serum stability
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Author(s):  
Rico Schwarz ◽  
Eric Zitzow ◽  
Adina Fiebig ◽  
Silvio Hering ◽  
Yvonne Humboldt ◽  
...  

Abstract Arginine auxotrophy is a metabolic defect that renders tumor cells vulnerable towards arginine-depleting substances, such as arginine deiminase (ADI) from Streptococcus pyogenes (SpyADI). Previously, we confirmed SpyADI susceptibility on patient-derived glioblastoma multiforme (GBM) models in vitro and in vivo. For application in patients, serum half-life of the enzyme has to be increased and immunogenicity needs to be reduced. For this purpose, we conjugated the S. pyogenes-derived SpyADI with 20 kDa polyethylene glycol (PEG20) moieties, achieving a PEGylation of seven to eight of the 26 accessible primary amines of the SpyADI. The PEGylation reduced the overall activity of the enzyme by about 50% without affecting the Michaelis constant for arginine. PEGylation did not increase serum stability of SpyADI in vitro, but led to a longer-lasting reduction of plasma arginine levels in mice. Furthermore, SpyADI-PEG20 showed a higher antitumoral capacity towards GBM cells in vitro than the native enzyme. Key points • PEGylation has no effect on the affinity of SpyADI for arginine • PEGylation increases the antitumoral effects of SpyADI on GBM in vitro • PEGylation prolongs plasma arginine depletion by SpyADI in mice


Author(s):  
Daniel Sandín ◽  
Javier Valle ◽  
Belén Chaves-Arquero ◽  
Guillem Prats-Ejarque ◽  
María Nieves Larrosa ◽  
...  

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Samira Komijani ◽  
Elham Bayat ◽  
Elham Rismani ◽  
Soma Hosseini ◽  
Reza Moazzami ◽  
...  

AbstractPseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni–NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.


Pharmaceutics ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 333
Author(s):  
Azmat Ali Khan ◽  
Amer M. Alanazi ◽  
Nawaf Alsaif ◽  
Nasser Algrain ◽  
Tanveer Ahmad Wani ◽  
...  

Candida albicans is the most studied species for the candidiasis infection and is becoming resistant towards existing antifungal drugs. Considering this, in the current study, we developed and characterized a fibrin liposome-based formulation encapsulating a novel thiosemicarbazone derivative, 2C, and evaluated its antifungal efficacy against murine candidiasis. The 2C-containing formulation was prepared by encapsulating 2C within the liposomes (2C-L) that were further encapsulated in the fibrin beads (2C-FL). The in-house synthesized 2C-FLs were spherical with a zeta potential of -34.12 ± 0.3 mV, an entrapment efficiency of 72.6 ± 4.7%, and a loading efficiency of 9.21 ± 2.3%, and they showed a slow and sustained release of 2C. Compared to free 2C, the formulation was non-toxic and exhibited serum stability, increased tissue specificity, and penetration. The 2C-FL formulation had a minimum inhibitory concentration (MIC) value of 4.92 ± 0.76 µg/mL and was able to induce apoptosis and necrosis in C. albicans in vitro. The administration of 2C-FL in C. albicans-infected mice prolonged their survival and antifungal effects when compared with the free 2C. The 2C-FL antifungal therapy significantly reduced the fungal burden and displayed an improved survival rate. In conclusion, the 2C thiosemicarbazone derivative possesses a potent antifungal activity that became more advantageous upon its encapsulation in the fibrin liposome delivery system.


Pharmaceutics ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 87
Author(s):  
Jaehong Park ◽  
Kyusik Kim ◽  
Sohee Jeong ◽  
Migyeom Lee ◽  
Tae-il Kim

In this work, highly osmotic oxidized sucrose-crosslinked polyethylenimine (SP2K) polymers were developed for gene delivery systems, and the transfection mechanism is examined. First, periodate-oxidized sucrose and polyethylenimine 2K (PEI2K) were crosslinked with various feed ratios via reductive amination. The synthesis was confirmed by 1H NMR and FTIR. The synthesized SP2K polymers could form positively charged (~40 mV zeta-potential) and nano-sized (150–200 nm) spherical polyplexes with plasmid DNA (pDNA). They showed lower cytotoxicity than PEI25K but concentration-dependent cytotoxicity. Among them, SP2K7 and SP2K10 showed higher transfection efficiency than PEI25K in both serum and serum-free conditions, revealing the good serum stability. It was found that SP2K polymers possessed high osmolality and endosome buffering capacity. The transfection experiments with cellular uptake inhibitors suggest that the transfection of SP2K polymers would progress by multiple pathways, including caveolae-mediated endocytosis. It was also thought that caveolae-mediated endocytosis of SP2K polyplexes would be facilitated through cyclooxygenase-2 (COX-2) expression induced by high osmotic pressure of SP2K polymers. Confocal microscopy results also supported that SP2K polyplexes would be internalized into cells via multiple pathways and escape endosomes efficiently via high osmolality and endosome buffering capacity. These results demonstrate the potential of SP2K polymers for gene delivery systems.


2021 ◽  
Vol 8 ◽  
Author(s):  
Na Wang ◽  
Jing Shi ◽  
Cong Wu ◽  
Weiwei Chu ◽  
Wanru Tao ◽  
...  

Objective: The aim of this study was to construct light and temperature dual-sensitive micellar carriers loaded with doxorubicin (DOX) and gold nanorods (DOX-GNRs-PNIPAM@PEG-PLA, DAPP) for melanoma therapy.Methods: The DAPP self-assembled using fine-tuned physicochemical properties in water. The DAPP structure, temperature- and photo-sensitivity, drug-release, in-vitro serum stability, and cytotoxicity against melanoma B16F10 cells were evaluated in detail. The corresponding in-vitro and in-vivo therapeutic mechanisms were then evaluated using a B16F10-melanoma bearing BALB/c nude mouse model (B16F10).Results: The light and temperature sensitive micellar drug-delivery system assembled from block copolymer and gold nanorods exhibited a narrow particle size and size distribution, good biocompatibility, well-designed photo-temperature conversion, controlled drug release, and high serum stability. Compared with the free DOX- and PBS-treated groups, the cell endocytosis-mediated cytotoxicity and intra-tumor accumulation of DAPP was markedly enhanced by the NIR-light exposure and induced potent in-vivo tumor inhibitory activity.Conclusion: The design of DAPP, a dual-functional micellar drug-delivery system with temperature- and light-sensitive properties, offers a new strategy for skin-cancer therapy with a potent therapeutic index.


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