A two-component drug delivery system using Her-2-targeting thermosensitive liposomes

We report on a new method for enhancing the specificity of drug delivery for tumor cells, using thermosensitive immunoliposomes. The liposomes are conjugated to the antibody trastuzumab (Herceptin^®), which targets the human epidermal growth factor receptor 2 (Her-2), a cell membrane receptor overexpressed in many human cancers. Being thermosensitive, the liposomes only release their contents when heated slightly above body temperature, allowing for the possibility of tissue targeting through localized hyperthermia. Using self-quenching calcein, we demonstrate the release of liposome contents into cell endosomes after brief heating to 42°C. To further increase targeting specificity, we incorporate the concept of a two-component delivery system that requires the interaction of two different liposomes within the same endosome for cytoplasmic delivery. Experimental evaluation of the technique using fluorescently labeled liposomes shows that a two-component delivery system, combined with intracellular disruption of liposomes by hyperthermia, significantly increases specificity for Her-2-overexpressing tumor cells.     

Gene delivery to Her-2 + breast cancer cells using a two-component delivery system to achieve specificity

Current liposomal gene delivery systems predominately utilize cationic lipids, which efficiently bind and deliver DNA plasmid, but also result in nonspecific gene expression in lung and liver tissue. To improve specificity, a two-component delivery strategy employing neutral liposomes was used to target breast cancers positive for the human epidermal growth factor receptor 2 (Her-2). The first component consisted of plasmid DNA condensed with cationic polyethylene glycol (PEG) modified polylysine (PL/DNA). The second component was a neutral Her-2 targeting liposome conjugated to the pore-forming protein, Listeriolysin O (LLO). Independently, PL/DNA delivery resulted in low expression of plasmid DNA. However, when PL/DNA and LLO/liposomes co-localized within an endosome, LLO disrupted endosome integrity, leading to cytoplasmic delivery and expression of the plasmid. When used to deliver a plasmid encoding the luciferase gene, this two-component system resulted in gene expression that was 268-fold greater in Her-2 positive cells than in Her-2 negative cells.From the Clinical EditorIn this paper a novel two-component gene delivery method is presented using PL/DNA and LLO liposomes, demonstrating strongly significant results in a model system.     

Efficiency of Cytoplasmic Delivery by pH-Sensitive Liposomes to Cells in Culture

The intracellular processing of pH-sensitive liposomes composed of cholesterylhemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) by eukaryotic cell lines has been compared to non-pH-sensitive liposomes made of CHEMS and dioleoylphosphatidylcholine (DOPC). The pH-sensitive liposomes can deliver encapsulated fluorescent molecules [calcein, fluoresceinated dextran, fluoresceinated polypeptide, and diphtheria toxin A chain (DTA)] into the cytoplasm. Cytoplasmic delivery can be blocked in the presence of ammonium chloride or EDTA, indicating that the process requires a low-pH environment and the presence of divalent cations. Inhibition of cellular protein synthesis by DTA delivery from the pH-sensitive liposome is orders of magnitude greater than from the non-pH-sensitive liposome composition. The delivery of DTA into the cytoplasm by pH-sensitive liposomes is at least 0.01% of cell-associated liposomal DTA. There is no significant difference in the degradation rate of bovine serum albumin (BSA) or the rate of acidification of pH-sensitive dye, 8-hydroxy-l,3,6-pyrene-trisulfonate (HPTS), when delivered to cells in pH-sensitive and non-pH-sensitive liposomes. Thus the efficiency of cytoplasmic delivery is less than 10% of the cell-associated liposome contents, which is the smallest difference that can be detected by these two assays. Based upon the various assays used to measure liposome content disposition in the cell, we conclude that the efficiency of cytoplasmic delivery by the CHEMS/DOPE liposomes is greater than 0.01% and less than 10% of the cell-associated liposomal contents.     

Cytosolic delivery via escape from the endosome using emulsion droplets and ultrasound

Vaporizing emulsion droplets may aid in endosomal rupture as a drug delivery route to the cytosol. Upon insonation, emulsion droplets formed from perfluorocarbon liquids may vaporize with sufficient expansion to disrupt liposomal or endosomal membranes. Emulsion droplets of perfluorohexane (PFC6) or perfluoropentane (PFC5) were prepared as free droplets in calcein or as droplets encapsulated within liposomes containing calcein. Folate-stimulated endocytosis created an experimental model, wherein calcein was self-quenched until released from the vesicles. Upon release, calcein was diluted below its self-quenching concentration and its release quantified by fluorescence. In this experimental model, folated emulsions or folated eLiposomes were incubated with folate-starved HeLa cells. Samples were exposed to two seconds of 20-kHz ultrasound (US) at 1?W/cm². Fluorescence microscopy identified released intracellular calcein. Upon insonation, both free emulsion samples and eLiposome samples produced calcein release to the cytosol. Calcein fluorescence was more intense in samples containing PFC5 compared to PFC6. Insonation of samples without emulsion droplets produced no cytosolic delivery. Likewise, cells that took up emulsion droplets but were not exposed to US did not exhibit fluorescence throughout the cell. These results suggest that vaporizing emulsion droplets are internalized into the cells and can produce endosomal escape of a therapeutic payload.     

Development of a bone targeted thermosensitive liposomal doxorubicin formulation based on a bisphosphonate modified non-ionic surfactant

Bone is among the most common sites of metastasis in cancer patients, so it is an urgent need to develop drug delivery systems targeting tumor bone metastasis with the feature of controlled release. This study aimed to delivery of thermosensitive liposomal doxorubicin to bone for tumor metastasis treatment. First, Brij78 (polyoxyethylene stearyl ether) was conjugated with Pamidronate (Pa). By incorporating Pa-Brij78 to DPPC/Chol liposomes, we developed Pa surface functionalized liposomes. The Pa-Brij78/DPPC/Chol liposomes (PB-liposomes) exhibited a stronger binding affinity to hydroxyapatite (HA), a major component of bone, than Brij78/DPPC/Chol liposomes (B-liposomes). Doxorubicin (Dox) was then encapsulated in PB-liposomes and the results demonstrated complete release of Dox from PB-liposomes or the complex of HA/PB-liposomes within 10?min at 42?°C. Next, human lung cancer A549 cells were treated with the thermosensitive complex of HA/PB-liposomes/Dox to mimic tumor bone metastasis treatment through bone targeted delivery of therapeutic agents. Pre-incubation of HA/PB-liposomes/Dox with mild heat at 42?°C induced subsequent higher cytotoxicity to A549 cells than incubation of the same complex at 37?°C, suggesting more active drug release triggered by heat. In conclusion, we synthesized a novel surfactant Pa-Brij78 and it has the potential to be used for development of a bone targeted thermosensitive liposome formulation for treatment of tumor bone metastasis.     

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.     

Near-Infrared Image-Guided Delivery and Controlled Release Using Optimized Thermosensitive Liposomes

Purpose

To engineer optimized near-infrared (NIR) active thermosensitive liposomes to potentially achieve image-guided delivery of chemotherapeutic agents.

Methods

Thermosensitive liposomes were surface-coated with either polyethylene glycol or dextran. Differential scanning calorimetry and calcein release studies were conducted to optimize liposomal release, and flow cytometry was employed to determine the in vitro macrophage uptake of liposomes. Indocyanine green (ICG) was encapsulated as the NIR dye to evaluate the in vivo biodistribution in tumor-bearing mice.

Results

The optimized thermosensitive liposome formulation consists of DPPC, SoyPC, and cholesterol in the 100:50:30 molar ratio. Liposomes with dextran and polyethylene glycol demonstrated similar thermal release properties; however in vitro macrophage uptake was greater with dextran. Non-invasive in vivo NIR imaging showed tumor accumulation of liposomes with both coatings, and ex vivo NIR imaging correlated well with actual ICG concentrations in various organs of healthy mice.

Conclusions

The optimized thermosensitive liposome formulation demonstrated stability at 37?°C and efficient burst release at 40 and 42?°C. Dextran exhibited potential for application as a surface coating in thermosensitive liposome formulations. In vivo studies suggest that liposomal encapsulation of ICG permits reliable, real-time monitoring of liposome biodistribution through non-invasive NIR imaging.     

Differential cytosolic delivery and presentation of antigen by listeriolysin O-liposomes to macrophages and dendritic cells

Delivery of antigenic protein to the cytosol of antigen-presenting cells (APCs), such as macrophages (MPhi) and dendritic cells (DCs), is required for an efficient CD8 T-cell-mediated immune response. We have previously shown that co-encapsulation of antigenic protein inside pH-sensitive liposomes with listeriolysin O (LLO), a pore-forming protein of Listeria monocytogenes, generates efficient major histocompatibility complex class I (MHC I)-restricted immune responses both in vitro and in vivo. In this study, we sought to analyze the relative efficiency of LLO-mediated cytosolic delivery of liposomal antigen in two important APCs, macrophages and dendritic cells, by examining the sequential steps involved in antigen presentation to T-cells in cultured mouse bone marrow-derived MPhis (BMMPhis) and DCs (BMDCs). BMMPhis overall presented liposomal antigen better than BMDCs at a given concentration of liposomal antigen incubated with cells, and the trend was also observed after the presentation was normalized by the uptake of antigen. When soluble antigen was directly introduced into the cytosol, however, BMDCs presented the antigen more efficiently than BMMPhis. In addition, when the APCs were externally loaded with the antigenic peptide of the protein, BMDCs displayed a higher level of cell surface MHC I-peptide complexes and presented the peptide more efficiently than BMMPhis. These results combined together suggest that LLO-mediated release of liposomal antigen from the endosomal/lysosomal compartment may be more pronounced in BMMPhis than in BMDCs, and further implicates differential activity of LLO and varying efficiency of LLO-mediated endosomal escape in different antigen-presenting cell types.     

Preparation and optimisation of anionic liposomes for delivery of small peptides and cDNA to human corneal epithelial cells

Drug delivery to corneal epithelial cells is challenging due to the intrinsic mechanisms that protect the eye. Here, we report a novel liposomal formulation to encapsulate and deliver a short sequence peptide into human corneal epithelial cells (hTCEpi). Using a mixture of Phosphatidylcholine/Caproylamine/Dioleoylphosphatidylethanolamine (PC/CAP/DOPE), we encapsulated a fluorescent peptide, resulting in anionic liposomes with an average size of 138.8?±?34?nm and a charge of ?18.2?±?1.3?mV. After 2?h incubation with the peptide-encapsulated liposomes, 66% of corneal epithelial (hTCEpi) cells internalised the FITC-labelled peptide, demonstrating the ability of this formulation to effectively deliver peptide to hTCEpi cells. Additionally, lipoplexes (liposomes complexed with plasmid DNA) were also able to transfect hTCEpi cells, albeit at a modest level (8% of the cells). Here, we describe this novel anionic liposomal formulation intended to enhance the delivery of small cargo molecules in situ.     

Visualizing in vivo liposomal drug delivery in real-time

Liposomes have tremendous potential for efficient small molecule delivery. Previous studies, however, have been hampered by an inability to monitor their distribution and release of contents. Here, the authors demonstrate the real time monitoring of small molecule delivery using luciferin as a model. To monitor the release of luciferin in vivo, luciferin was packaged in thermosensitive liposomes and delivered into transgenic mice that constitutively express luciferase. Their experiments show the thermally induced release of the liposomal content in real time. In addition, the model provides evidence that the thermosensitive liposomes are stable over a long period of time ( approximately 3 weeks), and still release their content upon heating. These data present a strategy to monitor liposomal drug delivery in vivo with luciferin.     

The Bifunctional Liposomes Constructed by Poly(2-ethyl-oxazoline)-cholesteryl Methyl Carbonate: an Effectual Approach to Enhance Liposomal Circulation Time,pH-Sensitivity and Endosomal Escape

Purpose

A novel bifunctional liposome with long-circulating and pH-sensitive properties was constructed using poly(2-ethyl-oxazoline)-cholesteryl methyl carbonate (PEtOz-CHMC) in this study.

Methods

PEtOz-CHMC was synthesized and characterized by TLC, IR and ¹H-NMR. The obtained PEtOz lipid was inserted into liposomes by the post-insertion method. Through a series of experiments, such as drug release, tumor cell uptake, cytotoxicity, calcium-induced aggregation, pharmacokinetic experiments, etc., the pH-sensitive and long-circulating properties of PEtOzylated liposomes was identified.

Results

PEtOz-CHMC modified liposomes (PEtOz-L) showed increased calcein release at low pH. Flow cytometric analysis results showed that the fusion and cellular uptake of PEtOz-L could be promoted significantly at pH 6.4 compared with those at pH 7.4. Confocal laser scanning microscope observations revealed that PEtOz-L could respond to low endosomal pH and directly released the fluorescent tracer into the cytoplasm. MTT assays in HeLa cells demonstrated that doxorubicin hydrochloride (DOX) loaded PEtOz-L exhibited stronger anti-tumor activity in a medium at pH 6.4 than in a medium pH 7.4. PEtOz-L remained stable when these liposomes were incubated in calcium chloride solution. The cumulative calcein release rate of PEtOz-L was significantly lower than that of CL when the liposomes were dialysed in PBS. The pharmacokinetic experiments of liposomes in rats showed that t _1/2 and AUC of PEtOz-L were 4.13 times and 4.71 times higher than those of CL.

Conclusions

PEtOzylated liposomes exhibits excellent long-circulating and pH-sensitive properties. Our results suggest that PEtOz is a promising biomaterial for the modification of liposome in drug delivery.     

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.     

Hepatocellular carcinoma targeting effect of PEGylated liposomes modified with lactoferrin

A hepatocellular carcinoma targeting lactoferrin (Lf) modified PEGylated liposome system was developed for improving drug efficacies to hepatic cancer cells. In this present work, PEGylated liposomes (PLS) were successfully prepared by the thin film hydration method combined with peglipid post insertion. Lf was covalently conjugated to the distal end of DSPE-PEG2000-COOH lipid by amide bound and loaded onto PEGylated liposomes surface as the targeting ligand. To confirm the targeting efficacies to hepatic cancer, coumarin-6 and DiR were encapsulated as fluorescent probes. The confocal microscopy and flow cytometry demonstrated that Lf conjugated PEGylated liposomes (Lf-PLS) were efficiently associated by HepG2 cells, while limited interaction was found for liposomes modified with a negative control protein. A similar pharmacokinetic behavior was observed in pharmacokinetics study of the liposomal formulations. Meanwhile, the in vivo imaging of liposomes in HepG2 tumor bearing mice indicated that Lf-PLS achieved more accumulation in tumor compared with PLS without Lf conjugated. The significant in vitro and in vivo results suggested that Lf-PLS might be a promising drug delivery system for hepatocellular carcinoma therapy with low toxicity.     

Estrogen-functionalized liposomes grafted with glutathione-responsive sheddable chotooligosaccharides for the therapy of osteosarcoma

An estrogen (ES)-functionalized cationic liposomal system was developed and exploited for targeted delivery to osteosarcoma. Natural biocompatible chotooligosaccharides (COS, MW2-5?KDa) were covalently tethered to the liposomal surface through a disulfate bond (-SS-) to confer reduction-responsive COS detachment, whereas estrogen was grafted via polyethylene glycol (PEG 2?K) chain to achieve estrogen receptor-targeting. The liposomal carriers were prepared by the ethanol injection method and fluorescent anticancer drug doxorubicin (DOX) was loaded with ammonium sulfate gradient. The physicochemical properties, reduction-sensitivity, and the roles of estrogen on cellular uptake and tumor-targeting were studied. The Chol-SS-COS/ES/DOX liposomes were spherical with an average size about 110?nm, and high encapsulation efficiency. The liposomes were stable in physiological condition but rapidly release the payload in response to tumoral intracellular glutathione (20?mM). MTT cytotoxicity assay confirmed that Chol-SS-COS/ES/DOX liposomes exhibited higher cytotoxicity to MG63 osteosarcoma cells than to liver cells (LO2). Flow cytometry (FCM) and confocal laser scanning microscopy revealed that cellular uptake of Chol-SS-COS/ES/DOX liposomes by MG63, than the free DOX or Chol-SS-COS/DOX. Ex vivo fluorescence distribution study showed that the multifunctional liposomes selectively accumulated in the MG63 xenografts versus the organs. Chol-SS-COS/ES/DOX liposomes strongly inhibited the tumor growth and enhanced the animal survival rate. Overall, the COS grafted estrogen-functionalized cationic liposomes, fortified with glutathione-responsiveness, showed great potential for specific intracellular drug delivery to estrogen receptor-expressing tumors such as osteosarcoma.     

"Smart" delivery of antisense oligonucleotides by anionic pH-sensitive liposomes

Antisense oligonucleotides are molecules that are able to inhibit gene expression being, therefore, potentially active for the treatment of viral infections cancer or inflammatory diseases. However, because of their poor stability in biological medium and their weak intracellular penetration, "smart" delivery systems such as anionic pH-sensitive liposomes were designed. Most known liposome formulations contain a specific phospholipid, phosphatidylethanolamine (PE), which undergoes a transition from lamellar to inverted micelles structures at low pH and allow fusion of liposomal and endosomal membranes and by consequence destabilization of the endosomes. Therefore, liposomes made of PE are able to release their contents in response to acidic pH within the endosomal system while remaining stable in plasma thus improving the cytoplasmic delivery of oligonucleotides after endocytosis. This review illustrates the advantages of this approach for the delivery of antisense oligonucleotides.     

Delivery of lipids and liposomal proteins to the cytoplasm and Golgi of antigen-presenting cells. mangala.rao@na.amedd.army.mil

Liposomes have the well-known ability to channel protein and peptide antigens into the MHC class II pathway of phagocytic antigen-presenting cells (APCs) and thereby enhance the induction of antibodies and antigen-specific T cell proliferative responses. Liposomes also serve as an efficient delivery system for entry of exogenous protein and peptide antigens into the MHC class I pathway and thus are very efficient inducers of cytotoxic T cell responses. Soluble antigens that are rendered particulate by encapsulation in liposomes are localized both in vacuoles and in the cytoplasm of bone marrow-derived macrophages. Utilizing fluorophore-labeled proteins encapsulated in liposomes we have addressed the question of how liposomal antigens enter the MHC class I pathway. After phagocytosis of the liposomes, the fluorescent liposomal protein and liposomal lipids enter the cytoplasm where they are processed by the proteasome complex. The processed liposomal protein is then transported via the TAP complex into the endoplasmic reticulum and the Golgi complex. Both the liposomal lipids and the liposomal proteins appear to follow the same intracellular route and they are processed as a protein-lipid unit. In the absence of a protein antigen (empty liposomes), there is no organelle-specific localization of the liposomal lipids. In contrast, when a protein is encapsulated in these liposomes, the distribution of the liposomal lipids is dramatically affected and the liposomal lipids localize to the trans-Golgi area. Localization of the protein in the trans-Golgi area requires liposomal lipids. Similarly, for the localization of liposomal lipids in the trans-Golgi area, there is an obligatory requirement for protein. Therefore, the intracellular trafficking patterns of liposomal lipids and liposomal protein are reciprocally regulated. Presence of both liposomal lipids and liposomal protein in the trans-Golgi therefore facilitates the entry of liposomal antigens into the MHC class I pathway. It is also possible that liposomal lipids are presented to T cells via the recently described CD1 pathway for lipid antigens. Because liposome-formulated vaccines have the potential to stimulate antibody as well as cellular immune responses to protein and lipid components, this approach could prove to be extremely useful in designing vaccine strategies.     

Endocytosis and transcytosis of an immunoliposome-based brain drug delivery system

Immunoliposomes conjugated with the OX26 monoclonal antibody to the rat transferrin receptor can be used for brain delivery of small molecules. In the present study the uptake of OX26-immunoliposomes by target cells as well as their transcytosis across the blood-brain barrier was investigated. Microscopy of RG2 rat glioma cells incubated with fluorescence labeled OX26-immunoliposomes revealed intracellular co-localization of liposomal cargo, the liposomal membrane bilayer and the OX26 monoclonal antibody. The distinct particulate staining pattern was indicative for accumulation of OX26-immunoliposomes within endosomal or lysosomal compartments. Prolonged incubations demonstrated endosomal release of the liposomal cargo propidium iodide to the cytoplasm. A maximum of 50% of propidium iodide was released from the endosomal compartment after 24 hours of incubation. Transcytosis was studied using an in vitro model of the blood-brain barrier consisting of immortalized RBE4 rat brain endothelial cells. OX26-immunoliposomes did permeate across the RBE4 cell monolayer and showed a permeability coefficient of P(app) = 1.6 x 10(-5) ml/s. Transport was inhibited at low temperature, by competition with free OX26 or by exchanging the OX26 monoclonal antibody for an unspecific isotype antibody. Transcytosis of OX26-immunolipsomes was confirmed in vivo by the brain perfusion and capillary depletion technique. OX26-immunoliposomes were detected within the post-vascular compartment of brain parenchyma (PS product = 2.4 microl/g/min.) and were not associated with the brain microvasculature.     

The Two Sweet Sides of Janus Lectin Drive Crosslinking of Liposomes to Cancer Cells and Material Uptake

A chimeric, bispecific Janus lectin has recently been engineered with different, rationally oriented recognition sites. It can bind simultaneously to sialylated and fucosylated glycoconjugates. Because of its multivalent architecture, this lectin reaches nanomolar avidities for sialic acid and fucose. The lectin was designed to detect hypersialylation—a dysregulation in physiological glycosylation patterns, which promotes the tumor growth and progression of several cancer types. In this study, the characteristic properties of this bispecific Janus lectin were investigated on human cells by flow cytometry and confocal microscopy in order to understand the fundamentals of its interactions. We evaluated its potential in targeted drug delivery, precisely leading to the cellular uptake of liposomal content in human epithelial cancer cells. We successfully demonstrated that Janus lectin mediates crosslinking of glyco-decorated giant unilamellar vesicles (GUVs) and H1299 lung epithelial cells. Strikingly, the Janus lectin induced the internalization of liposomal lipids and also of complete GUVs. Our findings serve as a solid proof of concept for lectin-mediated targeted drug delivery using glyco-decorated liposomes as possible drug carriers to cells of interest. The use of Janus lectin for tumor recognition certainly broadens the possibilities for engineering diverse tailor-made lectin constructs, specifically targeting extracellular structures of high significance in pathological conditions.     

Cytosolic Delivery of Liposomally Targeted Proteins Induced by Photochemical Internalization

Purpose The application of therapeutic proteins is often hampered by limited cell entrance and lysosomal degradation, as intracellular targets are not reached. By encapsulation of proteins into targeted liposomes, cellular uptake via endocytosis can be enhanced. To prevent subsequent lysosomal degradation and promote endosomal escape, photochemical internalization (PCI) was studied here as a tool to enhance endosomal escape. PCI makes use of photosensitising agents which localize in endocytic vesicles, inducing endosomal release upon light exposure. Materials and Methods The cytotoxic protein saporin was encapsulated in different types of targeted liposomes. Human ovarian carcinoma cells were incubated with the photosensitiser TPPS2a and liposomes. To achieve photochemical internalization, the cells were illuminated for various time periods. Cell viability was used as read-out. Illumination time and amount of encapsulated proteins were varied to investigate the influence of these parameters. Results The cytotoxic effect of liposomally targeted saporin was enhanced by applying PCI, likely due to enhanced endosomal escape. The cytotoxic effect was dependent on the amount of encapsulated saporin and the illumination time. Conclusion PCI is a promising technique for promoting cytosolic delivery of liposomally targeted saporin. PCI may also be applicable to other liposomally targeted therapeutic proteins with intracellular targets.     

Targeted delivery of doxorubicin via estrone-appended liposomes

Estrone-appended liposomal formulation of doxorubicin was designed to enhance the capability of clinically used liposomal doxorubicin formulation with the added advantage of delivery of doxorubicin to its destination site, i.e. cancerous cells over-expressing estrogen receptors (ERs). Estrone was conjugated with distearoyl phosphatidylethanolamine (DSPE) using succinic anhydride as a linker and the conjugate was characterized by IR and mass spectroscopies. Estrone-coupled liposomes were prepared with the composition of egg phosphatidylcholine/cholesterol/distearoyl phosphatidylethanolamine–estrone (PC/CHOL/DSPE–ES) at the molar and drug–lipid ratios of 7:3:0.5 and 0.1:1 (w/w), respectively. The average vesicle sizes of the conventional and estrone-appended liposomes were found to be 193 ± 24 and 207 ± 28 nm, respectively. The fluorescent microscopy studies were performed with estrone-appended liposomes loaded with 6-carboxyfluorescein (6-CF). Results of in vivo biodistribution studies showed that estrone-appended liposomes were effectively taken up by cells expressing ERs. The drug uptake study showed that accumulation of ligand-appended liposomes in the breast and uterus was 13.9 and 12.7 times higher when compared with plain drug, and 11.05 and 10.3 times higher when compared with conventional liposomes, respectively, after 8 h of tail vein intravenous administration. The findings are seminal for selective targeting of antineoplastic agents to the ER, which are frequently over-expressed on carcinoma of breast and uterine origin, and opens the promising possibilities for non-immunogenic, site-specific delivery of bioactive(s) to these sites.