LIGAND-TARGETED LIPOSOMES DIRECTED AGAINST PATHOLOGICAL VASCULATURE

ABSTRACT

The development of liposomes targeted to angiogenic endothelial cells offers exciting prospects for intervention in cancer and inflammation. Several proteins are (strongly) over-expressed on angiogenic endothelial cells as compared to the quiescent endothelium, and could potentially serve as targets for site-specific drug delivery. In this contribution particular attention is given to the design of targeted long-circulating liposomes directed against the alpha v beta 3-integrin protein.     

Enhanced Active Targeting via Cooperative Binding of Ligands on Liposomes to Target Receptors

To achieve effective active targeting in a drug delivery system, we previously developed dual-targeting (DT) liposomes decorated with both vascular endothelial growth factor receptor-1 (VEGFR-1)-targeted APRPG and CD13-targeted GNGRG peptide ligands for tumor neovessels, and observed the enhanced suppression of tumor growth in Colon26 NL-17 tumor-bearing mice by the treatment with the DT liposomes encapsulating doxorubicin. In this present study, we examined the binding characteristics of DT liposomes having a different couple of ligands, namely, APRPG and integrin α_vβ₃-targeted GRGDS peptides. These DT liposomes synergistically associated to stimulated human umbilical vein endothelial cells compared with single-targeting (ST) liposomes decorated with APRPG or GRGDS. The results of a surface plasmon resonance assay showed that ST liposomes modified with APRPG or GRGDS peptide selectively bound to immobilized VEGFR-1 or integrin α_vβ₃, respectively. DT liposomes showed a higher affinity for a mixture of VEGFR-1 and integrin α_vβ₃ compared with ST liposomes, suggesting the cooperative binding of these 2 kinds of ligand on the liposomal surface. In a biodistribution assay, the DT liposomes accumulated to a significantly greater extent in the tumors of Colon26 NL-17 tumor-bearing mice compared with other liposomes. Moreover, the intratumoral distribution of the liposomes examined by confocal microscopy suggested that the DT liposomes targeted not only angiogenic endothelial cells but also tumor cells due to GRGDS-decoration. These findings suggest that "dual-targeting" augmented the affinity of the liposomes for the target cells and would thus be useful for active-targeting drug delivery for cancer treatment.     

Antiangiogenic photodynamic therapy (PDT) by using long-circulating liposomes modified with peptide specific to angiogenic vessels

For the improvement of therapeutic efficacy in photodynamic therapy (PDT) by using a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), we previously prepared polyethylene glycol (PEG)-modified liposomes encapsulating BPD-MA (PEG-Lip BPD-MA). PEGylation of liposomes enhanced the accumulation of BPD-MA in tumor tissue at 3 h after injection of it into Meth-A-sarcoma-bearing mice, but, unexpectedly, decreased the suitability of the drug for PDT when laser irradiation was performed at 3 h after the injection of the liposomal photosensitizer. To improve the bioavailability of PEG-Lip BPD-MA, we endowed the liposomes with active-targeting characteristics by using Ala-Pro-Arg-Pro-Gly (APRPG) pentapeptide, which had earlier been isolated as a peptide specific to angiogenic endothelial cells. APRPG-PEG-modified liposomal BPD-MA (APRPG-PEG-Lip BPD-MA) accumulated in tumor tissue similarly as PEG-Lip BPD-MA and to an approx. 4-fold higher degree than BPD-MA delivered with non-modified liposomes at 3 h after the injection of the drugs into tumor-bearing mice. On the contrary, unlike the treatment with PEG-Lip BPD-MA, APRPG-PEG-Lip BPD-MA treatment strongly suppressed tumor growth after laser irradiation at 3 h after injection. Finally, we observed vasculature damage in the dorsal air sac angiogenesis model by APRPG-PEG-Lip BPD-MA-mediated PDT. The present results suggest that antiangiogenic PDT is an efficient modality for tumor treatment and that tumor neovessel-targeted, long-circulating liposomes are a useful carrier for delivering photosensitizer to angiogenic endothelial cells.     

Antiangiogenic photodynamic therapy (PDT) by using long-circulating liposomes modified with peptide specific to angiogenic vessels

For the improvement of therapeutic efficacy in photodynamic therapy (PDT) by using a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), we previously prepared polyethylene glycol (PEG)-modified liposomes encapsulating BPD-MA (PEG-Lip BPD-MA). PEGylation of liposomes enhanced the accumulation of BPD-MA in tumor tissue at 3 h after injection of it into Meth-A-sarcoma-bearing mice, but, unexpectedly, decreased the suitability of the drug for PDT when laser irradiation was performed at 3 h after the injection of the liposomal photosensitizer. To improve the bioavailability of PEG-Lip BPD-MA, we endowed the liposomes with active-targeting characteristics by using Ala-Pro-Arg-Pro-Gly (APRPG) pentapeptide, which had earlier been isolated as a peptide specific to angiogenic endothelial cells. APRPG-PEG-modified liposomal BPD-MA (APRPG-PEG-Lip BPD-MA) accumulated in tumor tissue similarly as PEG-Lip BPD-MA and to an approx. 4-fold higher degree than BPD-MA delivered with non-modified liposomes at 3 h after the injection of the drugs into tumor-bearing mice. On the contrary, unlike the treatment with PEG-Lip BPD-MA, APRPG-PEG-Lip BPD-MA treatment strongly suppressed tumor growth after laser irradiation at 3 h after injection. Finally, we observed vasculature damage in the dorsal air sac angiogenesis model by APRPG-PEG-Lip BPD-MA-mediated PDT. The present results suggest that antiangiogenic PDT is an efficient modality for tumor treatment and that tumor neovessel-targeted, long-circulating liposomes are a useful carrier for delivering photosensitizer to angiogenic endothelial cells.     

Mouse myodulin, a new potential angiogenic factor, functionally expressed in yeast

Myodulin is a new integral membrane protein down-regulated in skeletal muscle atrophy. A first characterization suggested that myodulin could be a skeletal muscle angiogenic factor operating through direct cell-to-cell interactions. Here, we show that mouse myodulin can be expressed at the plasma membrane of Saccharomyces cerevisiae and purified. Co-culture experiments of myoblasts and cardiac vascular endothelial cells reveal that myodulin, either presented in yeast membranes or in liposomes after purification, increases the invasive potential of endothelial cells with a similar efficiency as when over-expressed in skeletal muscle cells. Functional essays using myodulin expressed in yeast bring new information about the myodulin functional mechanism, suggesting that one or several muscle cell components could be necessary for myodulin to increase the invasive potential of endothelial cells. The yield of purified myodulin should allow structure-function relationships studies for a better understanding of myodulin functional mechanisms.     

Anginex-conjugated liposomes for targeting of angiogenic endothelial cells

Identification of a tumor angiogenesis specific ligand would allow targeting of tumor vasculature. Lipidic vehicles can be used to deliver therapeutic agents for treatment of disease or contrast agents for molecular imaging. A targeting ligand would allow specific delivery of such formulations to angiogenic sites, thereby reducing side effects and gaining efficiency. Anginex, a synthetic 33-mer angiostatic peptide, has been described to home angiogenically activated endothelium, suggesting an ideal candidate as targeting ligand. To investigate this application of anginex, fluorescently labeled paramagnetic liposomes were conjugated with anginex. Using phase contrast and fluorescence microscopy as well as magnetic resonance imaging (MRI), we demonstrate that anginex-conjugated liposomes bind specifically to activated endothelial cells, suggesting application as an angiogenesis targeting agent for molecular targeting and molecular imaging of angiogenesis-dependent disease.     

Interactions of antitumour Sialyl Lewis X liposomes with vascular endothelial cells

Recently, we showed that tetrasaccharide selectin ligand SiaLe^X provided targeted delivery of liposomes loaded in the bilayer with melphalan lipophilic prodrug to tumour endothelium followed by severe injury of tumour vessels in a Lewis lung carcinoma model. Here, we study the impact of SiaLe^X ligand on the interactions of liposomes with human umbilical vein endothelial cells (HUVEC) using flow cytometry, spectrofluorimetry and confocal microscopy. Liposomes composed of egg phosphatidylcholine/yeast phosphatidylinositol/1,2-dioleoyl glycerol ester of melphalan, 8:1:1, by mol, and varying percentages of lipophilic SiaLe^X conjugate were labelled with BODIPY-phosphatidylcholine. The increase in SiaLe^X content in liposomes led to a proportional increase in their uptake by cytokine-activated cells as opposed to non-activated HUVEC: for 10% SiaLe^X liposomes, binding avidity and overall accumulation increased 14- and 6-fold, respectively. The early stages of intracellular traffic of targeted liposomes in the activated cells were monitored by co-localisation with the trackers of organelles. Endocytosis of SiaLe^X liposomes occurred mostly via clathrin-independent pathways, which does not contradict the available literature data on E-selectin localisation in the plasma membrane. Using dual fluorescence labelling, with rhodamine-labelled phospholipid and calcein encapsulated at self-quenching concentrations, we found that SiaLe^X liposomes undergo rapid (within minutes) internalisation by activated HUVEC accompanied by the disruption of liposomes; non-activated cells consumed a negligible dose of liposomes during at least 1.5 h. Our data evidence the selective effect of SiaLe^X formulations on activated endothelial cells and indicate their potential for intracellular delivery of melphalan lipophilic prodrug.     

Cationic liposomes enhance targeted delivery and expression of exogenous DNA mediated by N-terminal modified poly(L-lysine)-antibody conjugate in mouse lung endothelial cells.

A new and improved system for targeted gene delivery and expression is described. Transfection efficiency of N-terminal modified poly(L-lysine) (NPLL) conjugated with anti-thrombomodulin antibody 34A can be improved by adding to the system a lipophilic component, cationic liposomes. DNA, antibody conjugate and cationic liposomes form a ternary electrostatic complex which preserves the ability to bind specifically to the target cells. At the same time the addition of liposomes enhance the specific transfection efficiency of antibody-polylysine/DNA binary complex by 10 to 20-fold in mouse lung endothelial cells in culture.     

Internalization of paramagnetic phosphatidylserine-containing liposomes by macrophages

ABSTRACT: BACKGROUND: Inflammation plays an important role in many pathologies, including cardiovascular diseases, neurological conditions and oncology, and is considered an important predictor for disease progression and outcome. In vivo imaging of inflammatory cells will improve diagnosis and provide a read-out for therapy efficacy. Paramagnetic phosphatidylserine (PS)-containing liposomes were developed for magnetic resonance imaging (MRI) and confocal microscopy imaging of macrophages. These nanoparticles also provide a platform to combine imaging with targeted drug delivery. RESULTS: Incorporation of PS into liposomes did not affect liposomal size and morphology up to 12 mol% of PS. Liposomes containing 6 mol% of PS showed the highest uptake by murine macrophages, while only minor uptake was observed in endothelial cells. Uptake of liposomes containing 6 mol% of PS was dependent on the presence of Ca2+ and Mg2+. Furthermore, these 6 mol% PS-containing liposomes were mainly internalized into macrophages, whereas liposomes without PS only bound to the macrophage cell membrane. CONCLUSIONS: Paramagnetic liposomes containing 6 mol% of PS for MR imaging of macrophages have been developed. In vitro these liposomes showed specific internalization by macrophages. Therefore, these liposomes might be suitable for in vivo visualization of macrophage content and for (visualization of) targeted drug delivery to inflammatory cells.     

A tumor vasculature targeted liposome delivery system for combretastatin A4: Design, characterization, and in vitro evaluation

The objective of this study was to develop an efficient tumor vasculature targeted liposome delivery system for combretastatin A4, a novel antivascular agent. Liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, distearoyl phosphoethanolamine-polyethylene-glycol-2000 conjugate (DSPE-PEG), and DSPE-PEG-maleimide were prepared by the lipid film hydration and extrusion process. Cyclic RGD (Arg-Gly-Asp) peptides with affinity for αvβ3-integrins expressed on tumor vascular endothelial cells were coupled to the distal end of PEG on the liposomes sterically stabilized with PEG (long circulating liposomes, LCL). The liposome delivery system was characterized in terms of size, lamellarity, ligand density, drug loading, and leakage properties. Targeting nature of the delivery system was evaluated in vitro using cultured human umbilical vein endothelial cells (HUVEC). Electron microscopic observations of the formulations revealed presence of small unilamellar liposomes of ∼120 nm in diameter. High performance liquid chromatography determination of ligand coupling to the liposome surface indicated that more than 99% of the RGD peptides were reacted with maleimide groups on the liposome surface. Up to 3 mg/mL of stable liposomal combretastatin A4 loading was achieved with ∼80% of this being entrapped within the liposomes. In the in vitro cell culture studies, targeted liposomes showed significantly higher binding to their target cells than non-targeted liposomes, presumably through specific interaction of the RGD with its receptors on the cell surface. It was concluded that the targeting properties of the prepared delivery system would potentially improve the therapeutic benefits of combretastatin A4 compared with nontargeted liposomes or solution dosage forms.     

Tumor angiogenic endothelial cell targeting by a novel integrin-targeted nanoparticle

Angiogenesis is an important process in cancer growth and metastasis. During the tumor angiogenic process, endothelial cells express various cell surface receptors which can be utilized for molecular imaging and targeted drug delivery. One such protein receptor of interest is the integrin alphav beta3. Our group is involved in the development of molecular imaging probes and drug delivery systems targeting alphav beta3. Based on extensive lead optimization study with the integrin antagonist compounds, we have developed a new generation of integrin alphav beta3 compound (IA) which has superior binding affinity to alphav beta3. Utilizing this IA as a targeting agent, we have developed a novel integrin-targeted nanoparticle (ITNP) system for targ alphav beta3 was observed. These ITNPs also were rapidly taken up by cells that express alphav beta3. The ITNPs accumulated in the angiogenic vessels, after systemic administration in a murine squamous cell carcinoma model. This novel intergrin targeted ITNP platform will likely have an application in targeted delivery of drugs and genes in vivo and can also be used for molecular imaging.     

Targeting of small molecule anticancer drugs to the tumour and its vasculature using cationic liposomes: lessons from gene therapy

Cationic (positively charged) liposomes have been tested in various gene therapy clinical trials for neoplastic and other diseases. They have demonstrated selectivity for tumour vascular endothelial cells raising hopes for both antiangiogenic and antivascular therapies. They are also capable of being selectively delivered to the lungs and liver when administered intravenously. These vesicles are being targeted to the tumour in various parts of the body by using advanced liposomal systems such as ligand-receptor and antibody-antigen combinations. At present, the transferrin receptor is commonly used for cancer-targeted drug delivery systems including cationic liposomes. This review looks at the growing utility of these vesicles for delivery of small molecule anticancer drugs.     

VCAM-1 directed immunoliposomes selectively target tumor vasculature in vivo

Targeting the tumor vasculature and selectively modifying endothelial functions is an attractive anti-tumor strategy. We prepared polyethyleneglycol modified immunoliposomes (IL) directed against vascular cell adhesion molecule 1 (VCAM-1), a surface receptor over-expressed on tumor vessels, and investigated the liposomal targetability in vitro and in vivo. In vitro, anti-VCAM-1 liposomes displayed specific binding to activated endothelial cells under static conditions, as well as under simulated blood flow conditions. The in vivo targeting of IL was analysed in mice bearing human Colo 677 tumor xenografts 30 min and 24 h post i.v. injection. Whereas biodistribution studies using [3H]-labelled liposomes displayed only marginal higher tumor accumulation of VCAM-1 targeted versus unspecific ILs, fluorescence microscopy evaluation revealed that their localisations within tumors differed strongly. VCAM-1 targeted ILs accumulated in tumor vessels with increasing intensities from 30 min to 24 h, while control ILs accumulated in the tumor tissue by passive diffusion. ILs that accumulated in non-affected organs, mainly liver and spleen, primarily co-localised with macrophages. This is the first morphological evidence for selective in vivo targeting of tumor vessels using ILs. VCAM-directed ILs are candidate drug delivery systems for therapeutic anti-cancer approaches designed to alter endothelial function.     

Angiogenesis at the interface between basic and clinical research

The field of antiangiogenesis has shown a remarkably rapid evolution from the discovery at the bench to translation into the clinic. Currently a wide variety of compounds are in clinical trial as inhibitors of angiogenesis, and new compounds are being frequently added. The target cell of most angiogenesis inhibitors is the endothelial cell, with inhibitors that selectively affect a number of endothelial cell functions acquired during angiogenesis, including activation, proliferation, migration, invasion and survival. The endothelial cell may also be targeted by chemotherapeutic agents currently in use. The high doses and intermittent treatment schedules used to fight resistant tumor cells may be altered towards lower doses and chronic administration to obtain selective inhibition of angiogenic factor-stimulated endothelial cells as adjuvant therapy. Finally, gene therapy is a promising route for the delivery of novel protein inhibitors of angiogenesis, and is actively being investigated.     

Liposomal glucocorticoids as tumor-targeted anti-angiogenic nanomedicine in B16 melanoma-bearing mice

This study evaluates whether the inhibitory effects of prednisolone phosphate (PLP) encapsulated in long-circulating liposomes (LCL-PLP) on tumor growth and tumor angiogenesis described previously can be generalized to other types of glucocorticoids (GC) encapsulated in LCL (LCL-GC). Four types of synthetic GC, i.e. budesonide disodium phosphate (BUP), dexamethasone disodium phosphate (DXP), methylprednisolone disodium phosphate (MPLP), and PLP, were selected based on the difference in their potency to activate the human glucocorticoid receptor. The effects of all LCL-GC on the production of angiogenic/inflammatory factors in vivo in the B16.F10 murine melanoma model as well as on the viability and proliferation of tumor cells and endothelial cells in vitro were investigated. Our results show that all four selected LCL-GC formulations inhibit tumor growth, albeit to different degrees. The differences in antitumor activity of LCL-GC correlate with their efficacy to suppress tumor angiogenesis and inflammation. The strongest antitumor effect is achieved by LCL-encapsulated BUP (LCL-BUP), due to the highest potency of BUP versus the other three GC types. The in vitro results presented herein suggest that LCL-BUP has strong cytotoxic effects on B16.F10 melanoma cells and the anti-proliferative effects of all LCL-GC towards angiogenic endothelial cells play a role in their antitumor activity.     

Anti-cancer activity of targeted pro-apoptotic peptides.

We have designed short peptides composed of two functional domains, one a tumor blood vessel 'homing' motif and the other a programmed cell death-inducing sequence, and synthesized them by simple peptide chemistry. The 'homing' domain was designed to guide the peptide to targeted cells and allow its internalization. The pro-apoptotic domain was designed to be nontoxic outside cells, but toxic when internalized into targeted cells by the disruption of mitochondrial membranes. Although our prototypes contain only 21 and 26 residues, they were selectively toxic to angiogenic endothelial cells and showed anti-cancer activity in mice. This approach may yield new therapeutic agents.     

脂质体包裹阿霉素的体外抗肿瘤作用

 我们用超声法制备了内部包裹阿霉素,表面有抗人胃癌细胞M85的单克隆抗体3Hll或非相关抗体IgG的阿霉素靶向脂质体和阿霉素非靶向脂质体,研究了这些脂质体和游离阿霉素抑制靶细胞M85和非靶细胞HeLa细胞生长的能力。结果表明,一方面,与游离阿霉素或阿霉素非靶向脂质体相比,阿霉素靶向脂质体抑制M85细胞生长的能力提高了2—4倍,而在抑制非靶细胞HeLa细胞的生长方面,阿霉素靶向脂质体的效力还不如游离阿霉素或阿霉素非靶向脂质体。过量的游离单克隆抗体3Hll可以有效地抑制阿霉素靶向脂质体对M85细胞的细胞毒作用,而另一种单克隆抗体3G9则不能。空靶向和空非靶向脂质体对M85细胞生长均无影响。因此,靶向脂质体可以把包裹的抗癌药物专一地送给胃癌细胞,井杀伤它们。     

The role of angiogenic growth factors in organogenesis

Angiogenic growth factors are a class of molecules which exert a fundamental role in the process of blood vessel formation. Besides vasculogenic and angiogenic properties, these compounds mediate a complex series of patterning activities during organogenesis. Angiogenic factors cooperate in the growth and development of embryo tissues in a cross-talk between endothelial cells and tissue cells. It is well established that many tissue-derived factors are involved in blood vessel formation, but there is now emerging evidence that angiogenic factors and endothelial cells themselves represent a crucial source of instructive signals to non-vascular tissue cells during organ development. Thus, angiogenic factors and endothelial cell signalling are currently believed to provide fundamental cues for cell fate specification, embryo patterning, organ differentiation and postnatal tissue remodelling. This review article will summarize some of the recent advances in our understanding of the role of angiogenic factors and endothelial cells as effectors in organ formation.     

Angiogenic activity of osteopontin-derived peptide SVVYGLR

Angiogenesis plays an important role in various pathological conditions as well as some physiological processes. Although a number of soluble angiogenic factors have been reported, extracellular matrix also has crucial effect on angiogenesis through interaction with endothelial cells. Since recent reports showed osteopontin had some angiogenic activity, the effect of the SVVYGLR peptide, novel binding motif in osteopontin molecule, on angiogenesis was examined in this study. Synthetic peptide SVVYGLR did not have proliferative effect on endothelial cells but adhesion and migration activity to endothelial cells. Furthermore, SVVYGLR had as potent activity for tube formation in three-dimensional collagen gel as vascular endothelial growth factor which is known to be the strongest angiogenic factor. Electron microscopical analysis showed a number of microvilli on the endothelial luminar surface and tight junction formation in the luminar intercellular border between endothelial cells, indicating SVVYGLR induced cell porarity and differentiation of endothelial cells. This small peptide might be expected to stimulate angiogenesis to improve some ischemic conditions in the future because of some advantages due to smaller molecular weight.