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BackgroundAntibody-based therapeutics is a rapidly growing field. Small engineered antibody fragments demonstrate similar antigen affinity compared with the parental antibody but have a shorter serum half-life and possess the ability to be conjugated to nanoparticles. The goal of this study was to engineer an anti-carbohydrate antigen 19-9 (CA19-9) cys-diabody fragment in hopes of targeting nanoparticles to pancreatic cancer.MethodsThe anti-CA19-9 cys-diabody was created by engineering a C-terminal cysteine residue into the DNA single-chain Fv construct of the anti-CA19-9 diabody and expressed in NS0 cells. Maleimide chemistry was used to conjugate the cys-diabody to polymerized liposomal nanoparticles (PLNs) through the cysteine residues. Flow cytometry was used to evaluate targeting of cys-diabody and cys-diabody-PLN conjugate to human pancreatic cancer cell lines. The cys-diabody was radiolabeled with a positron emitter ((124)I) and evaluated in a mouse model of CA19-9-positive and CA19-9-negative xenografts with micro-positron emission tomography/micro-computed tomography at successive time intervals after injection. Percentage of injected dose per gram of radioactivity was measured in blood and tumor to provide objective confirmation of the micro-positron emission tomographic images.ResultsTumor xenograft imaging of the anti-CA19-9 cys-diabody demonstrated an average tumor-to-blood ratio of 3.0 and positive-to-negative tumor ratio of 7.4. Successful conjugation of the cys-diabody to PLNs was indicated by flow cytometry showing specific binding of cys-diabody-PLN conjugate to human pancreatic cancer cells in vitro.ConclusionsOur results show that the anti-CA19-9 cys-diabody targets pancreatic cancer providing specific molecular imaging in tumor xenograft models. Furthermore, the cys-diabody-PLN conjugate demonstrates target-specific binding of human pancreatic cancer cells with the potential to deliver targeted treatment.

Osteosarcoma is the most common primary malignancy of bone in children, adolescents, and adults. Despite extensive surgery and adjuvant aggressive high-dose systemic chemotherapy with potentially severe bystander side effects, cure is attainable in about 70% of patients with localized disease and only 20%-30% of those patients with metastatic disease. Targeted therapies clearly are warranted in improving our treatment of this adolescent killer. However, a lack of osteosarcoma-associated/specific markers has hindered development of targeted therapeutics. We describe a novel osteosarcoma-associated cell surface antigen, ALCAM. We, then, create an engineered anti-ALCAM-hybrid polymerized liposomal nanoparticle immunoconjugate (α-AL-HPLN) to specifically target osteosarcoma cells and deliver a cytotoxic chemotherapeutic agent, doxorubicin. We have demonstrated that α-AL-HPLNs have significantly enhanced cytotoxicity over untargeted HPLNs and over a conventional liposomal doxorubicin formulation. In this way, α-AL-HPLNs are a promising new strategy to specifically deliver cytotoxic agents in osteosarcoma.

Research reveals that the addition of anionic groups to liposomes containing polyvalent monomeric sialyl Lewis (sup)x-like oligosaccharide groups enhance inhibitory effect on selectin binding. Data further show that additional anionic sulfate esters in the liposomes result in highly inhibitory membranes.

A nonreductive technique to derive oligosaccharides with allylamide groups efficiently at the glycosidic position involves three steps. The first step melts unprotected oligosaccharide in allylamine. This step is followed by the two-step reaction which starts with the attack of allylamine on the anomeric hydroxyl group of the oligosaccharide. The final steps involves 3'-acetic acid-Gal-beta-1,4-(Fuc alpha-1,3)Glc.

The sialyl Lewis X determinant NeuAc-alpha-2,3-Gal-beta-1,4-(Fuc-alpha-1,3)-GlcNAc was prepared from a trisaccharide treated with GDP-fucose utilizing the fucosyltransferase. On the other hand, beta-O-allyl Le(super x) was prepared from the treatment of beta-O-(2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl)trichloroacetimidate with the beta-O-allyl glycoside of a selectively protected GlcNAc derivative toyield beta-O-allyyl-N-acetyllactosamine. The fucose was inserted into the intermediate product via an enzymatic glycosylation reaction to finally give the target compound.

Neutrophils are the most abundant white blood cells, with a vital role in innate immune defense against bacterial and fungal pathogens. Although mostly associated with pathological processes directly related to immune defense, they can also play a detrimental role in inflammatory conditions and have been found to have a pro-metastatic role in the spread of cancer cells. Here, we explore ways to temporarily suppress these detrimental activities. We first examined the possibility of using siRNA and antisense oligonucleotides (ASOs) for transient knockdown of the human and mouse C5a receptor, an important chemoattractant receptor involved in neutrophil-mediated injury that is associated with myocardial infarction, sepsis, and neurodegenerative diseases. We found that siRNAs and ASOs transfected into cultured cell lines can eliminate 70–90% of C5a receptor mRNA and protein within 72 h of administration, a clinically relevant time frame after a cardiovascular event. Targeted drug delivery to specific cells or tissues of interest in a mammalian host, however, remains a major challenge. Here, using phage display technology, we have identified peptides that bind specifically to CD177, a neutrophil-specific surface molecule. We have attached these peptides to fluorescent, lipid-based nanoparticles and confirmed targeting and delivery to cultured cells ectopically presenting either human or mouse CD177. In addition, we have shown peptide-nanoparticle binding specifically to neutrophils in human and mouse blood. We anticipate that these or related tagged nanoparticles may be therapeutically useful for delivery of siRNAs or ASOs to neutrophils for transient knockdown of pro-inflammatory proteins such as the C5a receptor. [ABSTRACT FROM AUTHOR]

Probe sonication method helps prepare polymerizable liposomes capable of binding to influenza virus particles. These liposomes are biomolecular materials having a molecular recognition function, sialic acid, and a detection element, polydiacetylene backbone. Binding of these liposomes leads to a visible color change, and absorption spectroscopy helps quantify the color change. Competitive inhibition studies help confirm the specificity of the color change.

An investigation on polymerized liposomes containing alpha-C-glycosides of sialic acid is presented. A polymerizable sialoside lipid is synthesized to form liposome mixtures that are found to be inhibitors of influenza virus in vitro infectivity. Further studies have shown that the capacity of a sialoside to inhibit hemagglutination does not necessarily reflect its capacity to inhibit infectivity.

Identifies the novel P-selectin antagonists that would inhibit leukocyte attachment and rolling in vitro, and have potent anti-inflammatory effects in vivo in a murine model of allergic airway disease. Efficacy of formulation changes; Administration of polymerized lipid nanoparticle (PLNP) after establishing the leukocyte and selectin rolling interaction; Inhibition of peribronchial eosinophil recruitment by selectin PLNP.

In this study, the first mechanism-based monoclonal antibodies have been produced that recognize and differentiate diethoxy- and monoethoxyphosphorylated serine residues. Haptens were synthesized as the stable phosphonate form of phosphoserine esters to improve the immunoresponse. Following condensation with a glutaric anhydride to link the phosphoserine moieties to carrier protein, the hapten densities attached to bovine serum albumin and keyhole limpet henocyanin were determined by partial trypsin digestion and MALDI mass spectrometry, and confirmed using a fluorescent assay (FITC) to quantify unmodified lysine residues. The conjugation reactions were pH optimized to improve hapten density. Screening of subclones led to the identification of two monoclonal antibodies: (a) N257/25.11 that specifically recognizes (EtO) 2 P(O)-Ser as the phosphylated or inhibited form, and (b) N262/16 that recognizes (EtO)(HO)P(O)-Ser as the ‘aged’ form. Analysis of blood samples treated with paraoxon (EtO) 2 P(O)-OPhNO 2 showed a concentration dependent recognition of the phosphylated form. [ABSTRACT FROM AUTHOR]

The development of new therapies for surgical adhesions has proven to be difficult as there is no consistently effective way to assess treatment efficacy in clinical trials without performing a second surgery, which can result in additional adhesions. We have developed lipid microbubble formulations that use a short peptide sequence, CREKA, to target fibrin, the molecule that forms nascent adhesions. These targeted polymerized shell microbubbles (PSMs) are designed to allow ultrasound imaging of early adhesions for diagnostic purposes and for evaluating the success of potential treatments in clinical trials while acting as a possible treatment. In this study, we show that CREKA-targeted microbubbles preferentially bind fibrin over fibrinogen and are stable for long periods of time (∼48 h), that these bound microbubbles can be visualized by ultrasound, and that neither these lipid-based bubbles nor their diagnostic-ultrasound-induced vibrations damage mesothelial cells in vitro. Moreover, these bubbles show the potential to identify adhesionlike fibrin formations and may hold promise in blocking or breaking up fibrin formations in vivo.

Microbubbles have been used in ultrasound-assisted drug delivery to help target solid tumors via blood vessels in vivo; however, studies to understand the phenomena at the cellular level and to optimize parameters for ultrasound or microbubbles in vivo are challenging and expensive to perform. Here, we utilize microfluidic microvessels-on-a-chip that enable visualization of microbubble/ultrasound-dependent drug delivery to microvasculature. When exposed to pulsed ultrasound, microbubbles perfused through microvessels-on-a-chip were observed to stably oscillate. Minimal cellular damage was observed for both microbubbles and untargeted doxorubicin-encapsulating liposomes (DOX-liposomes) perfused through chip microvessels. In contrast, passive and ultrasound-assisted perfusion of integrin-targeted DOX-liposomes induced cytotoxicity, which was only significantly enhanced for ultrasound-assisted perfusion when microbubbles were coperfused. These results suggest that stably oscillating microbubbles enhance targeted DOX-liposome internalization/cytotoxicity largely by stimulating integrin receptor endocytosis. Furthermore, our study demonstrates the utility of our microvessels-on-a-chip as a screening platform for optimizing drug dosage, targeting ligands and drugs.

There is a critical need to formulate stable micron-sized oil droplets as hydrophobic drug carriers for efficient drug encapsulation, long-term storage, and sustained drug release. Microfluidic methods were developed to maximize the stability of micron-sized, oil-in-water (o/w) emulsions for potential use in drug delivery, using doxorubicin-loaded triacetin oil as a model hydrophobic drug formulation. Initial experiments examined multiple flow conditions for the dispersed (oil) and continuous (liposome aqueous) phases in a microfluidic device to establish the parameters that influenced droplet size. These data were fit to a mathematical model from the literature and indicate that the droplet sizes formed are controlled by the ratio of flow rates and the height of the device channel, rather than the orifice size. Next, we investigated effects of o/w emulsion production methods on the stability of the droplets. The stability of o/w emulsion produced by microfluidic flow-focusing techniques was found to be much greater (5 h vs 1 h) than for emulsions produced by mechanical agitation (vortexing). The increased droplet stability was attributed to the uniform size and lipid distribution of droplets generated by flow-focusing. In contrast, vortexed populations consisted of a wide size distribution that resulted in a higher prevalence of Ostwald ripening. Finally, the effects of shell polymerization on stability were investigated by comparing oil droplets encapsulated by a photopolymerizable diacetylene lipid shell to those with a nonpolymerizable lipid shell. Shell polymerization was found to significantly enhance stability against dissolution for flow-focused oil droplets but did not significantly affect the stability of vortexed droplets. Overall, results of these experiments show that flow-focusing is a promising technique for generating tunable, stable, monodisperse oil droplet emulsions, with potential applications for controlled delivery of hydrophobic drug formulations.

Monodisperse gas microbubbles, encapsulated with a shell of photopolymerizable diacetylene lipids and phospholipids, were produced by microfluidic flow focusing, for use as ultrasound contrast agents. The stability of the polymerized shell microbubbles against both aggregation and gas dissolution under physiological conditions was studied. Polyethylene glycol (PEG) 5000, which was attached to the diacetylene lipids, was predicted by molecular theory to provide more steric hindrance against aggregation than PEG 2000, and this was confirmed experimentally. The polymerized shell microbubbles were found to have higher shell-resistance than nonpolymerizable shell microbubbles and commercially available microbubbles (Vevo MicroMarker). The acoustic stability under 7.5 MHz ultrasound insonation was significantly greater than that for the two comparison microbubbles. The acoustic stability was tunable by varying the amount of diacetylene lipid. Thus, our polymerized shell microbubbles are a promising platform for ultrasound contrast agents., (© 2012 American Chemical Society)