RESUMO
Surfactin, which is composed of a ß-hydroxy fatty acid chain and a peptide ring, has drawn considerable attention due to its potential applications in the biomedicine, bioremediation, and petroleum industries. However, the low yield of surfactin from wild strains still restricts its industrial applications. In this study, eight genes relevant to the fatty acid biosynthesis pathway were targeted to enhance surfactin production, and high surfactin-yielding strains with potential industrial applications were obtained. When ldeHA and acc were co-overexpressed, the surfactin yield of recombinant strains TDS8 and TPS8 increased to 1.55- and 1.19-fold of their parental strains, respectively, again proving that the conversion of acetyl-coenzyme A (CoA) to malonyl-CoA is the rate-limiting step in fatty acid biosynthesis. Furthermore, changes in surfactin isoforms of recombinant strain TPS8 suggest that the fatty acid precursor synthesis pathway can be modified to improve the proportion of different isoforms. In addition, the deletion of lpdV, which is responsible for the conversion of α-ketoacyl-CoA precursors, resulted in a sharp decrease in surfactin production, further demonstrating the importance of branched-chain fatty acid biosynthesis in surfactin production. This work will facilitate the design and construction of more efficiently engineered strains for surfactin production and further extend industrial applications.
Assuntos
Bacillus subtilis , Ácidos Graxos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Ácidos Graxos/metabolismo , Engenharia Genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Lipopeptídeos/genética , Lipopeptídeos/metabolismo , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismoRESUMO
Background: Current immunotherapies with unexpected severe side effects and treatment resistance have not resulted in the desired outcomes for patients with melanoma, and there is a need to discover more effective medications. Cytotoxin (CTX) from Cobra Venom has been established to have favorable cytolytic activity and antitumor efficacy and is regarded as a promising novel anticancer agent. However, amphiphilic CTX with excellent anionic phosphatidylserine lipid-binding ability may also damage normal cells. Methods: We developed pH-responsive liposomes with a high CTX load (CTX@PSL) for targeted acidic-stimuli release of drugs in the tumor microenvironment. The morphology, size, zeta potential, drug-release kinetics, and preservation stability were characterized. Cell uptake, apoptosis-promoting effects, and cytotoxicity were assessed using MTT assay and flow cytometry. Finally, the tissue distribution and antitumor effects of CTX@PSL were systematically assessed using an in vivo imaging system. Results: CTX@PSL exhibited high drug entrapment efficiency, drug loading, stability, and a rapid release profile under acidic conditions. These nanoparticles, irregularly spherical in shape and small in size, can effectively accumulate at tumor sites (six times higher than free CTX) and are rapidly internalized into cancer cells (2.5-fold higher cell uptake efficiency). CTX@PSL displayed significantly stronger cytotoxicity (IC50 0.25 µg/mL) and increased apoptosis in than the other formulations (apoptosis rate 71.78±1.70%). CTX@PSL showed considerably better tumor inhibition efficacy than free CTX or conventional liposomes (tumor inhibition rate 79.78±5.93%). Conclusion: Our results suggest that CTX@PSL improves tumor-site accumulation and intracellular uptake for sustained and targeted CTX release. By combining the advantages of CTX and stimuli-responsive nanotechnology, the novel CTX@PSL nanoformulation is a promising therapeutic candidate for cancer treatment.
Assuntos
Antineoplásicos , Venenos Elapídicos , Lipossomos , Lipossomos/química , Concentração de Íons de Hidrogênio , Animais , Venenos Elapídicos/química , Venenos Elapídicos/farmacologia , Humanos , Linhagem Celular Tumoral , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Camundongos , Apoptose/efeitos dos fármacos , Liberação Controlada de Fármacos , Citotoxinas/química , Citotoxinas/farmacologia , Citotoxinas/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Distribuição Tecidual , Microambiente Tumoral/efeitos dos fármacos , Nanopartículas/químicaRESUMO
Selaginella doederleinii Hieron is a traditional Chinese medicinal herb widely used to treat different cancers. Previously, we showed that the total bioflavonoid extract of S. doederleinii (TBESD) exhibits anti-carcinogenic activities both in vitro and in vivo. However, the plasma protein binding and pharmacokinetics parameters of TBESD remain unclear. To investigate plasma protein binding, tissue distribution, and excretion of TBESD, rats were administered a single dose of TBESD (600 mg/kg) intragastrically and tissue distribution and excretion of TBESD components were determined by rapid high-performance liquid chromatography and tandem mass spectrometry. TBESD binding to human serum albumin (HSA) was assessed by fluorescence spectroscopy. TBESD components amentoflavone, delicaflavone, robustaflavone, 2â³,3â³-dihydro-3',3â´-biapigenin, and 3',3â´-binaringenin were rapidly absorbed and distributed in various tissues, mostly in the lungs, kidneys, and ovaries, without long-term accumulation. The excretion of bioflavonoids occurred mostly via the intestinal tract and constituted 30% of the administered dose up to 48 h. Spectral analysis indicated that TBESD had a dynamic quenching effect on HSA by binding to one HSA site through hydrophobic interactions and hydrogen bond formation. This is the first comprehensive report on the tissue distribution, excretion, and plasma protein binding of TBESD. This study provides important information on TBESD pharmacokinetics necessary for its further development into a therapeutic form for clinical applications.