Low-dose paclitaxel via hyaluronan-functionalized bovine serum albumin nanoparticulate assembly for metastatic melanoma treatment.

Due to the critical role of CD44 in mediating cell adhesion and migration, CD44-targeted drug delivery via hyaluronan has been extensively explored. Herein, cationic bovine serum albumin nanoparticles were assembled with hyaluronan (HA) of various molecular weights via simple electrostatic interaction to afford hierarchical nanoparticles (HNPs) with various size distributions and structures. Next, HNPs obtained using 49 kDa HA have been used to encapsulate paclitaxel (PTX-HNPs), which demonstrated selective lung accumulation due to both size effect and CD44-mediated targetability. Biodistribution studies showed that HNPs enhanced the lung specific accumulation of HNPs in the C57BL/6 mice melanoma lung metastasis model. In the antitumor studies, compared with the Taxol or bovine serum albumin nanoparticle (NP) groups, PTX-HNPs significantly inhibited B16F10 lung metastasis at a relatively low dose. Additionally, cell migration and invasion experiments in vitro further confirmed that PTX-HNPs significantly inhibited the migration of B16F10 cells compared to Taxol or paclitaxel-loaded NP groups. Overall, our results suggest that PTX-HNPs represent a highly promising strategy for the treatment of lung metastatic melanoma.

Sonochemical assisted synthesis of dual functional BSA nanoparticle for the removal of excessive bilirubin and strong anti-tumor effects.

In this study, we prepared a dual functional albumin-based nanoparticle (gal-BSA-NPs) by sonochemical method which allowed an efficient encapsulation for Bilirubin (BR) through its adsorption capacity and hydrophobic interaction. Our study provided a possibility that the blank gal-BSA-NPs can replace BSA with better ability for the adsorption of excessive BR. Additionally, we unearthed the potential anti-tumor activity of BR on HepG2 cells and developed GSH-responsive BR-loaded gal-BSA-NPs for the treatment of liver cancer. The results showed BR-loaded gal-BSA-NPs effectively enhanced cellular uptake and exerted strong inhibition on tumor cell proliferation and migration. In vivo anti-tumor study revealed BR-loaded gal-BSA-NPs showed strong anti-tumor effects. Our study not only revealed the anti-tumor potency of BR, but also brought conventional BSA with novel application in liver cancer treatment.

Multicolored Protein Nanoparticles: Synthesis, Characterization, and Cell Uptake.

Synthesis, characterization, and applications of strongly fluorescent, multicolored protein nanoparticles (GlowDots) are reported here. Bovine serum albumin was cross-linked under controlled conditions to form nanoparticles, where particle size was controlled from 20 to 100 ± 10 nm by choosing appropriate reaction conditions. The absorption as well as the emission wavelengths were controlled without changing the particle size, unlike quantum dots. Each GlowDot was loaded with up to 214 ± 50 chromophores, and hence, the particles have high molar absorptivities (106 M-1 cm-1) as well as high brightness (105 to 106 M-1 cm-1). A large number of functional groups cover the particle surface and these are further functionalized to enhance cellular uptake. GlowDots that were labeled with fluorescein and functionalized with taurine, for example, were quickly taken up by HeLa, MDA-MB-231, PC3, and L6 myoblast cells, as interrogated by fluorescence imaging studies. GlowDots were biocompatible, size tunable, biodegradable, strongly fluorescent, and stable for months at room temperature, and they may serve as substitutes for quantum dots in a variety of practical applications.

Synthetic and immunological studies on trimeric MUC1 immunodominant motif antigen-based anti-cancer vaccine candidates.

Therapeutic vaccines have been regarded as a very promising treatment modality against cancer. Tumor-associated MUC1 is a promising antigen for the design of antitumor vaccines. However, body's immune tolerance and low immunogenicity of MUC1 glycopeptides limited their use as effective antigen epitopes of therapeutic vaccines. To solve this problem, we chose the immune dominant region of MUC1 VNTRs. We designed and synthesized its linear trivalent glycopeptide fragments and coupled the fragments with BSA. Immunological evaluation indicated that the antibodies induced by glycosylated MUC1 based vaccine 11 had a stronger binding than non-glycosylated 10. The novel constructed antigen epitopes have the potential to overcome the weak immunogenicity of natural MUC1 glycopeptides and deserve further research.

Bovine Serum Albumin as a Versatile Platform for Cancer Imaging and Therapy.

BACKGROUND: Due to the good biocompatibility, biodegradability, facile surface functionalization and high water solubility, Bovine serum albumin has gain increasing attention in the nanomedicine. OBJECTIVE: Despite there are many reviews on albumin based nanoparticles, most of them focus on one aspect of the albumin functionality, e.g., drug delivery, cancer theranostics or half-life extension in vivo. This review aims to comprehensively summary bovine serum albumin as a versatile platform in the applications of cancer imaging and therapy. METHODS: We review the extensive applications of bovine serum albumin in drug carrier, surface engineering and biomimetic synthesis for cancer imaging and therapy. CONCLUSION: Based on the studies reviewed, variety of in vitro and in vivo studies show good performance of bovine serum albumin as the drug carrier, surface modification agent and biomimetic template in cancer imaging and therapy. Nevertheless, there are still some issues to be solved, e.g., the technological parameters for enhancing the drug loading efficiency and controlling drug release, optimizing surface modification process to provide more stable nanoagents, investigation of the biomimetic mechanism, in-depth study of their toxicity, further exploring their bioapplications, etc.

Tailored Multivalent Neo-Glycoproteins: Synthesis, Evaluation, and Application of a Library of Galectin-3-Binding Glycan Ligands.

Galectin-3 (Gal-3), a member of the ß-galactoside-binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galß1,4GlcNAc; LacNAc type 2)-based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N',N″-diacetyllactosamine (GalNAcß1,4GlcNAc; LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich enzyme-linked immunosorbent assay format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer-related biomedical research.

Synthesis and biological assay of erlotinib analogues and BSA-conjugated erlotinib analogue.

A series of erlotinib analogues that have structural modification at 6,7-alkoxyl positions is efficiently synthesized. The in vitro anti-tumor activity of synthesized compounds is studied in two non-small cell lung cancer (NSCLC) cell lines (A549 and H1975). Among the synthesized compounds, the iodo compound 6 (ETN-6) exhibits higher anti-cancer activity compared to erlotinib. An efficient method is developed for the conjugation of erlotinib analogue-4, alcohol compound, with protein, bovine serum albumin (BSA), via succinic acid linker. The in vitro anti-tumor activity of the protein attached erlotinib analogue, 8 (ETN-4-Suc-BSA), showed stronger inhibitory activity in both A549 and H1975 NSCLC cell lines.

Vinyl sulfone-based ferrocenylation reagents: applications in conjugation and bioconjugation.

The easy vinyl sulfone derivatization of ferrocene allows the preparation of some effective, versatile and valuable ferrocenylation reagents. The applicability of such compounds in conjugation and bioconjugation of amine and/or thiol containing molecules and biomolecules through Michael-type addition under mild conditions that preserve the biological function of the latter is described. The feasibility of the methodology is demonstrated by the preparation of a variety of conjugates and bioconjugates (ferrocenyl terminated dendrimers and ferrocene-sugar, ferrocene-cyclodextrin, ferrocene-peptide and ferrocene-protein conjugates).

[Synthesis of conjugates of bovine serum albumin with water-soluble ytterbium porphyrins]. / Sintez kon''iugatov bych'ego syvorotochnogo al'bumina s vodorastvorimym itterbiebym porfirinom.

The ytterbium complex of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin was synthesized as an IR-fluorescent label and covalently bound to bovine serum albumin. The resulting conjugate fluoresces at 985 nm and is of interest for use in IR-fluorescent tumor diagnostic, immunoassay, and energy transfer studies. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

Surfactant-free microspheres of poly(epsilon-caprolactone)/poly (ethylene glycol)/poly(epsilon-caprolactone) triblock copolymers as a protein carrier.

The aim of this study is to prepare biodegradable microspheres without the use of surfactants or emulsifiers for a novel sustained delivery carriers of protein drugs. A poly(epsilon-caprolactoney poly(ethylene glycol)/poly(epsilon-caprolactone) (CEC) triblock copolymer was synthesized by the ring-opening of epsilon-caprolactone with dihydroxy poly (ethylene glycol) to prepare surfactant-free microspheres. When dichloromethane (DCM) or ethyl formate (EF) was used as a solvent, the formation of microspheres did not occur. Although the microspheres could be formed prior to lyophilization under certain conditions, the morphology of microspheres was not maintained during the filtration and lyophilization process. Surfactant-free microspheres were only formed when ethyl acetate (EA) was used as the organic solvent and showed good spherical microspheres although the surfaces appeared irregular. The content of the protein in the microsphere was lower than expected, probably because of the presence of water channels and pores. The protein release kinetics showed a burst release until 2 days and after that sustained release pattern was showed. Therefore, these observations indicated that the formation of microsphere without the use of surfactant is feasible, and, this the improved process, the protein is readily incorporated in the microsphere.

Efecto de la sustitución de yema de huevo por albúmina sérica bovina, suero equino o suero bovino en el diluyente de congelación sobre la viabilidad posdescongelación del espermatozoide equino / Effects of the substitution of bovine serum albumin, equine serum or bovine serum in the freezing extender on posthawed viability of equine sperm

Se colectaron 14 eyaculados equinos, de cada uno de ellos, previa centrifugación, se obtuvieron 7 alícuotas que fueron incorporadas a los diluyentes de congelación, cuya base general no difiere de ninguno de los tratamientos estudiados, excepto en el componente proteínico, ya que en el grupo testigo se utilizó la yema de huevo al 20 por ciento (YH), la cual se sustituyó, en los grupos experimentales por suero equino al 3 por ciento (SE3), suero equino al 6 por ciento (SE6), suero bovino al 3 por ciento (SB3), suero bovino al 6 por ciento (SB6), albúmina sérica bovina al 2 por ciento (AS2) o albúmina sérica bovina al 3 por ciento (AS3). El proceso de congelación-descongelación fue el mismo para todos los tratamientos. Se utilizó el sistema PAS (Programa para Análisis de Semen) con el fin de evaluar la motilidad, velocidad y linealidad del recorrido de las muestras en posdescongelación. En general se notó un efecto detrimental considerable de todos los sustitutos al momento de la descongelación ya que sólo el 46 por ciento de las muestras en estudio tuvieron viabilidad detectable por la computadora. En cambio, todas las muestras testigo (YH) se pudieron analizar. La motilidad no presentó diferencias entre los tratamientos, a excepción del diluyente SB6 con 36 ñ 5 por ciento vs el diluyente SB3 con 14 ñ 4 por ciento (P< 0.01). En cuanto a la motilidad también analizada visualmente, se encontró que el diluyente YH resultó ser el de mayor motilidad, 30 ñ 3 por ciento, y el SB3 y SE6 los de menor motilidad con 13 ñ 3 por ciento (P< 0.001). El diluyente YH presentó el mejor índice de velocidad con 64 ñ 2 µm/seg, y el SB3, SB6 y SE3 con 49, 45 y 52 µm/seg respectivamante, como los de manor velocidad (P< 0.001). La linealidad resultó mejor para el diluyente YH y SB6 que para los demás diluyentes (P< 0.001). Se concluye que el uso de la yema de huevo en el diluyente de congelación de semen equino provee una mejor protección al espermatozoide ante dicho proceso, que los sueros de equino y bovino y la albúmina sérica bovina

Photosensitizer targeting in photodynamic therapy. I. Conjugates of haematoporphyrin with albumin and transferrin.

Conjugates of haematoporphyrin (HP) with serum albumin and transferrin were prepared, purified by gel filtration and characterized by high performance liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE) and spectroscopy. Although the fluorescence was somewhat quenched, the conjugates had similar singlet oxygen quantum yields to free porphyrin. The albumin conjugate (HP-BSA) could be divided into monomeric and cross-linked fractions. In NIH 3T3 and HT29 cells, native albumin could not compete with the uptake of HP-BSA and the uptake was greatly enhanced in the absence of serum and in the presence of poly-L-lysine. We infer that the conjugate was mostly associated with the plasma membrane in these cells. The uptake of HP-transferrin showed evidence of a receptor-mediated component in that it was partially inhibited by native protein and increased when transferrin receptors were upregulated by an iron chelator. J774 macrophage-like cells accumulated fluorescence from HP-BSA to a much higher degree than HT29 cells, even though the protein was extensively degraded (HT29 cells did not appear to degrade the protein). The time course of the photocytotoxicity of HP-BSA was prolonged in J774 cells, although their response to free porphyrins was similar to that seen in HT29 cells. Chloroquine inhibited protein degradation without having an effect on the fluorescence uptake. J774 cells acquired more fluorescence and degraded more protein when supplied with cross-linked HP-BSA compared with monomeric fraction. For a given fluorescence uptake, the cross-linked fraction was also more photocytotoxic. We conclude that macrophages can acquire photosensitizer-protein conjugates avidly and that these are delivered to the lysosomes. These types of conjugate may have applications in targeting fluorescent molecules for diagnostic imaging and for the photodynamic treatment of macrophage malignancies.

Preparation of a neoglycoprotein using a homobifunctional reagent and its applicability for protein blotting and electron microscopy.

A new method for the preparation of a neoglycoprotein (chemically mannosylated bovine serum albumin, D-Man.BSA) is described using the homobifunctional reagent divinylsulphone.D-Man.BSA purified by affinity chromatography on ConA-Sepharose 4B shows microheterogeneity as demonstrated by immunoaffinity electrophoresis with free ConA in the first-dimension gel. The dissociation constant K for the neoglycoprotein-ConA complex has been calculated to be 2.5.10(-5) M. Biotinylated D-Man.BSA is a useful reagent to detect carbohydrate binding proteins of L1210 leukemia cells on blots. The neoglycoprotein labelled with colloidal gold may be used to demonstrate L1210 cell surface D-Man binding proteins by preembedding electron microscopy.

Production of macrophage-derived cytotoxic factor by N-[3-[(carbamoylmethyl)thio]propionylated] neoglycoproteins.

6-Phosphomannosylated bovine serum albumin (Man6P-BSA), a neoglycoprotein endocytosed by macrophages, bearing either 3-(2-pyridyldithio)propionyl or 3-[(carbamoylmethyl)thio]propionyl residues coming from alkylation of thiol residues by iodoacetamide were prepared and tested for their immunomodulator properties. The supernatants of mouse peritoneal macrophages incubated with Man6P-BSA bearing 3-[(carbamoylmethyl)thio]propionyl groups, and by a lesser extent 3-(2-pyridyldithio)propionyl groups, were cytotoxic to L929 cells, suggesting the presence of a tumor necrosis factor like compound. This macrophage-activation process is linked to the capacity of Man6P-BSA to be endocytosed via membrane lectins of macrophages, because the supernatants of macrophages incubated with unglycosylated conjugates were not cytotoxic. The cytotoxic activity induced by 3-[(carbamoylmethyl)thio]propionyl groups bound onto Man6P-BSA was similar to that induced by Man6P-BSA bearing muramyl dipeptide, indicating that endocytosed neoglycoproteins bearing 3-[(carbamoylmethyl)thio]propionyl residues are potent macrophage activators.