• Bulletin of the Korean Chemical Society
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• v.34 no.10
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• pp.2921-2924
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• 2013

Immunoliposomes (antibody-conjugated liposomes) are highly useful as both a drug carrier in drug delivery and 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 derivatized antibody to liposomes, and separation of the unbound antibodies from immunoliposomes. Recently, liposome preparation steps have simplified by using microfluidic devices (${\mu}FDs$) where liposomes are formed when a stream 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 ${\mu}FD$. The presence of antibody on the liposome was verified by observing the binding of immunoliposomes to rabbit IgG on the surface. The results suggest that immunoliposomes can be easily prepared through sequential mixing of antibody, conjugation reagents, preformed liposomes using microvalve-controlled ${\mu}FD$.

• Food Science of Animal Resources
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• v.29 no.4
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• pp.533-538
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• 2009

To suggest an improved diagnostic method for Salmonella spp., analyses were conducted with immunoliposomes and compared with the results from a commercial test kit. One sample out of 36 samples of eggshell was Salmonella-positive via immunoliposomes. In the case of the use of the commercial test kit, six samples out of 36 samples were Salmonellapositive. These Salmonella-positive samples were subjected to biochemical identification tests that confirmed that they were Salmonella-negative. As for the egg content samples, they were Salmonella-negative in both analyses with immunoliposomes and the commercial test kit. The Salmonella analysis with immunoliposomes reduced detection time, by 24 h compared to the commercial test kit. Bacteria, including Acinetobacter baumanni, Chryseomonas luteola, Enterobacter cloacae, Escherichia coli, Escherichia hermannii, Klebsiella pneumonia, Pantoea spp., and Pasteurella pneumotropica, were isolated from the eggshells. Other than Acinetobacter baumanni and Pasteurella pneumotropica most of the isolates were known to frequently appear during egg production processing.

• Biomedical Science Letters
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• v.28 no.4
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• pp.247-259
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• 2022

Immunotherapy, which uses an immune mechanism in the body, has received considerable attention for cancer treatment. Suberoylanilide hydroxamic acid (SAHA), also known as a histone deacetylase inhibitor (HDACi), is used as a cancer treatment to induce active immunity by increasing the expression of T cell-induced chemokines. However, this SAHA treatment has the disadvantage of causing PD-L1 overexpression in tumor cells. In this study, we prevented PD-L1 overexpression by blocking the PD-1/PD-L1 pathway using PD-L1 siRNA. We designed two types of liposomes, the neutral lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholin (POPC) for SAHA, and 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) for siRNA. To effectively target PD-L1 in cancer cells, we conjugated PD-L1 antibody with liposomes containing SAHA or PD-L1 siRNA. These immunoliposomes were also evaluated for cytotoxicity, gene silencing, and T-cell-induced chemokine expression in human non-small cell lung cancer A549 cells. It was confirmed that the combination of the two immunoliposomes increased the cancer cell suppression efficacy through Jurkat T cell induction more than twice compared to SAHA alone treatment. In conclusion, this combination of immunoliposomes containing a drug and nucleic acid has promising therapeutic potential for non-small-cell lung carcinoma (NSCLC).

• BMB Reports
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• v.31 no.1
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• pp.95-100
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• 1998

Two innovative methods to prepare target-sensitive immunoliposomes containing doxorubicin by coupling monoclonal antibodies (mAb DH2, SH1) specific to cancer cell surface antigens ($G_{M3}$, $Le^X$) have been developed and are described here. Firstly, liposomes containing N-glutaryl phosphatidylethanolamine (NGPE) were prepared, followed by the encapsulation of doxorubicin, DH2 or SH1 antibodies were conjugated to NGPE in the liposomes (direct coupling). Secondly, liposomes were prepared with NGPE/mAb conjugates by the detergent dialysis method (conjugate insertion), and then doxorubicin was encapsulated by proton gradient. The immunoliposomes prepared by both methods were able to specifically bind to the surface of the tumor cells - B16BL6 mouse melanoma cells. The efficiencies of doxorubicin-entrapping into liposomes prepared by direct coupling and conjugate insertion was about 98% and 25%, respectively. These types of liposomal formulation are sensitive to target cells, which can be useful for various clinical applications.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.509-516
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• 2003

A model system for the immnunochemical detection of Escherichia coli O157:H7 using a combined immunomagnetic separation (IMS) and test-strip liposome immunoassay (LIA) procedure was developed. Immunomagnetic beads coated with anti-E. coli O157 IgG antibodies were used to separate the E. coli O157 (including the H7 serotype) from culture. Immunoliposomes, whose surface was conjugated to goat anti-E. coli O157:H7 IgG and which encapsulated the marker dye, sulforhodamine B, were used as a detection label. The test strip, onto which antibodies to goat IgG were immobilized, was the immunosensor capturing immunoliposomes that did not bind to E. coli O157:H7 on the immunomagnetic bead-E. coli O157:H7 complexes. In experiments, pure cell culture suspensions of $10^5 E.$ coli O157:H7 organisms per ml produced a measurable signal inhibition, whereas a weak yet detectable signal inhibition occurred with $10^3CFU/ml$. The inhibition signals increased, when the incubation time for IMS was extended to 90 min and higher IgG-tag density (0.4mol%) was used on the liposomes. With 0.2 and 0.4mol% IgG-tagged liposomes, the IMS-LIA procedure showed more improved signal inhibitions than those of a direct (no IMS) LIA. The combined assay, which measures the instantaneous signal from immunoliposomes, can be completed within 90 min, making it significantly faster than conventional plating methods and enzyme-linked immunosorbent assay (ELISA). Accordingly, it is quite feasible to use the combined immunoassay format of IMS and dye-loaded immunoliposomes for the detection of E. coli O157:H7.

• Archives of Pharmacal Research
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• v.27 no.12
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• pp.1263-1269
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• 2004

The objective of this study was to characterize immunoliposomes carrying plasmid DNA with optimal encapsulation efficiency and antibody density. Plasmid DNA was encapsulated by the freezing/thawing method into liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycerol- 3-phosphocholine), DDAB (didodecyl dimethyl ammonium bromide), DSPE-PEG 2000 (distearoyl phosphatidyl ethanolamine polyethylene glycol 2000) and DSPE-PEG 2000-maleimide. The liposomes carrying plasmid DNA were extruded through two stacked polycarbonate filters, of different pore size, to control the liposome size. Then, rat IgG molecules were conjugated to the liposomes. The immunoliposomes containing plasmid DNA were separated from the free plasmid DNA and unconjugated IgG by Sepharose CL-4B column chromatography. The DNA amount encapsulated was affected by DDAB (cationic lipid) concentration, the initial amount of plasmid DNA between 10 ${\mu}g$ and 200 ${\mu}g$, the total lipid amount and plasmid DNA size, but not significantly by liposome size. By varying the ratio of DSPE-PEG 2000-maleimide to IgG, the number of IgG molecules per liposome was changed significantly.

• Journal of Pharmaceutical Investigation
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• v.20 no.3
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• pp.135-144
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• 1990

The effects of bile salts (BS) on the stability of dioleoylphosphatidylethanolamine (DOPE) liposomes were investigated, observing apparent absorbance of vacant liposomes and calcein release from entrapped liposomes. Unilamellar liposomes were prepared by using a small quantity of palmitoly-immunoglobulin G(IgG) ($2.5{\times}10^{-4}$ mo1/lipid mol) to stabilize the bilayer phase of the unsaturated DOPE which by itself does not form stable liposomes. The destabilization of PE immunoliposomes by papain, clearly demonstrates that the IgG is essential for stabilization of PE bilayer. Approximately 4% of the entrapped calcein was released from the PE liposomes after 1 hr from liposome formation. Calcein release and absorbance of liposomes depended on the BS/lipid ratio because of the solubilization of lipid molecule in bilayer and the formation of mixed micelles. At very low BS concentrations, the incorporation of BS induced BS/lipid aggregates in the outer vesicles monolayer, while high BS concentrations, mixed micelles were formed. Chelate and its conjugates as $3{\alpha},\;7{\alpha},\;12{\alpha}-trihydroxy$ BS induce the concentration of the $3{\alpha}$, $12{\alpha}-dihydroxy$ BS at half-maximal solubilization of immunoliposomes to approximately 2.5-, or 5-fold. Conjugation of BS with glycine or taurine slightly enhanced their capacities to perturb membranes.

• Bulletin of the Korean Chemical Society
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• v.33 no.2
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• pp.651-656
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• 2012

Cationic immunoliposomes were prepared by conjugation of Fab' fragments of the recombinant humanized monoclonal antibody (HuCC49) against tumor-associated glycoprotein (TAG)-72 to sterically unilamella liposomes. The cationic immunoliposomes are composed of cationic lipid (O,O'-dimyristyl-N-lysyl aspartate, DMKD), cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)$_{2000}$] (DPPE-PEG-maleimide) with a molar ratio of 0.5:0.47:0.03. Plasmid DNA was effectively condensed by addition of transferrin (Tf) during the formation of anti-TAG-72 PEG-immunolipoplexes (PILPs). These anti-TAG-72 PILPs were able to adhere to the surface of TAG-72-overexepressing LS174T human colon cancer cells more effectively than conventional liposomes, thereby facilitating gene delivery in vitro. Furthermore, intravenous administration of the anti-TAG-72 PILPs into the tumor-carrying mice exhibited efficient localization of the reporter gene in the tumor tissues.

• Journal of Pharmaceutical Investigation
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• v.21 no.3
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• pp.161-170
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• 1991

The storage stabilities of immunoliposomes incorporated with variable amounts of ganglioside $G_{M1}$ were investigated as a function of time. temperature. and composition by observing absorbance of visible light and calcein release. In the column chromatographe, the layer of unsaturated PE(dioleoylphosphatidylethanolamine : DOPE). unable to form stable liposomes at physiological temperature and pH, were formed when palmitoyl-immunoglobulin G(IgG) $(2.5{\times}10^{-4}\;mol/DOPE\;mol)$ added. The incorporation of ganglioside $G_{M1}$ into immunoliposome. enhanced the stabilities of bilayers during the extended period of storage. The turbidities of immunoliposomes coated with ganglioside $G_{M1}$ exhibited the maximum near 20 mol% $G_{M1}/DOPE$ mol. probably because of the disturbance of the bilayer characteristics, i.e., layer transition or reorientation of interaction sites. At low temperature. the higher stability was achieved than at elevated temperatures. After one week of storage. the redispersed liposomal solutions at lower temperatures maintained the original elution patterns in chromatography but broader distribution at elevated temperatures. During the storage, it is suggested the aggregation is the more dominant phenomena for liposomes kept at $5^{\circ}C$ than the fusion. while he fusion is at elevated temperatures.

• Food Science of Animal Resources
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• v.23 no.3
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• pp.278-283
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• 2003

Feasibility tests on using liposomes for detecting food-borne pathogenic bacteria were studied with E. coli 0157:H7 as a model analyte. lmmunoliposomes, whose surface was conjugated with anti-E. coli 0157:H7 IgG and which encapsulated the marker dye, sulforhodamine B, were used for the detection label. Among the feasibility tests, the first test was to use a test-strip on which antibodies to anti-E. coli O157:H7 IgG were immobilized. In this format, immunoliposomes that did not bind to E. coli O157:H7 in sample were captured and then exhibited a visible signal which was inversely related with the number of E. coli O157:H7 in sample. The second test was a direct liposome assay followed by immunomagnetic separation. In this format, immunoliposomes which were bound to E. coli O157:H7 were lysed with detergent and produced a signal which was proportionally related with the number of E. coli O157:H7 in sample. The results from both formats indicate that liposomes can be utilized as a detection label.