Liposome Conjugates Research Articles

Synthesis of SulfoCy5‐ and SulfoCy7‐αGalCer Probes as Chemical Tools for Investigating the Uptake of Liposomal αGalCer Conjugates by Antigen Presenting Cells

Abstractα‐Galactosylceramide (αGalCer) is a synthetic glycolipid that, when loaded into the CD1d receptor of antigen presenting cells, activates iNKT cells to coordinate the generation of both innate and adaptive immune responses. In vaccines, the αGalCer adjuvant is generally delivered in liposomes, but interestingly, little is known about the intracellular fate of αGalCer‐antigen conjugates resulting in the absence of clear “design rules” for this type of compounds and their formulations. To unravel the uptake mechanism of liposomal αGalCer conjugates and understand the fate of its components, we synthesized sulfoCy7‐αGalCer 1 and sulfoCy5‐αGalCer 2. The fluorescent probes were obtained from a linker‐functionalized αGalCer, which was then modified with sulfoCy7/sulfoCy5. After formulation in liposomes, the cellular presentation was studied in a murine dendritic cell line using fluorescence imaging and flow cytometry. Imaging indicates that liposomes loaded with sulfoCy5‐αGalCer are taken up by DC2.4 cells in a heterogeneous and concentration‐dependent manner. Additionally, fluorescence antibody staining confirms that αGalCer is ultimately presented by CD1d receptors, but importantly is cleaved from sulfoCy5 before reaching the cell surface. These results validate our synthetic probes as useful in mechanistic studies of the uptake of αGalCer‐antigen conjugates as shown here for DC2.4 cells.

Phage-liposome nanoconjugates for orthopedic biofilm eradication

Infection by multidrug-resistant (MDR) bacteria has become one of the biggest threats to public health worldwide. One reason for the difficulty in treatment is the lack of proper delivery strategies into MDR bacterial biofilms, where the thick extracellular polymeric substance (EPS) layer impedes the penetration of antibiotics and nanoparticles. Here, we propose a novel bioactive nanoconjugate of drug-loaded liposomes and bacteriophages for targeted eradication of the MDR biofilms in orthopedic infections. Phage Sb-1, which has the ability to degrade EPS, was conjugated with antibiotic-loaded liposomes. Upon encountering the biofilm, phage Sb-1 degrades the EPS structure, thereby increasing the sensitivity of bacteria to antibiotics and allowing the antibiotics to penetrate deeply into the biofilm. As a result, effective removal of MDR bacterial biofilm was achieved with low dose of antibiotics, which was proved in this study by both in vitro and in vivo investigations. Notably, in the rat prosthetic joint infection (PJI) model, we found that the liposome-phage nanoconjugates could effectively decrease the bacterial load in the infected area and significantly promote osteomyelitis recovery. It is therefore believed that the conjugation of bacteriophage and liposomes could open new possibilities for the treatment of orthopedic infections, possibly other infections in the deep tissues.

Liposome nanoparticle conjugation and cell penetrating peptide sequences (CPPs) enhance the cellular delivery of the tau aggregation inhibitor RI-AG03.

Given the pathological role of Tau aggregation in Alzheimer's disease (AD), our laboratory previously developed the novel Tau aggregation inhibitor peptide, RI-AG03. As Tau aggregates accumulate intracellularly, it is essential that the peptide can traverse the cell membrane. Here we examine the cellular uptake and intracellular trafficking of RI-AG03, in both a free and liposome-conjugated form. We also characterize the impact of adding the cell-penetrating peptide (CPP) sequences, polyarginine (polyR) or transactivator of transcription (TAT), to RI-AG03. Our data show that liposome conjugation of CPP containing RI-AG03 peptides, with either the polyR or TAT sequence, increased cellular liposome association three-fold. Inhibition of macropinocytosis modestly reduced the uptake of unconjugated and RI-AG03-polyR-linked liposomes, while having no effect on RI-AG03-TAT-conjugated liposome uptake. Further supporting macropinocytosis-mediated internalization, a 'fair' co-localisation of the free and liposome-conjugated RI-AG03-polyR peptide with macropinosomes and lysosomes was observed. Interestingly, we also demonstrate that RI-AG03-polyR detaches from liposomes following cellular uptake, thereby largely evading organellar entrapment. Collectively, our data indicate that direct membrane penetration and macropinocytosis are key routes for the internalization of liposomes conjugated with CPP containing RI-AG03. Our study also demonstrates that peptide-liposomes are suitable nanocarriers for the cellular delivery of RI-AG03, furthering their potential use in targeting Tau pathology in AD.

Turmeric and curcumin as adjuncts in controlling Helicobacter pylori-associated diseases: a narrative review.

Non-antibiotic adjuncts may improve Helicobacter pylori infection control. Our aim was to emphasize curcumin benefits in controlling H. pylori infection. We discussed publications in English mostly published since 2020 using keyword search. Curcumin is the main bioactive substance in turmeric. Curcumin inhibited H. pylori growth, urease activity, three cag genes, and biofilms through dose- and strain-dependent activities. Curcumin also displayed numerous anticancer activities such as apoptosis induction, anti-inflammatory and anti-angiogenic effects, caspase-3 upregulation, Bax protein enhancement, p53 gene activation, and chemosensitization. Supplementing triple regimens, the agent increased H. pylori eradication success in three Iranian studies. Bioavailability was improved by liposomal preparations, lipid conjugates, electrospray-encapsulation, and nano-complexation with proteins. The agent was safe at doses of 0.5->4g daily, the most common (in 16% of the users) adverse effect being gastrointestinal upset. Notably, curcumin favorably influences the intestinal microbiota and inhibits Clostridioides difficile. Previous reports showed the inhibitory effect of curcumin on H pylori growth. Curcumin may become an additive in the therapy of H. pylori infection, an adjunct for gastric cancer control, and an agent beneficial to the intestinal microbiota. Further examination is necessary to determine its optimal dosage, synergy with antibiotics, supplementation to various eradication regimens, and prophylactic potential.

An antibody fragment-decorated liposomal conjugate targets Philadelphia-like acute lymphoblastic leukemia

Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is an aggressive B-ALL malignancy associated with high rates of relapse and inferior survival rate. While targeted treatments against the cell surface proteins CD22 or CD19 have been transformative in the treatment of refractory B-ALL, patients may relapse due to antigen loss, necessitating targeting alternative antigens. Cytokine receptor-like factor 2 (CRLF2) is overexpressed in half of Ph-like ALL cases conferring chemoresistance and enhancement of leukemia cell survival. Therefore, targeting CRLF2 may reduce the likelihood of relapse associated with antigen loss. We developed a CRLF2-targeting single-chain variable fragment modified by the fragment crystallizable region (CRLF2 scFv-Fc) conjugated to a drug maytansinoid 1 (DM1)-DOPC liposomal conjugate, creating homogeneous CRLF2-targeted liposomes (CRLF2-DM1 LIP). Cellular association and internalization studies in a Ph-like ALL cell line, MHH-CALL-4, compared to its lentivirally transduced CRLF2-knockdown counterpart (KD-CALL-4) revealed excellent CRLF2-targeting efficiency of CRLF2-DM1 LIP. Moreover, CRLF2-DM1 LIP showed selective association and internalization ex vivo using Ph-like ALL patient-derived xenograft (PDX) cells with minimal reactivity with non-target cells. Cell apoptosis assays demonstrated the CRLF2-dependent potency of CRLF2-DM1 LIP in Ph-like ALL cell lines. This study is the first to highlight the therapeutic potential of a CRLF2-directed scFv-Fc-liposomal conjugate for targeting Ph-like ALL.

QCM-D Investigations on Cholesterol-DNA Tethering of Liposomes to Microbubbles for Therapy.

Lipid-shelled microbubbles (MBs) offer potential as theranostic agents, capable of providing both contrast enhancement in ultrasound imaging as well as a route for triggered drug release and improved localized drug delivery. A common motif in the design of such therapeutic vehicles is the attachment of the drug carrier, often in the form of liposomes, to the microbubble. Traditionally, such attachments have been based around biotin-streptavidin and maleimide-PDP chemistries. Comparatively, the use of DNA-lipid tethers offers potential advantage. First, their specificity permits the construction of more complex architectures that might include bespoke combinations of different drug-loaded liposomes and/or targeting groups, such as affimers or antibodies. Second, the use of dual-lipid tether strategies should increase the strength of the individual tethers tethering the liposomes to the bubbles. The ability of cholesterol-DNA (cDNA) tethers for conjugation of liposomes to supported lipid bilayers has previously been demonstrated. For in vivo applications, bubbles and liposomes often contain a proportion of polyethylene glycol (PEG) to promote stealth-like properties and increase lifetimes. However, the associated steric effects may hinder tethering of the drug payload. We show that while the presence of PEG reduced the tethering affinity, cDNA can still be used for the attachment of liposomes to a supported lipid bilayer (SLB) as measured via QCM-D. Importantly, we show, for the first time, that QCM-D can be used to study the tethering of microbubbles to SLBs using cDNA, signified by a decrease in the magnitude of the frequency shift compared to liposomes alone due to the reduced density of the MBs. We then replicate this tethering interaction in the bulk and observe attachment of liposomes to the shell of a central MB and hence formation of a model therapeutic microbubble.

DOPE/CHEMS-Based EGFR-Targeted Immunoliposomes for Docetaxel Delivery: Formulation Development, Physicochemical Characterization and Biological Evaluation on Prostate Cancer Cells

Docetaxel (DTX) is a non-selective antineoplastic agent with low solubility and a series of side effects. The technology of pH-sensitive and anti-epidermal growth factor receptor (anti-EGFR) immunoliposomes aims to increase the selective delivery of the drug in the acidic tumor environment to cells with EFGR overexpression. Thus, the study aimed to develop pH-sensitive liposomes based on DOPE (dioleoylphosphatidylethanolamine) and CHEMS (cholesteryl hemisuccinate), using a Box-Behnken factorial design. Furthermore, we aimed to conjugate the monoclonal antibody cetuximab onto liposomal surface, as well as to thoroughly characterize the nanosystems and evaluate them on prostate cancer cells. The liposomes prepared by hydration of the lipid film and optimized by the Box-Behnken factorial design showed a particle size of 107.2 ± 2.9 nm, a PDI of 0.213 ± 0.005, zeta potential of -21.9 ± 1.8 mV and an encapsulation efficiency of 88.65 ± 20.3%. Together, FTIR, DSC and DRX characterization demonstrated that the drug was properly encapsulated, with reduced drug crystallinity. Drug release was higher in acidic pH. The liposome conjugation with the anti-EGFR antibody cetuximab preserved the physicochemical characteristics and was successful. The liposome containing DTX reached an IC50 at a concentration of 65.74 nM in the PC3 cell line and 28.28 nM in the DU145 cell line. Immunoliposome, in turn, for PC3 cells reached an IC50 of 152.1 nM, and for the DU145 cell line, 12.60 nM, a considerable enhancement of cytotoxicity for the EGFR-positive cell line. Finally, the immunoliposome internalization was faster and greater than that of liposome in the DU145 cell line, with a higher EGFR overexpression. Thus, based on these results, it was possible to obtain a formulation with adequate characteristics of nanometric size, a high encapsulation of DTX and liposomes and particularly immunoliposomes containing DTX, which caused, as expected, a reduction in the viability of prostate cells, with high cellular internalization in EGFR overexpressing cells.

Liposome functionalized reduced graphene oxide for rapid electrochemical sensing of bacteria.

Pathogenic bacteria represent a severe threat to global public health, particularly with the growing rate of antibiotic resistance, and, therefore, indicate a critical need for developing efficient sensing platforms. Liposome-based sensors are collocating interest due to their intrinsic fusogenic ability to fuse with the outer membrane of bacteria. However, the lack of a conducting property limits their applicability for developing biosensing platforms. In this study, we report conjugation of liposomes with reduced graphene oxide (rGO) for fabricating a rapid and sensitive biosensor for electrochemical detection of Escherichia coli (E. coli). The large surface area of rGO facilitated binding of liposomes with their surface, and the intrinsic electrical and biocompatible properties assisted electrochemical sensing of bacteria. The electrochemical response of the liposome and the rGO-liposome coated electrode shows nonconducting and conducting characteristics, respectively. A significant change in the peak current of differential pulse voltammetry with the gradual variation of bacterial density in the electrolyte was observed for the glassy carbon electrode rGO-liposome (GCE-L-rGO) surface only. The detection sensitivity of GCE-L-rGO sensors was ∼26 μA/106 cells per ml of electrolyte for varying cell densities from 3 × 103 to 3 × 104 cells/ml. The proposed sensing technique can serve as an alternative to conventional methodologies for rapid and in situ detection of bacterial load in different samples, laying the foundation for new applications in clinical diagnostics.

Cyclodextrin-mediated conjugation of macrophage and liposomes for treatment of atherosclerosis.

Current pharmacological treatments of atherosclerosis often target either cholesterol control or inflammation management, to inhibit atherosclerotic progression, but cannot lead to direct plaque lysis and atherosclerotic regression, partly due to the poor accumulation of medicine in the atherosclerotic plaques. Due to enhanced macrophage recruitment during atheromatous plaque progression, a macrophage-liposome conjugate was facilely constructed for targeted anti-atherosclerosis therapy via synergistic plaque lysis and inflammation alleviation. Endogenous macrophage is utilized as drug-transporting cell, upon membrane-modification with a β-cyclodextrin (β-CD) derivative to form β-CD decorated macrophage (CD-MP). Adamantane (ADA) modified quercetin (QT)-loaded liposome (QT-NP), can be conjugated to CD-MP via host-guest interactions between β-CD and ADA to form macrophage-liposome conjugate (MP-QT-NP). Thus, macrophage carries liposome "hand-in-hand" to significantly increase the accumulation of anchored QT-NP in the aorta plaque in response to the plaque inflammation. In addition to anti-inflammation effects of QT, MP-QT-NP efficiently regresses atherosclerotic plaques from both murine aorta and human carotid arteries via CD-MP mediated cholesterol efflux, due to the binding of cholesterol by excess membrane β-CD. Transcriptome analysis of atherosclerotic murine aorta and human carotid tissues reveal that MP-QT-NP may activate NRF2 pathway to inhibit plaque inflammation, and simultaneously upregulate liver X receptor to promote cholesterol efflux.

Disrupting the Interplay between Programmed Cell Death Protein 1 and Programmed Death Ligand 1 with Spherical Nucleic Acids in Treating Cancer.

Disrupting the interplay between programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) is a powerful immunotherapeutic approach to cancer treatment. Herein, spherical nucleic acid (SNA) liposomal nanoparticle conjugates that incorporate a newly designed antisense DNA sequence specifically against PD-L1 (immune checkpoint inhibitor SNAs, or IC-SNAs) are explored as a strategy for blocking PD-1/PD-L1 signaling within the tumor microenvironment (TME). Concentration-dependent PD-L1 silencing with IC-SNAs is observed in MC38 colon cancer cells, where IC-SNAs decrease both surface PD-L1 (sPD-L1) and total PD-L1 expression. Furthermore, peritumoral administration of IC-SNAs in a syngeneic mouse model of MC38 colon cancer leads to reduced sPD-L1 expression in multiple cell populations within the TME, including tumor cells, dendritic cells, and myeloid derived suppressor cells. The treatment effectively increases CD8+ T cells accumulation and functionality in the TME, which ultimately inhibits tumor growth and extends animal survival. Taken together, these data show that IC-SNA nanoconstructs are capable of disrupting the PD-1/PD-L1 interplay via gene regulation, thereby providing a promising avenue for cancer immunotherapy.

Development of tumor-specific liposomes containing quantum dots-photosensitizer conjugate used for radiotherapy

This study aims to develop a multifunctional liposomal radiosensitizer to destroy more tumor cells by using lower radiation doses compared to clinically used 6 MV X-ray doses. To achieve this aim, first Chlorine-e6 (Ce6) was covalently bound to functional groups of outer surfaces of quantum dots (QDs) through EDC/NHS reactions. Then, QDs-Ce6 conjugate loaded, nanosized, PEG-coated, and tumor-specific folic acid-modified immunoliposome dispersions were prepared by film method. Enhanced anti-proliferation activity of free and liposomal conjugate against 4T1 (murine breast cancer) cell lines was investigated at different X-ray doses (5, 10, 15, and 20 Gy). As a result, the best radiosensitizer effect was observed at a 5 Gy X-ray dose and it was found that following the X-ray irradiation, immunoliposome dispersions containing QDs-Ce6 conjugate killed 26.8 ± 1.7% more cancer cells than radiation alone.

Efficient Entrapment of Carbonic Anhydrase in Alginate Hydrogels Using Liposomes for Continuous-Flow Catalytic Reactions.

A versatile approach to entrap relatively small enzymes in hydrogels allows their diverse biotechnological applications. In the present work, bovine carbonic anhydrase (BCA) was efficiently entrapped in calcium alginate beads with the help of liposomes. A mixture of sodium alginate (3 wt %) and carbonic anhydrase–liposome conjugates (BCALs) was dripped into a Tris-HCl buffer solution (pH = 7.5) containing 0.4 M CaCl2 to induce the gelation and curing of the dispersed alginate-rich droplets. The entrapment efficiency of BCALs, which was defined as the amount of catalysts entrapped in alginate beads relative to that initially charged, was 98.7 ± 0.2% as determined through quantifying BCALs in the filtrate being separated from the beads. When free BCA was employed, on the other hand, a significantly lower entrapment efficiency of 27.2 ± 4.1% was obtained because free BCA could pass through alginate matrices. Because the volume of a cured alginate bead (10 μL) entrapped with BCALs was about 2.5 times smaller than that of an original droplet, BCALs were densely present in the beads to give the concentrations of lipids and BCA of 4.6–8.3 mM and 1.1–1.8 mg/mL, respectively. Alginate beads entrapped with BCALs were used to catalyze the hydrolysis of 1.0 mM p-nitrophenyl acetate (p-NA) at pH = 7.5 using the wells of a microplate or 10 mL glass beakers as batch reactors. Furthermore, the beads were confined in a column for continuous-flow hydrolysis of 1.0 mM p-NA for 1 h at a mean residence time of 8.5 or 4.3 min. The results obtained demonstrate that the conjugation of BCA to liposomes gave an opportunity to achieve efficient and stable entrapment of BCA in alginate hydrogels for applying to catalytic reactions in bioreactors.

PREPARATION OF A LIPOSOMAL CONJUGATE OF THE MUTANT EXTRACELLULAR DOMAIN OF THE MOUSE PD-1 RECEPTOR

Liposomal conjugate of the mutant extracellular domain of the mouse PD-1 receptor obtained to inhibit the PD-1/PD-L1 signaling pathway.

Preparation, characterization and evaluation of cytotoxic activity of Tamoxifen bound liposomes against breast cancer cell line

The goal of this study was to determine whether neutrally or positively charged liposomes encapsulated with tamoxifen could inhibit the proliferation of human breast cancer MCF-7 cell proliferation compared to free tamoxifen by estimating the potential effects of these complexes on the cell death of human breast carcinoma (MCF-7) cell lines. The formulated tamoxifen liposomal conjugate was characterized by analytical techniques to determine its size, size-distribution, thermotropic changes and conformational changes along with possible cytotoxicity towards MCF-7 (breast cancer cell line) in Vitro. The mean particle diameter was estimated to be 64.70±49.75 nm, 73.72±23.8 nm and 119±54.7 nm for blank liposomes, neutral and positively charged liposomes bound tamoxifen, respectively. As tamoxifen is introduced into positive liposomes, it results in noticeable broadening and shift to lower temperature at 135 C compared to the main characteristic endothermic peak (Tm) of pure liposomes that exists at 140C, while the introduction of tamoxifen into neutral liposomes contributes to the disappearance of the main endothermic peak of pure liposomes. FTIR analysis revealed structure changes in vesicles after tamoxifen was incorporated into liposomes. The IC50 value for tamoxifen in the cytotoxic assay with MCF-7 treated cells was 0.34 μg/ml, while tamoxifen-loaded neutrally or positively charged liposomes showed an increase of IC50 value to 119.62 and 101.23 µg /ml, respectively. Current data suggests a new treatment regimen in where free tamoxifen is substituted with liposomal tamoxifen to improve anticancer activity against MCF-7 cancer cell lines.

Bioorthogonal supramolecular cell-conjugation for targeted hitchhiking drug delivery

Cells possess inherent advantages to facilitate targeted payload delivery. Current strategies to conjugate payload carriers to the surface of cells are either via covalent bonds that not only involve complicated synthetic process but also often impair cellular functions, or via biological ligand-receptor interactions that are only specific to particular types of cells. Herein, we report a facile, bioorthogonal supramolecular conjugation strategy to prepare targeted cell-hitchhiking delivery systems, mediated via artificial host–guest interactions between β-cyclodextrin and adamantane, respectively anchored (via insertion) on the surfaces of live cells and payload carriers. In a paw swelling inflammation mouse model, supramolecularly conjugated macrophage-carriers (either cell–cell or cell-nanoparticle systems) were efficiently delivered hand-in-hand to the swelling paw, driven by the inflammatory tropism of macrophage. Furthermore, in an acute lung inflammation model of mouse, supramolecular conjugation of peritoneal macrophage and quercetin-loaded liposomes significantly improved targeting efficiency of the liposomes, and effectively alleviated the lung inflammation through the anti-inflammatory and anti-oxidative effects of quercetin. The cell-friendly, facile, host–guest interactions mediated cellular conjugation may provide the very first general strategy for preparing various cell-hitchhiking delivery systems to meet the needs of diverse biomedical applications.

Synthesis, biocompatibility and gene encapsulation of poly(2-Ethyl 2-Oxazoline)-dioleoyl phosphatidylethanolamine (PEtOx-DOPE) and post-modifications with peptides and fluorescent dye coumarin

Liposome surface modifications serve great potential applications of liposomes, for instance, increasing stability, bioactive liposome conjugates, and targeted drug, gene, and image agent delivery. In this study, novel targeted lipopolymers, peptide 18/peptide 563-poly(2-ethyl-2-oxazoline)-dioleoylphosphatidyl-ethanolamine (P18/P563-PEtOx-DOPE), have been demonstrated to be successfully synthesized. The structures of P18/P563-PEtOx-DOPE were confirmed by FT-IR spectroscopy, GPC, and 1H-NMR. In this strategy, poly(2-ethyl 2-oxazoline)-modified liposomes were firstly constructed with molecular weights of 3,500 and 5,800 Da. Then, we chose PEtOx5800-DOPE because it has been obtained better particle size (88.74 ± 0.6816) according to the DLS results. Then, peptides- and dye-PEtOx lipid-based nanovesicle (LN) were prepared by peptide-18, peptide-563, and 7-mercapto-4-methyl coumarin. Genetic material (pDNA) was encapsulated into the liposomes and evaluated the encapsulation of plasmid DNA with migration by using agarose gel electrophoresis. In vitro cytotoxicity experiment results on prostate cancer and breast cancer cell lines, parallelly with the healthy prostate (PNT1A) and breast (MCF10A) epithelial cell lines, cells showed insignificant toxic effects. Thus, we can suggest a novel PEtOx phospholipid thanks to this article and its integration with ligands, which great potential for gene transfer system.

Dual functional liposomes carrying antioxidants against tau hyperphosphorylation and apoptosis of neurons

Quercetin (QU) and rosmarinic acid (RA) were loaded in phosphatidic acid-liposomes (QU/RA-PA-liposomes) with surface apolipoprotein E (ApoE) using a process of thin-film hydration, followed by covalent crosslinking to activate biological pathways for penetrating the blood–brain barrier (BBB) and redeeming the neuronal apoptosis from attack of β-amyloid 1-42 (Aβ1-42) and neurofibrillary tangles. The conjugation of liposomes with PA improved the activity of QU and RA against neurotoxicity of Aβ1-42. The fluorescent images of brain capillaries revealed that surface modification with ApoE improved the permeation ability of QU/RA-PA-ApoE-liposomes across the BBB. In addition, the highest therapeutic efficacy was obtained in the case of QU/RA-PA-ApoE-liposomes, compared to other QU/RA formulations studied using in vivo Aβ1-42-insulted rats mimicking Alzheimer’s disease (AD). The cellular and molecular evidence from AD rats included the decrease in Aβ1-42 plaque formation and interleukin-6 secretion, increase in the neuronal count in Nissl staining, and reduction in the expression of phosphorylated extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, p38 kinase and tau protein at serine 202 as well as caspase-3. The use of PA-ApoE-liposomes as a dual targeting formulation enhances the QU and RA ability to infiltrate the BBB, docks Aβ1-42 plaques and can be a potent approach to rescue degenerated neurons from AD.

Use of Target-Specific Liposome and Magnetic Nanoparticle Conjugation for the Amplified Detection of Norovirus.

Viral diseases are one of the most life-threatening diseases as they can erupt unpredictably and spread rapidly in any medium with a very small number of particles. Therefore, the key for lethal virus detection should be highly sensitive in the early-stage detection, which can help increase the chance of survival. Amplification of the detecting signal is one of the most promising mechanisms for the detection of low-concentration analytes. A proper amplification can develop such a kind of system where a small number of particles can produce intense signals for a prominent detection. Keeping this in mind, in this report, we have presented a fluorometric method to detect norovirus (NoV) by a newly developed fluorophore-labeled liposome and a magnetically modified Fe3O4 combined system. Homogeneously distributed amine-functionalized liposomes have been constructed filled with a strong fluorophore of calcein. Simultaneously, (3-aminopropyl)-triethoxysilane (APTES)-functionalized Fe3O4 nanoparticles are also synthesized by the standard silanization process, and these two separately synthesized nanoparticles were functionalized with an antibody to achieve specificity. The Fe3O4 and calcein-liposome system has been applied for NoV detection, which was magnetically separated from the analyte medium and then externally burst to release the fluorophores from the core of the liposome. The easiness, rapidity, and sensitivity in a wide linear range can offer a huge potential of this method in point-of-care diagnostics.

Utilization of natural stabilizer to prepare liposomal conjugate for the newly developed aptamer

Background: Aptamers are peptide or oligonucleotides that have the capacity to bind to a specific target molecule in human body. One of the newly developed aptamers named BAS aptamer. For targeting improvement of the aptamer, liposomal conjugate was adapted utilizing phosphatidylcholine as a natural lipid stabilizer in a comparison to synthetic one. Method: Two types of blank DSPE-PEG 2000-maleimide liposomes were prepared using two types of stabilizers; synthetic (Dipalmitoylphosphatidylcholine; DPPC) and natural lipids (phosphatidylcholine) then conjugated with BAS-aptamer through introducing thiol moiety. The liposomal conjugates then evaluated by H1-NMR and urea polyacrylamide gel electrophoresis and its efficacy on binding affinity, anticancer activity against MCF-7 breast cancer as well as its stability in plasma and buffers were estimated. Results: The binding affinity study revealed a significantly higher affinity of the naturally stabilized BAS aptamer conjugated liposomes towards drug receptor (SIRT1) than the synthetically stabilized one (KD= 38.5 ± 0.985 and 68± 0.967; respectively). The cytotoxic effect of the liposomal conjugates was higher than those of pure BAS aptamer and its thiol derivative in all concentrations used. The stability study in plasma showed that naturally stabilized liposomes remained stable for up to 6 days while the synthetically prepared liposomes showed partial degradation after 5 days and both gave higher stability than the pure BAS aptamer while the stability study in phosphate buffer at pH 7.4 and 5.5 revealed non-significant decline in the absorbance of the drug started after 6 days in its naturally stabilized conjugate and after 3 days (at pH5.5) its synthetically stabilized liposomal. Conclusion: Using natural stabilizer for liposomal conjugates gave better physical properties as well as significant binding affinity for the aptamer conjugated liposomes to the receptor (SIRT1) and higher cytotoxic effect as well as better stability in plasma and buffers.

Abstract 3628: Comparison of CD44 expression in pancreatic cancer, pancreatic stem and normal pancreatic cells: Development of CLENs for tumor targeting and therapy using cell models

Abstract Purpose: The incidence of drug resistance in pancreatic cancer correlates with poor prognosis, increased disease progression and unfavorable treatment outcomes. CD44, a surface membrane proteoglycan has been identified to be directly involved in pancreatic cancer drug resistance and metastasis. As such, the aim of this research was to screen various pancreatic cells for their expression of CD44, and to begin the early design and development of cell membrane lipid-extracted nanoliposmes (CLENs) for enhanced targeting and therapy. Methods: Three pancreatic cancer cells- Panc-1, MiaPaCa 2, MS1 VEGF and a normal cell line- hTERT- were employed for this study. Stem cell, Mia PaCa-2, served as a positive control for screening purposes. Four liposome compositions were prepared using various ratios of DOPC, Chol, DPPE-PEG5000, and cellular membrane lipid-extracted (LE) was derived from target cell, Panc 1. To evaluate CD44 glycoprotein expression, cells were seeded in 48-well plates and incubated overnight. Fluorescence measurements were determined at excitation wavelength of 485/25 and emission wavelength at 528/20 nm. G44-26 monoclonal antibody was used for CLENs antibody conjugation studies. A Sephadex G-25 column was used for separation of bound and unbound antibody material, and fluorescence detection was used for cell culture analysis. Fluorescence detection was performed at excitation and emission wavelengths of 530/25 and 590/25, respectively. Results: The relative level of CD44 expression for each of the cell lines was as follows. Panc1>Mia PaCa-2 = hTERT>MS1-VEGF. There was a statistically significant difference between Panc-1 and Mia Paca 2 (P<0.001), between hTERT and MS1 VEGF (P<0.05), and between Panc-1 and hTERT (P<0.0001). The inclusion of Panc1-LE in combination with additional liposome components did not increase cellular uptake significantly. The conjugation of G44-26 monoclonal antibody to the liposomes increased average size of all 4 preparations. Average size for CLENs and G44-26 monoclonal antibody -CLENs increased from 140.75 ± 3.22 nm to 250.23 ± 15.31 nm. Conclusion: Future studies are required to demonstrate the effect of G44-26 monoclonal antibody liposome conjugates to further exploit the optimal expression of CD44 for pancreatic drug targeting. Citation Format: Dotun D. Adegunle, Eugene Boakye Ansah, Danny Nguyen, Kanchi Sanghvi, Drew Goodrich, Robert Campbell. Comparison of CD44 expression in pancreatic cancer, pancreatic stem and normal pancreatic cells: Development of CLENs for tumor targeting and therapy using cell models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3628.