Antibody-conjugated Liposomes Research Articles

Site-Selective Lysine Acetylation of Human Immunoglobulin G for Immunoliposomes and Bispecific Antibody Complexes.

Site-selective acetylation of a single lysine residue in a protein that reaches a lysine acetyltransferase's accuracy, precision, and reliability is challenging. Here, we report a peptide-guided, proximity-driven group transfer reaction that acetylates a single lysine residue, Lys 248, of the fragment crystallizable region (Fc region) in the heavy chain of the human Immunoglobulin G (IgG). An Fc-interacting peptide bound with the Fc domain and positioned a phenolic ester close to Lys 248, which induced a nucleophilic reaction and resulted in the transfer of an acetyl group to Lys 248. The acetylation reaction proceeded to a decent yield under the physiological condition without the need for deglycosylation, unnatural amino acids, or catalysts. Along with acetylation, functional moieties such as azide, alkyne, fluorescent molecules, or biotin could also be site-selectively installed on Lys 248, allowing IgG's further derivatization. We then synthesized an antibody-lipid conjugate and constructed antibody-conjugated liposomes (immunoliposomes), targeting HER2-positive (HER2+) cancer cells. We also built a bispecific antibody complex (bsAbC) covalently linking an anti-HER2 antibody and an anti-CD3 antibody. The bsAbC showed in vitro effector-cell-mediated cytotoxicity at nanomolar concentrations. Compared with bispecific antibodies (bsAbs), bsAbCs are constructed based on native IgGs and contain two antigen-binding sites to each antigen, twice that of bsAbs. Altogether, this work reports a method of site-selective acetylation of native antibodies, highlights a facile way of site-selective IgG functionalization, and underscores the potential of bsAbCs in cancer immunotherapy.

Antibody-conjugated liposomes loaded with indocyanine green for oral targeted photoacoustic imaging-guided sonodynamic therapy of Helicobacter pylori infection

Helicobacter pylori is a causative factor of various gastrointestinal tract diseases. As clinical antibiotic-based therapy for H. pylori infection might induce bacterial drug resistance, the in vivo eradication of H. pylori remains a huge challenge. In the present study, monoclonal antibody-conjugated liposomes loaded with indocyanine green (ICG) (HpAb-LiP-ICG) were successfully developed for targeted photoacoustic (PA) imaging-guided sonodynamic therapy (SDT) of H. pylori infection in vivo. HpAb-LiP-ICG showed high stability and favorable biocompatibility in acidic environment (pH 1.5) and was used for treating H. pylori-infected mice through oral administration. PA imaging showed that HpAb-LiP-ICG could precisely recognize and target H. pylori in the stomach. Following the targeting of HpAb-LiP-ICG to H. pylori, ICG was activated to generate singlet oxygen (1O2) for eliminating H. pylori under ultrasound (US) irradiation. Pathological analysis revealed that the HpAb-LiP-ICG-mediated SDT eradicated H. pylori without unintended toxicity to normal tissues. In conclusion, the HpAb-LiP-ICG-mediated SDT might shed new light on treating H. pylori infection, indicating the clinical translational prospects of this therapy in near future. Statement of significanceTraditional antibiotic-based therapy for Helicobacter pylori infections suffers from the risk of drug resistance. To meet this challenge, a monoclonal antibody-conjugated nanoliposome loaded with indocyanine green (ICG) (HpAb-LiP-ICG) was successfully developed, and efficient eradication of H. pylori was achieved in vivo by visual sonodynamic therapy (SDT).HpAb-LiP-ICG exhibited biocompatibility, targeting, and stability in the acidic microenvironment. Under ultrasound (US) irradiation in vitro, the HpAb-LiP-ICG nanoliposomes accumulated on the surface of H. pylori were activated to produce adequate singlet oxygen (1O2) to eliminate H. pylori.Gastric mucous tissues infected with H. pylori recovered to the normal state after HpAb-LiP-ICG-mediated SDT without side effects, thus highlighting the clinical translational prospects of the prepared HpAb-LiP-ICG nanoliposome in near future.

Group V Phospholipase A2 Mediates Endothelial Dysfunction and Acute Lung Injury Caused by Methicillin-Resistant Staphylococcus Aureus.

Lung endothelial dysfunction is a key feature of acute lung injury (ALI) and clinical acute respiratory distress syndrome (ARDS). Previous studies have identified the lipid-generating enzyme, group V phospholipase A2 (gVPLA2), as a mediator of lung endothelial barrier disruption and inflammation. The current study aimed to determine the role of gVPLA2 in mediating lung endothelial responses to methicillin-resistant Staphylococcus aureus (MRSA, USA300 strain), a major cause of ALI/ARDS. In vitro studies assessed the effects of gVPLA2 inhibition on lung endothelial cell (EC) permeability after exposure to heat-killed (HK) MRSA. In vivo studies assessed the effects of intratracheal live or HK-MRSA on multiple indices of ALI in wild-type (WT) and gVPLA2-deficient (KO) mice. In vitro, HK-MRSA increased gVPLA2 expression and permeability in human lung EC. Inhibition of gVPLA2 with either the PLA2 inhibitor, LY311727, or with a specific monoclonal antibody, attenuated the barrier disruption caused by HK-MRSA. LY311727 also reduced HK-MRSA-induced permeability in mouse lung EC isolated from WT but not gVPLA2-KO mice. In vivo, live MRSA caused significantly less ALI in gVPLA2 KO mice compared to WT, findings confirmed by intravital microscopy assessment in HK-MRSA-treated mice. After targeted delivery of gVPLA2 plasmid to lung endothelium using ACE antibody-conjugated liposomes, MRSA-induced ALI was significantly increased in gVPLA2-KO mice, indicating that lung endothelial expression of gVPLA2 is critical in vivo. In summary, these results demonstrate an important role for gVPLA2 in mediating MRSA-induced lung EC permeability and ALI. Thus, gVPLA2 may represent a novel therapeutic target in ALI/ARDS caused by bacterial infection.

Oral Antibody-Conjugated Liposomes Loaded with Indocyanine Green for Targeted Photoacoustic Imaging Directed Sonodynamic Therapy of <i>H.Pylori</i>

Clinical antibiotic-based therapy for H. pylori infections can induce bacterial drug resistance. How to eradicate H. Pylori in vivo exists great challenges. Herein we report that monoclonal antibody conjugated liposomes filled with ICG(HpAb-LiP-ICG) was successfully developed and used for targeted photoacoustic imaging directed sonodynamic therapy (SDT) of H. pylori in vivo. After prepared HpAb-LiP-ICG was orally administered to H. pylori infected mice, photoacoustic imaging clearly showed prepared ICG liposomes could recognize and target actively H. Pylori in gastric organ in mice models, then under ultrasonic radiation on the stomach of mice in vitro, ICG in HpAb-LiP-ICG combined on the surface of H. pylori was activated to produce enough singlet oxygen ( 1O2 )to kill H. Pylori. Pathological analysis showed that H. pylori were killed under antibacterial effect of SDT, gastric mucous tissues recovered to normal status after a month of SDT. In conclusion, prepared HpAb-LiP-ICG nanoprobes-based SDT provides a novel strategy to treat H. pylori infection in vivo, own clinical translational prospects in near future. Funding Information: This work was supported from National Key Research and Development Program of China (2017FYA0205304), National Natural Science Foundation of China (No. 81225010, 81028009, 81921002, 8202010801, 31170961), Shanghai Jiao Tong University Medicine-Engineering Cross budget (No. YG2016ZD10), Shanghai Engineering Research Center for Intelligent diagnosis and treatment instrument (No.15DZ2252000). Declaration of Interests: The authors declare that they have no competing interest. Ethics Approval Statement: All animal experiments were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University.

Effectiveness of a Controlled 5-FU Delivery Based on FZD10 Antibody-Conjugated Liposomes in Colorectal Cancer In vitro Models.

The use of controlled delivery therapy in colorectal cancer (CRC) reduces toxicity and side effects. Recently, we have suggested that the Frizzled 10 (FZD10) protein, a cell surface receptor belonging to the FZD protein family that is overexpressed in CRC cells, is a novel candidate for targeting and treatment of CRC. Here, the anticancer effect of novel immuno-liposomes loaded with 5-Fluorouracil (5-FU), decorated with an antibody against FZD10 (anti-FZD10/5-FU/LPs), was evaluated in vitro on two different CRC cell lines, namely metastatic CoLo-205 and nonmetastatic CaCo-2 cells, that were found to overexpress FZD10. The anti-FZD10/5-FU/LPs obtained were extensively characterized and their preclinical therapeutic efficacy was evaluated with the MTS cell proliferation assay based on reduction of tetrazolium compound, scratch test, Field Emission Scanning Electron Microscopes (FE-SEM) investigation and immunofluorescence analysis. The results highlighted that the cytotoxic activity of 5-FU was enhanced when encapsulated in the anti-FZD10 /5-FU/LPs at the lowest tested concentrations, as compared to the free 5-FU counterparts. The immuno-liposomes proposed herein possess a great potential for selective treatment of CRC because, in future clinical applications, they can be encapsulated in gastro-resistant capsules or suppositories for oral or rectal delivery, thereby successfully reaching the intestinal tract in a minimally invasive manner.

Imaging of amyloid-β in Alzheimer's disease transgenic mouse brains with ToF-SIMS using immunoliposomes.

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been proven to successfully image different kinds of molecules, especially a variety of lipids, in biological samples. Proteins, however, are difficult to detect as specific entities with this method due to extensive fragmentation. To circumvent this issue, the authors present in this work a method developed for detection of proteins using antibody-conjugated liposomes, so called immunoliposomes, which are able to bind to the specific protein of interest. In combination with the capability of ToF-SIMS to detect native lipids in tissue samples, this method opens up the opportunity to analyze many different biomolecules, both lipids and proteins, at the same time, with high spatial resolution. The method has been applied to detect and image the distribution of amyloid-β (Aβ), a biologically relevant peptide in Alzheimer's disease (AD), in transgenic mouse brain tissue. To ensure specific binding, the immunoliposome binding was verified on a model surface using quartz crystal microbalance with dissipation monitoring. The immunoliposome binding was also investigated on tissue sections with fluorescence microscopy, and compared with conventional immunohistochemistry using primary and secondary antibodies, demonstrating specific binding to Aβ. Using ToF-SIMS imaging, several endogenous lipids, such as cholesterol and sulfatides, were also detected in parallel with the immunoliposome-labeled Aβ deposits, which is an advantage compared to fluorescence microscopy. This method can thus potentially provide further information about lipid-protein interactions, which is important to understand the mechanisms of neurodegeneration in AD.

In Vivo Biomolecule Corona around Blood-Circulating, Clinically Used and Antibody-Targeted Lipid Bilayer Nanoscale Vesicles.

The adsorption of proteins and their layering onto nanoparticle surfaces has been called the "protein corona". This dynamic process of protein adsorption has been extensively studied following in vitro incubation of many different nanoparticles with plasma proteins. However, the formation of protein corona under dynamic, in vivo conditions remains largely unexplored. Extrapolation of in vitro formed protein coronas to predict the fate and possible toxicological burden from nanoparticles in vivo is of great interest. However, complete lack of such direct comparisons for clinically used nanoparticles makes the study of in vitro and in vivo formed protein coronas of great importance. Our aim was to study the in vivo protein corona formed onto intravenously injected, clinically used liposomes, based on the composition of the PEGylated liposomal formulation that constitutes the anticancer agent Doxil. The formation of in vivo protein corona was determined after the recovery of the liposomes from the blood circulation of CD-1 mice 10 min postinjection. In comparison, in vitro protein corona was formed by the incubation of liposomes in CD-1 mouse plasma. In vivo and in vitro formed protein coronas were compared in terms of morphology, composition and cellular internalization. The protein coronas on bare (non-PEGylated) and monoclonal antibody (IgG) targeted liposomes of the same lipid composition were also comparatively investigated. A network of linear fibrillary structures constituted the in vitro formed protein corona, whereas the in vivo corona had a different morphology but did not appear to coat the liposome surface entirely. Even though the total amount of protein attached on circulating liposomes correlated with that observed from in vitro incubations, the variety of molecular species in the in vivo corona were considerably wider. Both in vitro and in vivo formed protein coronas were found to significantly reduce receptor binding and cellular internalization of antibody-conjugated liposomes; however, the in vivo corona formation did not lead to complete ablation of their targeting capability.

Continuous Production of Immunoliposomes using a Microvalve-controlled Microfluidic Device (μFD)

Department of Food Science and Technology, Yeungnam University, Gyeongsan-si, Gyeongsangbuk-do 712-749, KoreaReceived May 12, 2013, Accepted July 8, 2013Immunoliposomes (antibody-conjugated liposomes) are highly useful as both a drug carrier in drug deliveryand as a reporting probe in immunodiagnostics. However, antibody conjugation is lengthy and cumbersome,because this includes several steps such as derivatization of the antibody, conjugation of the derivatizedantibody to liposomes, and separation of the unbound antibodies from immunoliposomes. Recently, liposomepreparation steps have simplified by using microfluidic devices (µFDs) where liposomes are formed when astream of lipids in solvent is hydrodynamically focused between two oblique buffer streams in a microchannel.Herein, we report a simple method for the production of immunoliposomes (rabbit IgG-conjugated liposomes)using microvalve-controlled µFD. The presence of antibody on the liposome was verified by observing thebinding of immunoliposomes to rabbit IgG on the surface. The results suggest that immunoliposomes can beeasily prepared through sequential mixing of antibody, conjugation reagents, preformed liposomes usingmicrovalve-controlled µFD.Key Words : Liposomes, Immunoliposomes, Microfluidic device, SRBIntroductionLiposomes can be described as spherical vesicles consist-ing of one or more phospholipid bilayers surrounding anaqueous cavity. Their main application is drug delivery fortargeting particular types of cells, such as cancer cells.

Non-muscle myosin light chain kinase isoform is a viable molecular target in acute inflammatory lung injury.

Acute lung injury (ALI) and mechanical ventilator-induced lung injury (VILI), major causes of acute respiratory failure with elevated morbidity and mortality, are characterized by significant pulmonary inflammation and alveolar/vascular barrier dysfunction. Previous studies highlighted the role of the non-muscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory response, with variants in the MYLK gene that contribute to ALI susceptibility. To define nmMLCK involvement further in acute inflammatory syndromes, we used two murine models of inflammatory lung injury, induced by either an intratracheal administration of lipopolysaccharide (LPS model) or mechanical ventilation with increased tidal volumes (the VILI model). Intravenous delivery of the membrane-permeant MLC kinase peptide inhibitor, PIK, produced a dose-dependent attenuation of both LPS-induced lung inflammation and VILI (~50% reductions in alveolar/vascular permeability and leukocyte influx). Intravenous injections of nmMLCK silencing RNA, either directly or as cargo within angiotensin-converting enzyme (ACE) antibody-conjugated liposomes (to target the pulmonary vasculature selectively), decreased nmMLCK lung expression (∼70% reduction) and significantly attenuated LPS-induced and VILI-induced lung inflammation (∼40% reduction in bronchoalveolar lavage protein). Compared with wild-type mice, nmMLCK knockout mice were significantly protected from VILI, with significant reductions in VILI-induced gene expression in biological pathways such as nrf2-mediated oxidative stress, coagulation, p53-signaling, leukocyte extravasation, and IL-6-signaling. These studies validate nmMLCK as an attractive target for ameliorating the adverse effects of dysregulated lung inflammation.

Fibrin Targeting with Ultrasound Active Antibody-Conjugated Liposomes

Open AccessConference Report Fibrin Targeting with Ultrasound Active Antibody-Conjugated Liposomes by E. E. J. MARXER 1,*, J. BRÜßLER 1, A. BECKER 2, C. NIMSKY 2 and U. BAKOWSKY 1 1 Department of Pharmaceutical Technology and Biopharmaceutics, Philipps-Universität, Marburg, Germany 2 Department of neuro surgery, Universitätsklinikum Gießen und Marburg GmbH, Philipps-Universität, Marburg, Germany * Author to whom correspondence should be addressed. Sci. Pharm. 2010, 78(3), 675; https://doi.org/10.3797/scipharm.cespt.8.PNM05 Received: 12 July 2010 / Accepted: 12 July 2010 / Published: 12 July 2010 Download PDF Citation Export BibTeX EndNote RIS Cite This Paper

Enhanced Loading Efficiency and Retention of225Ac in Rigid Liposomes for Potential Targeted Therapy of Micrometastases

Targeted alpha-particle emitters are promising therapeutics for micrometastatic disease. Actinium-225 has a 10-day half-life and generates a total of four alpha-particles per parent decay rendering (225)Ac an attractive candidate for alpha-therapy. For cancer cells with low surface expression levels of molecular targets, targeting strategies of (225)Ac using radiolabeled carriers of low specific radioactivities (such as antibodies) may not deliver enough alpha-particle emitters at the targeted cancer cells to result in killing. We previously proposed and showed using passive (225)Ac entrapment that liposomes can stably retain encapsulated (225)Ac for long time periods, and that antibody-conjugated liposomes (immunoliposomes) with encapsulated (225)Ac can specifically target and become internalized by cancer cells. However, to enable therapeutic use of (225)Ac-containing liposomes, high activities of (225)Ac need to be stably encapsulated into liposomes. In this study, various conditions for active loading of (225)Ac in preformed liposomes (ionophore-type, encapsulated buffer solution, and loading time) were evaluated, and liposomes with up to 73 +/- 9% of the initial activity of (225)Ac (0.2-200 microCi) were developed. Retention of radioactive contents by liposomes was evaluated at 37 degrees C in phosphate buffer and in serum-supplemented media. The main fraction of released (225)Ac from liposomes occurs within the first two hours of incubation. Beyond this two hour point, the encapsulated radioactivity is released from liposomes slowly with an approximate half-life of the order of several days. In some cases, after 30 days, (225)Ac retention as high as 81 +/- 7% of the initially encapsulated radioactivity was achieved. The (225)Ac loading protocol was also applied to immunoliposome loading without significant loss of targeting efficacy. Liposomes with surface-conjugated antibodies that are loaded with (225)Ac overcome the limitations of low specific activity for molecular carriers and are expected to be therapeutically useful against tumor cells having a low antigen density.

A novel assay for monitoring internalization of nanocarrier coupled antibodies

BackgroundDiscovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA.ResultsThe specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv) (F5) antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol)-linked lipid (DSPE-PEG-NTA-Ni) was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay.ConclusionThe CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers.

Drug transport to brain with targeted liposomes

Antibody-conjugated liposomes or immunoliposomes are particulate drug carriers that can be used to direct encapsulated drug molecules to diseased tissues or organs. The present review discusses examples of successful applications of this technology to achieve drug transport across the blood-brain barrier. In addition, information will be provided on practical aspects such as phospholipid compositions of liposomes, antibody coupling technologies, large-scale production of liposomes, and obstacles related to drug loading of the carrier. Prospects of future uses of immunoliposome-based drug delivery systems such as gene therapy of the brain and clinical trials are discussed.

Drug Transport to Brain with Targeted Liposomes

Antibody-conjugated liposomes or immunoliposomes are particulate drug carriers that can be used to direct encapsulated drug molecules to diseased tissues or organs. The present review discusses examples of successful applications of this technology to achieve drug transport across the blood-brain barrier. In addition, information will be provided on practical aspects such as phospholipid compositions of liposomes, antibody coupling technologies, large-scale production of liposomes, and obstacles related to drug loading of the carrier. Prospects of future uses of immunoliposome-based drug delivery systems such as gene therapy of the brain and clinical trials are discussed.

In Vitro Targeting of Antibody-Conjugated Echogenic Liposomes for Site-Specific Ultrasonic Image Enhancement

Tissue-specific ultrasonic enhancement can be used for the detection and characterization of atherosclerosis. We have previously demonstrated the generation of inherently echogenic (acoustically reflective) liposomes solely by varying lipid composition and controlling the method of production. In this study, echogenic liposomes composed of phosphatidylcholine (PC), 4-(p-maleimidophenyl) butyryl phosphatidyl-ethanolamine (MPB-PE), phosphatidylglycerol (PG), and cholesterol were conjugated to human gamma globulin to determine the effect of antibody conjugation on liposomal acoustic reflectivity. The liposomes remained highly echogenic following antibody conjugation. Echogenic liposomes were also conjugated to rabbit antihuman fibrinogen to study their ability to target fibrin. Antibody-conjugated liposomes were targeted to fibrin-coated filter paper and slides, thrombi made in vitro, and segments of atheroma in an animal model of atherosclerosis. Liposomes were detected by scanning electron microscopy, radiolabeling, and imaging with intravascular ultrasound. Electron microscopy revealed attachment of antibody-conjugated liposomes to fibrin on slides and to the fibrous plaques of the arterial segments, whereas unconjugated liposomes did not attach. Similarly, conjugated liposomes did not attach to normal arteries, indicating their binding to the arterial segment is directed towards a component of the fibrous plaque. Ultrasound imaging of the thrombi demonstrated surface attachment of the acoustic conjugated liposomes. 125I-Labeled liposomes conjugated to rabbit anti-human were targeted to fibrin-coated paper. Counting specifically bound radioactivity showed that >84% of applied liposomes remained attached to the fibrin after washing with saline. These results demonstrate the potential of acoustically reflective liposomes for site-specific targeting and acoustic enhancement.

Targeting of stealth liposomes to erbB-2 (Her/2) receptor: in vitro and in vivo studies.

Long-circulating (stealth) liposomes coated with polyethylene glycol (PEG), which show reduced uptake by the reticuloendothelial system (RES) and enhanced accumulation in tumours, were used for conjugation to monoclonal antibodies (MAbs) as a drug-targeting device. A MAb (N-12A5) directed against erbB-2 oncoprotein, a functional surface antigen, was used. Amplification and overexpression of the erbB-2 gene product, being unique to malignancy, confer onto this antibody-mediated therapy high tumour specificity. In vitro binding of [3H]cholesteryl ether ([3H]Chol ether) labelled anti-erbB-2 conjugated liposomes to N-87 cells (erbB-2-positive human gastric carcinoma) was compared with the binding of non-targeted liposomes and indicated a 16-fold increase in binding for the targeted liposomes. No difference in binding to OV1063 cells (erbB-2-negative human ovary carcinoma) was observed. These results indicate highly selective binding of antibody-targeted liposomes to erbB-2-overexpressing cells. Despite increased cell binding, doxorubicin (DOX) loaded in anti-erbB-2-conjugated liposomes did not cause increased in vitro cytotoxicity against N-87 cells, suggesting lack of liposome internalisation. In vivo, the critical factor needed to decrease the non-specific RES uptake and prolong the circulation time of antibody-conjugated liposomes is a low protein to phospholipid ratio ( < 60 micrograms mumol-1). Using these optimised liposome preparations loaded with DOX and by monitoring the drug levels and the [3H]Chol ether label, biodistribution studies in nude mice bearing subcutaneous implants of N-87 tumours were carried out. No significant differences in liver and spleen uptake between antibody-conjugated and plain liposomes were observed. Nevertheless, there was no enhancement of tumour liposome levels over plain liposomes. Both liposome preparations considerably enhanced DOX concentration in the tumour compared with free drug administration. Therapeutic experiments with N-87 tumour-bearing nude mice indicated that anti-tumour activity of targeted and non-targeted liposomes was similar, although both preparations had an increased therapeutic efficacy compared with the free drug. These studies suggest that efficacy is dependent on drug delivery to the tumour and that the rate-limiting factor of liposome accumulation in tumours is the liposome extravasation process, irrespective of liposome affinity or targeting to tumour cells.

Improved in vitro growth inhibitory effect of N-(phosphonacetyl)- l-maspartic acid in immunoliposomes

The use of liposome-encapsulated N-(phosphonacetyl)- l-maspartic acid (PALA) for the possible treatment of human ovarian cancer has been investigated in vitro. Protein A or tumor-specific antibodies were conjugated to liposomes via the reaction of a maleimide derivatized phospholipid (MPB-PE) with a thiol introduced into the protein by a heterobifunctional cross-linking agent, N-succimidyl 3-(2-pyridyldithio) propionate (SPDP). Antibody-conjugated PALA-containing liposomes were separated from free antibodies by ultracentrifugation in discontinuous metrizamide gradients. PALA in Protein A-conjugated liposomes was found to be over 400-fold more effective (IC 50 = 0.04 μM) than free drug (IC 50 = 18 μM) for growth inhibition of L929 cells in vitro, when the cells were pretreated with 20–40 μg of 11-4.1 monoclonal antibody for 30 min. PALA in tumor-specific antibody-conjugated liposomes was 60-fold more effective (IC 50 = 0.2 μM) than free drug (IC 50 = 12 μM) for growth inhibition of HEY 1B human ovarian cancer cells. Anti-c- erbB2 antibody (454C11) and anti-transferrin receptor antibody (454A12) were particularly effective in this regard. For growth inhibition of SKOV-3 cells, a human ovarian cancer cell line that grows more slowly than HEY 1B, PALA in antibody-conjugated liposomes was also about 60-fold more effective (IC 50 = 0.9 μM) than free drug (IC 50 = 50 μM). Antibody against a high molecular weight glycoprotein (2G3) and anti-transferrin receptor antibody (454A12) were the most effective antibodies among those tested for their ability to inhibit growth of SKOV-3 cells. These results demonstrate that PALA is a good candidate for drug delivery to ovarian cancer cells by immunoliposomes, and that the c- erbB2 oncogene product, a high molecular weight glycoprotein, and the transferrin receptor are suitable ligands, through which to target the delivery of PALA.

Magnetofluorescent liposomes for increased sensitivity of immunofluorescence.

Immunofluorescence and immunomagnetism are important technologies for analysis and sorting of cells according to specific marker molecules. Due to the limited sensitivity at least several thousands of antigens per cell are required for optical detection. Molecules expressed at low copy numbers cannot be analysed, although they may be of considerable functional importance. Development of a magnetic and fluorescent staining reagent for analysis and sorting of cells according to antigens expressed at low number. To this end, uniformly sized, antibody-conjugated liposomes loaded with large amounts of dye molecules and small magnetic particles were generated. A method for the preparation of homogeneously sized large unilamellar liposomes which contain carboxyfluorescein, magnetic particles and surface-bound antibodies had to be developed. These liposomes were then tested for their ability to enhance immunofluorescence compared to conventional staining in a model system and by staining of CD25 on resting B and T cells. Large unilamellar liposomes, homogeneous in size and loaded with fluorescein and magnetic beads can be prepared by combining membrane extrusion and magnetic filtration. Hapten-specific antibodies conjugated to their surface make them a universal tool for immunofluorescence. With such liposomes, intensity of fluorescent staining can be increased 100-1000-fold without increased background fluorescence, compared to conventional fluorochrome-conjugated antibodies. Due to the simultaneous magnetic labelling, stained cells can easily be isolated by MACS. The magnetofluorescent liposomes proved to be useful for improvement of sensitivity of detection and physical separation in general and to visualize and sort cells according to antigens expressed at low levels. The high affinity IL2 receptor CD25 is expressed in low copy number on a significant fraction of resting B and T lymphocytes in human peripheral blood, as can be shown exclusively by the magnetofluorescent liposomes.

モノクローナル抗体結合リポソームを用いての癌ターゲティング療法の開発

Monoclonal antibodies against human α-fetoprotein (AFP) or carcinoembryonic antigen (CEA) were conjugated to liposomes containing adriamycin (ADM), and their therapeutic effects were experimentally studied in vivo on AFP or CEA positive human solid tumors maintained in BALB/c nu/nu mice. The anti-tumor effect of antibody-conjugated liposomes containing ADM was immunologically specific and was greater than that of unconjugated liposomes containing ADM or that of ADM alone. Furthermore, tissue distribution studies revealed the selective delivery of ADM to tumors occurred with immunoliposomes. However, it was also revealed the liposomes are preferentially accumulated to the reticuloendothelial cells. To improve the therapeutic effects, we further prepared the following three types of liposomes and their therapeutic activities were assessed. (1) Fab′ fragment was introduced instead of whole molecule of antibody. These liposomes are expected to keep the antigen-binding activity of antibody intact. Actually, the therapeutic effect of liposomes with Fab′ fragment was greater than that of liposomes with whole molecule. (2) GMI gangliosides were incorporated into liposomes and with these liposomes the accumulation of ADM to reticuloendothelial cells was reduced. (3) Temperature-sensitive liposomes were prepared with dipalmitoylphosphatidylcholine. Combination of temperature sensitivity and monoclonal antibodies for targeting therapy is now in progress.

Immunomagnetic Particle Induced Lysis of Antibody-Conjugated Liposomes

AbstractWe have prepared liposomes of dioleoylphosphatidylethanolamine (DOPE) which have been stabilized by addition of 9-12 mol% N-biotinyl- phosphatidylethanolamine. Liposomes composed of DOPE/N-biotinyl-PE are quite stable and non-leaky although they exhibit strong temperature-dependent leakage following incorporation of palmitylated murine monoclonal antibodies as a targeting ligand. Addition of magnetic chromium dioxide particles coated with anti-mouse antibody to these immunoliposomes lead to their aggregation and the release of entrapped calcein. The lytic event was biphasic with an initial rapid release of 20% dye within 5 min. followed by a slower rate which reached nearly 40% release after 80 min. The rapid release phase was dependent upon the concentration of the liposomes and that of the multivalent particles. Lysis was immunospecific since no release was observed upon addition of nonspecific immunomagnetic particles to the immunoliposomes or if no antibody was incorporated into the liposome. ...