspension. Formulations were characterized by UV-visible and fluorescent measurements, liposomal particle size, and zeta potential. The targeting and imaging ability of FR-targeted liposomes were investigated against the human uterine cervix cancer cell line (HeLa). Furthermore, the cytotoxicity of QD liposomes was evaluated by HeLa cells incubated with FR-targeted QD liposomes, nontargeted QD liposomes, and free QDs. The results showed that FR-targeted QD liposomes were spherically shaped with high fluorescence yield, excellent photochemical stability, good cancer targeting, and minimal cytotoxicity. The average size of FR-targeted fluorescence liposomes was ~105?nm, and their size distribution was rather narrow. After storage at 4°C for 11 months, QD liposomes maintained similar size and did not show any leakage of QDs. FR-targeted CdTe QD liposomes, which can target tumor cells via FR-mediated endocytosis, would become an attractive probe for tumor cell or tissue imaging for a long-time monitoring.     

Anticancer Drug Delivery with Transferrin Targeted Polymeric Chitosan Vesicles

Purpose. The study reports the initial biological evaluation of targeted polymeric glycol chitosan vesicles as carrier systems for doxorubicin (Dox). Methods. Transferrin (Tf) was covalently bound to the Dox-loaded palmitoylated glycol chitosan (GCP) vesicles using dimethylsuber- imidate (DMSI). For comparison, glucose targeted niosomes were prepared using N-palmitoyl glucosamine. Biological properties were studied using confocal microscopy, flow cytometry, and cytotoxicity assays as well as a mouse xenograft model. Results. Tf vesicles were taken up rapidly with a plateau after 1-2 h and Dox reached the nucleus after 60-90 min. Uptake was not increased with the use of glucose ligands, but higher uptake and increased cytotoxicity were observed for Tf targeted as compared to GCP Dox alone. In the drug-resistant A2780AD cells and in A431 cells, the relative increase in activity was significantly higher for the Tf-GCP vesicles than would have been expected from the uptake studies. All vesicle formulations had a superior in vivo safety profile compared to the free drug. Conclusions. The in vitro advantage of targeted Tf vesicles did not translate into a therapeutic advantage in vivo. All vesicles reduced tumor size on day 2 but were overall less active than the free drug.     

Preparation of a TK/GCV administration system mediated by transferrin modified pro-cationic liposomes

Transferrin modified pro-cationic liposomes were prepared and used to investigate the effect of targeting therapeutic genes to human hepatoma carcinoma cells in vitro. The main lipid CHETA, cholest-5-en-3beta-yl[2-[[4-[(carboxymethyl)dithio]-1-iminobutyl]amino]ethyl] carbamate (C36H61N3O4S2), was synthesized and used to prepare pro-cationic liposomes. The thymidine kinase (TK) gene loaded pro-cationic liposomes were prepared by first mixing the plasmid DNA and protamine together, and then incubating the resulted polyplexes with blank pro-cationic liposomes preformed by the thin film dispersion-sonication method. Transferrin (Tf) was adsorbed on the surface of pro-cationic liposomes via electrostatic interactions to form transferrin modified pro-cationic liposomes. Cellular association was measured by fluorimetry at excitation and emission wavelengths of 490 and 520 nm, respectively. The viability of TK gene infected cells following administration of ganciclovir (GCV) was investigated by MTT assay. The transferrin modified TK gene pro-cationic liposomes had a mean diameter of 240 +/- 12 nm and zeta potential of -24.10 +/- 2.5 mV (n = 3). The transmission electron microscopy image indicated that most of the liposomes were relatively regular and spherical with a condensed core inside. Cell-associated fluorescence of Tf-liposomes and unmodified liposomes (without transferrin) was 7.8 x 10(6), and 3.2 x 10(6) per milligram protein, respectively. Compared to Lipofectamine 2000 (Invitrogen, USA) the pro-cationic liposomes and transferrin modified pro-cationic liposomes had less cytotoxicity to cells. The transduced TK gene HepG2 cells were more sensitive to GCV than the un-transduced TK gene ones and the human normal Chang liver cells were not affected by the TK/GCV system mediated by procationic liposomes.     

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.     

Mannosylated liposomes improve therapeutic effects of paclitaxel in colon cancer models

Mannose receptor (MR) is a highly effective endocytic receptor. It is closely related to tumour immune escape and metastasis. We found that MR was highly expressed in some colon cancer cell lines such as CT26 and HCT116 cells. Therefore, MR might be a potential target in colon cancer therapy. In this study, we aimed to develop mannosylated liposomes containing anticancer drug paclitaxel and investigate the potential effects on targeted therapy for colon cancer. Mannosylated liposomes were prepared by film dispersion method. Characterisation, drug release behaviour, cytotoxicity, cellular uptake, anti-tumour efficacy and safety profiles of liposomes were investigated. The results showed that mannosylated liposomes had a higher CT26 cells uptake efficiency and tumour inhibition rate, which might be due to the target effect to MR. And no notable toxicity was observed. Taken together, these data demonstrated that mannosylated liposomes could target colon cancer and improve the efficacy of chemotherapy.     

Intracellular delivery of redox cycler-doxorubicin to the mitochondria of cancer cell by folate receptor targeted mitocancerotropic liposomes

Cancer cells reflect higher level of ROS in comparison to the normal cell, so they become more vulnerable to further oxidative stress induced by exogenous ROS-generating agents. Through this a novel therapeutic strategy has evolved, which involves the delivery of redox cycler-doxorubicin (DOX) to the mitochondria of cancer cell where it acts as a source of exogenous ROS production. The purpose of this study is to develop a liposomal preparation which exhibits a propensity to selectively target cancer cell along with the potential of delivering drug to mitochondria of cell. We have rendered liposomes mitocancerotropic (FA-MTLs) by their surface modification with dual ligands, folic acid (FA) for cancer cell targeting and triphenylphosphonium (TPP) cations for mitochondria targeting. The cytotoxicity, ROS production and cell uptake of doxorubicin loaded liposomes were evaluated in FR (+) KB cells and found to be increased considerably with FA-MTLs in comparison to folic acid appended, mitochondria targeted and non-targeted liposomes. As confirmed by confocal microscopy, the STPP appended liposomes delivered DOX to mitochondria of cancer cell and also showed higher ROS production and cytotoxicity in comparison to folic acid appended and non-targeted liposomes. Most importantly, mitocancerotropic liposomes showed superior activity over mitochondria targeted liposomes which confirm the synergistic effect imparted by the presence of dual ligands - folic acid and TPP on the enhancement of cellular and mitochondrial delivery of doxorubicin in KB cells.     

Targeting of folate-conjugated liposomes with co-entrapped drugs to prostate cancer cells via prostate-specific membrane antigen (PSMA)

Folate-targeted liposomes (FTL) were tested as drug delivery vehicles to PSMA-positive cancer cells. We used FL with co-entrapped mitomycin C lipophilic prodrug (MLP) and doxorubicin (DOX), and the LNCaP prostate cancer cell line which expresses PSMA but is negative for folate receptor. A major increase in cell drug levels was observed when LNCaP cells were incubated with FTL as compared to non-targeted liposomes (NTL). MLP was activated to mitomycin C, and intracellular and nuclear fluorescence of DOX was detected, indicating FTL processing and drug bioavailability. PMPA (2-(phosphonomethyl)-pentanedioic acid), a specific inhibitor of PSMA, blocked the uptake of FTL into LNCaP cells, but did not affect the uptake of FTL into PSMA-deficient and folate receptor-positive KB cells. The cytotoxic activity of drug-loaded FTL was found significantly enhanced when compared to NTL in LNCaP cells. FTL may provide a new tool for targeted therapy of cancers that over-express the PSMA receptor.     

Heterogeneous liposome membranes with pH-triggered permeability enhance the in vitro antitumor activity of folate-receptor targeted liposomal doxorubicin

The killing efficacy of doxorubicin from liposome-based delivery carriers has been shown to correlate strongly with its intracellular trafficking and, in particular, its fast and extensive release from the delivery carrier. However, previously explored pH-triggered mechanisms that were designed to become activated during liposome endocytosis have also been shown to interfere with the liposome stability in vivo. We have designed pH-triggered gel-phase liposomes with heterogeneous membranes for the delivery of doxorubicin. These liposomes are triggered to form "leaky" interfacial boundaries between gel-gel phase separated domains on the membrane bilayer with lowering pH. The pH-triggered mechanism does not compromise liposome stability in vivo and results in superior in vitro killing efficacy of delivered doxorubicin when liposomes are endocytosed by a clathrin-mediated pathway. In the present work, we evaluate the general applicability of these liposomes when targeted to the folate receptor (FR) of KB cancer cells in vitro and become endocytosed by a less acidic pathway: the caveolae pathway. FR-targeting liposomes exhibit almost 50% decrease in cell association for increase in liposome size from 120 to 280 nm in diameter after relatively short incubation times (up to 4 h). The fraction of internalized vesicles, however, is approximately 60% of the cell associated vesicles independent of their size. Our findings demonstrate that, for the same doxorubicin uptake per cancer cell, the killing effect of doxorubicin delivered by pH-triggered lipid vesicles is greater (IC(50) = 0.032 mM for a 6 h incubation) than when delivered by a conventional non-pH-responsive composition (IC(50) = 0.194 mM). These findings suggest higher bioexposure of cells to the therapeutic agent possibly via faster and more extensive release from the carrier. Animal studies of FR-targeting non-pH-responsive liposomal doxorubicin report stronger therapeutic potential for the targeted approach relative to nontargeted liposomes and to free doxorubicin. The findings of the present study suggest that the targeted pH-triggered liposomes could potentially further enhance the therapeutic outcomes of doxorubicin in vivo.     

Effect of transferrin receptor-targeted liposomal doxorubicin in P-glycoprotein-mediated drug resistant tumor cells

The over-expression of P-glycoprotein (P-gp) has been associated with the development of multidrug resistance (MDR) in cancer cells. In this study, we examined whether transferrin receptor (Tf-R) targeted liposomes can efficiently deliver encapsulated doxorubicin (DXR) into MDR cells (SBC-3/ADM) via Tf-R-mediated endocytosis thus overcoming MDR by by-passing P-gp-mediated drug efflux. We prepared four types of liposome, i.e. untargeted and Tf-R-targeted, made of either egg-PC/cholesterol or hydrogenated egg PC/cholesterol. Only with the targeted EPC-liposome we achieved significant delivery of encapsulated DXR and increased cytotoxicity of encapsulated DXR on the MDR cells (3.5-fold higher than free DXR). Confocal microscopy and an intracellular drug-accumulation assay indicated that the targeted liposomes efficiently delivered DXR into cells where it readily accumulated in the nucleus, in both drug-sensitive and MDR cells. These findings suggest that the targeted liposomes are rapidly internalized via Tf-R-mediated endocytosis followed by release of their contents into the cytoplasm. The rapid internalization and content release, most likely facilitated by the higher fluidity of the EPC-based liposomes, may explain why only targeted EPC-liposomes were able to prevent drug efflux by P-gp and to consequently circumvent MDR. Our results indicate that in order to achieve MDR circumvention by means of liposomal encapsulation of DXR the liposomes not only need to be targeted, but also to have the proper physicochemical properties for adequate release of the drug. Furthermore, these in vitro results suggest that Tf-R targeted EPC-liposomes are a potentially useful drug delivery system to circumvent P-gp-mediated MDR of tumors.     

Physicochemical properties and antioxidant activity of gamma-oryzanol-loaded liposome formulations for topical use

The objective of this study is to prepare the γ-oryzanol-loaded liposomes and investigate their physicochemical properties and antioxidant activity intended for cosmetic applications. Liposomes, Composing phosphatidylCholine (PC) and Cholesterol (Chol), CHAPS or sodium taurocholate (NaTC) were prepared by sonication method. γ-oryzanol-loaded liposomes were prepared by using 3, 5 and 10% γ-oryzanol as an initial concentration. The formulation factors in a particular type and composition of lipid and initial drug loading on the physicochemical properties (i.e., particle size, zeta potential, entrapment efficiency, drug release) and antioxidant activity were studied. The particle sizes of bare liposomes were in nanometer range. The γ-oryzanol-loaded liposomes in formulations of PC/CHAPS and PC/NaTC liposomes were smaller than PC/Chol liposomes. The incorporation efficiency of 10% γ-oryzanol-loaded PC/Chol liposomes was less than γ-oryzanol-loaded PC/CHAPS liposomes and PC/NaTC liposomes allowing higher in vitro release rate due to higher free γ-oryzanol in buffer solution. The antioxidant activity of γ-oryzanol-loaded liposomes was not different from pure γ-oryzanol. Both γ-oryzanol-loaded PC/CHAPS liposomes and PC/NaTC liposomes were showed to enhance the antioxidant activity in NHF cells. γ-oryzanol-loaded PC/Chol liposomes demonstrated the lowest cytotoxicity in NHF cells. It was conceivably concluded that liposomes prepared in this study are suitable for γ-oryzanol incorporation without loss of antioxidant activity.     

Synthesis and evaluation of a novel ligand for folate-mediated targeting liposomes

Folate receptors (FRs) have been identified as cellular surface markers for cancer and leukemia. Liposomes containing lipophilic derivatives of folate have been shown to effectively target FR-expressing cells. Here, we report the synthesis of a novel lipophilic folate derivative, folate-polyethylene glycol-cholesterol hemisuccinate (F-PEG-CHEMS), and its evaluation as a targeting ligand for liposomal doxorubicin (L-DOX) in FR-expressing cells. Liposomes containing F-PEG-CHEMS, with a mean diameter of 120+/-20 nm, were synthesized by polycarbonate membrane extrusion and were shown to have excellent colloidal stability. The liposomes were taken up selectively by KB cells, which overexpress FR-alpha. Compared to folate-PEG-cholesterol (F-PEG-Chol), which contains a carbamate linkage, F-PEG-CHEMS better retained its FR-targeting activity during prolonged storage. In addition, F-PEG-CHEMS containing liposomes loaded with DOX (F-L-DOX) showed greater cytotoxicity (IC(50)=10.0muM) than non-targeted control L-DOX (IC(50)=57.5 microM) in KB cells. In ICR mice, both targeted and non-targeted liposomes exhibited long circulation properties, although F-L-DOX (t(1/2)=12.34 h) showed more rapid plasma clearance than L-DOX (t(1/2)=17.10h). These results suggest that F-PEG-CHEMS is effective as a novel ligand for the synthesis of FR-targeted liposomes.     

In vitro assessment of transferrin-conjugated liposomes as drug delivery systems for inhalation therapy of lung cancer

Most human tumours over-express receptors for growth factors and peptide hormones, which are being increasingly studied as a means to selectively deliver cytotoxic agents. An example being the transferrin receptor (TfR, CD71). Here, we studied expression levels and location of TfR in different lung epithelial cell types (i.e., bronchial and alveolar epithelial cells) by flow-cytometry and confocal laser scanning microscopy (CLSM). Furthermore, we assessed uptake levels and cytotoxicity of transferrin (Tf)-conjugated liposomes in vitro. TfR was found to be expressed at a significantly higher level in bronchial epithelial cells compared with their alveolar counterparts. Cells of cancerous origin (i.e., A549 cell line) showed a higher TfR expression level than healthy alveolar epithelial type II cells in primary culture. CLSM revealed TfR to be located primarily at the basolateral aspect of cells, with the exception of cells undergoing mitotic proliferation, which also showed TfR at their apical membranes, due to their loss of cell polarity. Higher expression levels of TfR correlated well with enhanced uptake of Tf-liposomes and increased levels of cytotoxicity. Liposome uptake was temperature-dependent and inhibitable by excess free Tf. Tf-conjugated liposomes appear as good candidates for an approach to deliver cytostatic drugs to sites of lung cancer by inhalation.     

Disaccharide-modified liposomes and their in vitro intracellular uptake

Sterically stabilized liposomes (SSL) were known to be accumulated passively in cancer due to the effect of enhanced permeability and retention (EPR). However, drug delivery via SSL to cancer seemed to show an insufficient improvement of chemotherapeutic efficacy. Herein, carbohydrate-binding proteins (lectins) of cell surface, which express on the plasmic membrane of many malignant cells, can be a good model of surface-modified liposomes. In this study, we investigated the in vitro characteristics of liposomes of which the surface was modified with a disaccharide molecule, sucrose or maltose. The disaccharide-modified lipids such as sucrose-modified lipid and maltose-modified lipid, in which the disaccharide was conjugated to the one end of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol)-2000 (DSPE-PEG2000), was synthesized. The disaccharide-modified liposomes were prepared by thin film-hydration method and then doxorubicin (DOX), an anticancer drug, was loaded to the prepared liposomes by the remote loading method with ammonium ion gradient. Flow cytometry and confocal microscopy analyses showed that the disaccharide-modified liposomes enhanced the intracellular uptake of liposomes into various cancer cell lines via lectin-mediated endocytosis. The disaccharide-modified liposomes in which DOX was loaded inside of liposomes exhibited higher cytotoxicity against various cancer cells than DOX-loaded SSL did. These results suggest that disaccharide-modified liposomes may be promising cancer targeting carriers which can enhance intracellular uptake and cytotoxicity of the drug-loaded liposomes via lectin-mediated endocytosis.     

Coupling of metal containing homing devices to liposomes via a maleimide linker: use of TCEP to stabilize thiol-groups without scavenging metals

Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe(3+)), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe(3+)). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2-3 h coupling to the liposomes, Fe(3+) is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes. Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe(3+) from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself. In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe(3+) from Tf. This method can be applied to other metal-containing homing devices as well.     

Targeting liposomes with protein drugs to the blood-brain barrier in vitro.

In this study, we aim to target pegylated liposomes loaded with horseradish peroxidase (HRP) and tagged with transferrin (Tf) to the BBB in vitro. Liposomes were prepared with the post-insertion technique: micelles of polyethylene glycol (PEG) and PEG-Tf were inserted into pre-formed liposomes containing HRP. Tf was measured indirectly by measuring iron via atomic absorption spectroscopy. All liposomes were around 100 nm in diameter, contained 5-13 microg HRP per mumol phospholipid and 63-74 Tf molecules per liposome (lipo Tf) or no Tf (lipo C). Brain capillary endothelial cells (BCEC) were incubated with liposomes at 4 degrees C (to determine binding) or at 37 degrees C (to determine association, i.e. binding+endocytosis) and the HRP activity, rather than the HRP amount was determined in cell lysates. Association of lipo Tf was two- to three-fold higher than association of lipo C. Surprisingly, the binding of lipo Tf at 4 degrees C was four-fold higher than the association of at 37 degrees C. Most likely this high binding and low endocytosis is explained by intracellular degradation of endocytosed HRP. In conclusion, we have shown targeting of liposomes loaded with protein or peptide drugs to the BCEC and more specifically to the lysosomes. This is an advantage for the treatment of lysosomal storage disease. However, drug targeting to other intracellular targets also results in intracellular degradation of the drug. Our experiments suggest that liposomes release some of their content within the BBB, making targeting of liposomes to the TfR on BCEC an attractive approach for brain drug delivery.     

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.