Lactoferrin/pectin nanocomplex encapsulating ciprofloxacin and naringin as a lung targeting antibacterial nanoplatform with oxidative stress alleviating effect.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium with adaptive metabolic abilities. It can cause hospital-acquired infections with significant mortality rates, particularly in people with already existing medical conditions. Its ability to develop resistance to common antibiotics makes managing this type of infections very challenging. Furthermore, oxidative stress is a common consequence of bacterial infection and antibiotic therapy, due to formation of reactive oxygen species (ROS) during their mode of action. In this study we aimed to alleviate oxidative stress and enhance the antibacterial efficacy of ciprofloxacin (CPR) antibiotic by its co-encapsulation with naringin (NAR) within a polyelectrolyte complex (PEX). The PEX comprised of polycationic lactoferrin (LF) and polyanionic pectin (PEC). CPR/NAR-loaded PEX exhibited spherical shape with particle size of 237 ± 3.5 nm, negatively charged zeta potential (-23 ± 2.2 mV) and EE% of 61.2 ± 4.9 for CPR and 76.2 ± 3.4 % for NAR. The LF/PEC complex showed prolonged sequential release profile of CPR to limit bacterial expansion, followed by slow liberation of NAR, which mitigates excess ROS produced by CPR's mechanism of action without affecting its efficacy. Interestingly, this PEX demonstrated good hemocompatibility with no significant in vivo toxicity regarding hepatic and renal functions. In addition, infected mice administrated this nanoplatform intravenously exhibited significant CFU reduction in the lungs and kidneys, along with reduced immunoreactivity against myeloperoxidase. Moreover, this PEX was found to reduce the lungs´ oxidative stress via increasing both glutathione (GSH) and catalase (CAT) levels while lowering malondialdehyde (MDA). In conclusion, CPR/NAR-loaded PEX can offer a promising targeted lung delivery strategy while enhancing the therapeutic outcomes of CPR with reduced oxidative stress.

Prodigiosin/celecoxib-loaded into zein/sodium caseinate nanoparticles as a potential therapy for triple negative breast cancer.

Nowadays, breast cancer is considered one of the most upsetting malignancies among females. Encapsulation of celecoxib (CXB) and prodigiosin (PDG) into zein/sodium caseinate nanoparticles (NPs) produce homogenous and spherical nanoparticles with good encapsulation efficiencies (EE %) and bioavailability. In vitro cytotoxicity study conducted on human breast cancer MDA-MB-231 cell lines revealed that there was a significant decline in the IC50 for encapsulated drugs when compared to each drug alone or their free combination. In addition, results demonstrated that there is a synergism between CXB and PDG as their combination indices were 0.62251 and 0.15493, respectively. Moreover, results of scratch wound healing assay revealed enhanced antimigratory effect of free drugs and fabricated NPs in comparison to untreated cells. Furthermore, In vitro results manifested that formulated nanoparticles exhibited induction of apoptosis associated with reduced angiogenesis, proliferation, and inflammation. In conclusion, nanoencapsulation of multiple drugs into nanoparticles might be a promising approach to develop new therapies for the managing of triple negative breast cancer.

Carbon dots labeled Lactiplantibacillus plantarum: a fluorescent multifunctional biocarrier for anticancer drug delivery.

A carbon dots (CDs)-biolabeled heat-inactivated Lactiplantibacillus plantarum (HILP) hybrid was investigated as a multifunctional probiotic drug carrier with bioimaging properties using prodigiosin (PG) as anticancer agent. HILP, CDs and PG were prepared and characterized using standard methods. CDs-labeled HILP (CDs/HILP) and PG loaded CDs/HILP were characterized by transmission electron microscopy (TEM), laser scanning confocal microscopy (LSCM) and for entrapment efficiency (EE%) of CDs and PG, respectively. PG-CDs/HILP was examined for stability and PG release. the anticancer activity of PG-CDs/HILP was assessed using different methods. CDs imparted green fluorescence to HILP cells and induced their aggregation. HILP internalized CDs via membrane proteins, forming a biostructure with retained fluorescence in PBS for 3 months at 4°C. Loading PG into CDs/HILP generated a stable green/red bicolor fluorescent combination permitting tracking of both drug carrier and cargo. Cytotoxicity assay using Caco-2 and A549 cells revealed enhanced PG activity by CDs/HILP. LCSM imaging of PG-CDs/HILP-treated Caco-2 cells demonstrated improved cytoplasmic and nuclear distribution of PG and nuclear delivery of CDs. CDs/HILP promoted PG-induced late apoptosis of Caco-2 cells and reduced their migratory ability as affirmed by flow cytometry and scratch assay, respectively. Molecular docking indicated PG interaction with mitogenic molecules involved in cell proliferation and growth regulation. Thus, CDs/HILP offers great promise as an innovative multifunctional nanobiotechnological biocarrier for anticancer drug delivery. This hybrid delivery vehicle merges the physiological activity, cytocompatibility, biotargetability and sustainability of probiotics and the bioimaging and therapeutic potential of CDs.

Nano zinc oxide-functionalized nanofibrous microspheres: A bioactive hybrid platform with antimicrobial, regenerative and hemostatic activities.

Bioactive hybrid constructs are at the cutting edge of innovative biomaterials. PLA nanofibrous microspheres (NF-MS) were functionalized with zinc oxide nanoparticles (nZnO) and DDAB-modified nZnO (D-nZnO) for developing inorganic/nano-microparticulate hybrid constructs (nZnO@NF-MS and D-nZnO@NF-MS) merging antibacterial, regenerative, and haemostatic functionalities. The hybrids appeared as three-dimensional NF-MS frameworks made-up entirely of interconnecting nanofibers embedding nZnO or D-nZnO. Both systems achieved faster release of Zn2+ than their respective nanoparticles and D-nZnO@NF-MS exhibited significantly greater surface wettability than nZnO@NF-MS. Regarding bioactivity, D-nZnO@NF-MS displayed a significantly greater and fast-killing effect against Staphylococcus aureus. Both nZnO@NF-MS and D-nZnO@NF-MS showed controllable concentration-dependent cytotoxicity to human gingival fibroblasts (HGF) compared with pristine NF-MS. They were also more effective than pristine NF-MS in promoting migration of human gingival fibroblasts (HGF) in the in vitro wound healing assay. Although D-nZnO@NF-MS showed greater in vitro hemostatic activity than nZnO@NF-MS (blood-clotting index 22.82 ± 0.65% vs.54.67 ± 2.32%), both structures exhibited instant hemostasis (0 s) with no blood loss (0 mg) in the rat-tail cutting technique. By merging the multiple therapeutic bioactivities of D-nZnO and the 3D-structural properties of NF-MS, the innovative D-nZnO@NF-MS hybrid construct provides a versatile bioactive material platform for different biomedical applications.

A molecular study on recombinant pullulanase type I from Metabacillus indicus.

Despite the great potential of cold-adapted pullulanase type I in tremendous industrial applications, the majority of commercialized pullulnases type I are of mesophilic and thermophilic origin so far. Hence, the present study underlines cloning, heterologous expression in Escherichia coli, characterization, and in silico structural modeling of Metabacillus indicus open reading frame of cold-adapted pullulanase type I (Pull_Met: 2133 bp & 710 a.a) for the first time ever. The predicted Pull_Met tertiary structure by I-TASSER, was structurally similar to PDB 2E9B pullulanase of Bacillus subtilis. Purified to homogeneity Pull_Met showed specific activity (667.6 U/mg), fold purification (31.7), molecular mass (79.1 kDa), monomeric subunit and Km (2.63 mg/mL) on pullulan. Pull_Met had optimal pH (6.0) and temperature (40 oC). After 10 h pre-incubation at pH 2.6-6.0, Pull_Met maintained 47.12 ± 0.0-35.28 ± 1.64% of its activity. After 120 min pre-incubation at 30 oC, the retained activity was 51.11 ± 0.29%. At 10 mM Mn2+, Na2+, Ca2+, Mg2+, and Cu2+ after 30 min preincubation, retained activity was 155.89 ± 8.97, 134.71 ± 1.82, 97.64 ± 7.06, 92.25 ± 4.18, and 71.28 ± 1.10%, respectively. After 30 min pre-incubation with Tween-80, Tween-20, Triton X-100, and commercially laundry detergents at 0.1% (v/v), the retained activity was 141.15 ± 3.50, 145.45 ± 0.20, 118.12 ± 11.00, and 90%, respectively. Maltotriose was the only end product of pullulan hydrolysis. Synergistic action of CA-AM21 (α-amylase) and Pull_Met on starch liberated 16.51 g reducing sugars /g starch after 1 h at 40 oC. Present data (cold-adeptness, detergent stability, and ability to exhibit starch saccharification of Pull_Met) underpins it as a promising pullulanase type I for industrial exploitation.

Prodigiosin-Functionalized Probiotic Ghosts as a Bioinspired Combination Against Colorectal Cancer Cells.

Lactobacillus acidophilus ghosts (LAGs) with the unique safety of a probiotic, inherent tropism for colon cells, and multiple bioactivities offer promise as drug carriers for colon targeting. Our objective was to evaluate LAGs functionalized with prodigiosin (PG), apoptotic secondary bacterial metabolite, as a bioinspired formulation against colorectal cancer (CRC). LAGs were prepared by a chemical method and highly purified by density gradient centrifugation. LAGs were characterized by microscopic and staining techniques as relatively small-sized uniform vesicles (≈1.6 µm), nearly devoid of cytoplasmic and genetic materials and having a negatively charged intact envelope. PG was highly bound to LAGs envelope, generating a physiologically stable bioactive entity (PG-LAGs), as verified by multiple microscopic techniques and lack of PG release under physiological conditions. PG-LAGs were active against HCT116 CRC cells at both the cellular and molecular levels. Cell viability data highlighted the cytotoxicity of PG and LAGs and LAGs-induced enhancement of PG selectivity for HCT116 cells, anticipating dose reduction for PG and LAGs. Molecularly, expression of the apoptotic caspase 3 and P53 biomarkers in HCT116 intracellular proteins was significantly upregulated while that of the anti-apoptotic Bcl-2 (B-cell lymphoma 2) was downregulated by PG-LAGs relative to PG and 5-fluorouracil. PG-LAGs provide a novel bacteria-based combination for anticancer biomedicine.

Recombinant acetylxylan esterase of Halalkalibacterium halodurans NAH-Egypt: molecular and biochemical study.

Acetylxylan esterase plays a crucial role in xylan hydrolysis as the acetyl side-groups restrict endoxylanase action by stearic hindrance. In this study, an acetylxylan esterase (AXE-HAS10: 960 bp & 319 a.a) putative ORF from Halalkalibacterium halodurans NAH-Egypt was extensively studied through heterologous overexpression in Escherichia coli, biochemical characterization, and structural modeling. The AXE-HAS10 tertiary structure was predicted by the Local Meta Threading Server. AXE-HAS10 belongs to the carbohydrate esterase Family 7. Purified to homogeneity AXE-HAS10 showed specific activity (36.99 U/mg), fold purification (11.42), and molecular mass (41.39 kDa). AXE-HAS10 showed optimal pH (8.5) and temperature (40 oC). After 15 h of incubation at pH 7.0-9.0, AXE-HAS10 maintained 100% activity. After 120 min at 35 and 40 oC, the retained activity was 80 and 50%, respectively. At 10 mM Mn2+, Fe3+, K+, and Ca2+ after 30 min, retained activity was 329 ± 15, 212 ± 5.2, 123 ± 1.4, and 120 ± 3.0%, respectively. After 30 min of preincubation with triton x-100, SDS, and CTAB at 0.1% (v/v), the retained activity was 150 ± 19, 88 ± 4, and 82 ± 7%, respectively. At 6.0 M NaCl after 30 min, retained activity was 58%. A 1.44-fold enhancement of beechwood xylan hydrolysis was achieved by AXE-HAS10 and Penicillium chrysogenum DSM105774 ß-xylanase concurrently. Present data underpins AXE-HAS10 as a promising AXE for industrial exploitation.

Cloning, expression, and in silico structural modeling of cholesterol oxidase of Acinetobacter sp. strain RAMD in E. coli.

Cholesterol oxidases (CHOXs) are flavin-adenine dinucleotide-dependent oxidoreductases with a range of biotechnological applications. There remains an urgent need to identify novel CHOX family members to meet the demands of enzyme markets worldwide. Here, we report the cloning, heterologous expression, and structural modeling of the cholesterol oxidase of Acinetobacter sp. strain RAMD. The cholesterol oxidase gene was cloned and expressed in pGEM®-T and pET-28a(+) vectors, respectively, using a gene-specific primer based on the putative cholesterol oxidase ORF of Acinetobacter baumannii strain AB030 (GenBank [gb] locus tag: IX87_05230). The obtained nucleotide sequence (1671 bp, gb: MK575469.2), translated to a protein designated choxAB (556 amino acids), was overexpressed as inclusion bodies (IBs) (MW ˜ 62 kDa) in 1 mm IPTG-induced Escherichia coli BL21 (DE3) Rosetta cells. The optimized expression conditions (1 mm IPTG with 2% [v/v] glycerol and at room temperature) yielded soluble active choxAB of 0.45 U·mL-1 , with 56.25-fold enhancement. The recombinant choxAB was purified to homogeneity using Ni2+ -affinity agarose column with specific activity (0.054 U·mg-1 ), yield (8.1%), and fold purification (11.69). Capillary isoelectric-focusing indicated pI of 8.77 for choxAB. LC-MS/MS confirmed the IBs (62 kDa), with 82.6% of the covered sequence being exclusive to A. baumannii cholesterol oxidase (UniProtKB: A0A0E1FG24). The 3D structure of choxAB was predicted using the LOMETS webtool with the cholesterol oxidase template of Streptomyces sp. SA-COO (PDB: 2GEW). The predicted secondary structure included 18 α-helices and 12 ß-strands, a predicted catalytic triad (E220 , H380 , and N514 ), and a conserved FAD-binding sequence (GSGFGGSVSACRLTEKG). Future studies should consider fusion to solubilization tags and switching to the expression host Pichia pastoris to reduce IB formation.