Cationic dextrin nanoparticles for effective intracellular delivery of cytochrome C in cancer therapy.

Intracellular protein delivery shows promise as a selective and specific approach to cancer therapy. However, a major challenge is posed by delivering proteins into the target cells. Despite the development of nanoparticle (NP)-based approaches, a versatile and biocompatible delivery system that can deliver active therapeutic cargo into the cytosol while escaping endosome degradation remains elusive. In order to overcome these challenges, a polymeric nanocarrier was prepared using cationic dextrin (CD), a biocompatible and biodegradable polymer, to encapsulate and deliver cytochrome C (Cyt C), a therapeutic protein. The challenge of endosomal escape of the nanoparticles was addressed by co-delivering the synthesized NP construct with chloroquine, which enhances the endosomal escape of the therapeutic protein. No toxicity was observed for both CD NPs and chloroquine at the concentration tested in this study. Spectroscopic investigations confirmed that the delivered protein, Cyt C, was structurally and functionally active. Additionally, the delivered Cyt C was able to induce apoptosis by causing depolarization of the mitochondrial membrane in HeLa cells, as evidenced by flow cytometry and microscopic observations. Our findings demonstrate that an engineered delivery system using CD NPs is a promising platform in nanomedicine for protein delivery applications.

Nucleic Acid Delivery Nanotechnologies for In Vivo Cell Programming.

In medicine, it is desirable for clinicians to be able to restore function and imbue novel function into selected cells for therapy and disease prevention. Cells damaged by disease, injury, or aging could be programmed to restore normal or lost functions, such as retinal cells in inherited blindness and neuronal cells in Alzheimer's disease. Cells could also be genetically programmed with novel functions such as immune cells expressing synthetic chimeric antigen receptors for immunotherapy. Furthermore, knockdown or modification of risk factor proteins can mitigate disease development. Currently, nucleic acids are emerging as a versatile and potent therapeutic modality for achieving this cellular programming. In this review, we highlight the latest developments in nanobiomaterials-based nucleic acid therapeutics for cellular programming from a biomaterial design and delivery perspective and how to overcome barriers to their clinical translation to benefit patients.

MoS2 nanosheets effectively bind to the receptor binding domain of the SARS-CoV-2 spike protein and destabilize the spike-human ACE2 receptor interactions.

The use of nanotechnology is becoming increasingly significant as a tool that can provide a range of options for the identification, inactivation, and therapy of coronavirus disease 2019 (COVID-19). The potential of nanoparticles as an alternative therapeutic agent to inactivate SARS-CoV-2 is continually being investigated. Herein, we have explored the interaction of 2D molybdenum disulfide (MoS2) nanosheets with the SARS-CoV-2 spike protein, human ACE2 receptor and the complex formed between them through molecular docking and atomistic simulations. The results indicated that MoS2 nanosheets can effectively bind to the receptor binding domain (RBD) of the spike protein with good docking energies. It is interesting to note that this also applied to the extensively glycosylated spike protein and its variations, Kappa and Delta. A significant loss of secondary structures was observed when MoS2 nanosheets interacted with the RBD of the spike protein. The nanosheets interacted strongly with the proteins through a number of hydrogen bonds and van der Waals interactions. Moreover, the binding of the MoS2 nanosheets at different locations of the RBD or ACE2 in the spike-RBD/ACE2 complex resulted in significant conformational changes. Detailed energetics and solvent accessibility calculations revealed that, when present at the interface, MoS2 nanosheets can be a potential inhibitory agent. The findings were supported by de-wetting calculations, indicating strong adherence of the RBD of spike protein on the MoS2 nanosheet and de-stability of the spike-ACE2 interaction. Thus, the findings clearly demonstrate the antiviral potential of 2D MoS2 nanosheets, prompting its further exploration for combating COVID-19.

Antimicrobial mechanisms of biomaterials: from macro to nano.

Overcoming the global concern of antibiotic resistance is one of the biggest challenges faced by scientists today, and the key to tackling this issue of emerging infectious diseases is the development of next-generation antimicrobials. The rapid emergence of multi-drug resistant microbes, superbugs and mutated strains of viruses has fuelled the search for new and alternative antimicrobial agents with broad-spectrum biocidal activity. Biomaterials, ranging from macroscopic polymers, proteins, and peptides to nanoscale materials such as nanoparticles, nanotubes and nanosheets have emerged as effective antimicrobials. An extensive body of research has established the antibacterial and antiviral efficiencies of different types of biomaterials. What make these materials unique are the different modes through which they interact and exert their antimicrobial activity. This review provides a comprehensive and detailed overview of the diverse modes of interaction between biomaterials and bacteria and viruses, and sheds light on how different biomaterials influence and modulate antimicrobial mechanisms to achieve a high degree of therapeutic efficacy without resistance generation.

An oxidant and organic solvent tolerant alkaline lipase by P. aeruginosa mutant: downstream processing and biochemical characterization

An extracellular alkaline lipase from Pseudomonas aeruginosa mutant has been purified to homogeneity using acetone precipitation followed by anion exchange and gel filtration chromatography and resulted in 27-fold purification with 19.6% final recovery. SDS-PAGE study suggested that the purified lipase has an apparent molecular mass of 67 kDa. The optimum temperature and pH for the purified lipase were 45°C and 8.0, respectively. The enzyme showed considerable stability in pH range of 7.0-11.0 and temperature range 35-55 °C. The metal ions Ca2+, Mg2+ and Na+ tend to increase the enzyme activity, whereas, Fe2+ and Mn2+ ions resulted in discreet decrease in the activity. Divalent cations Ca+2 and Mg+2 seemed to protect the enzyme against thermal denaturation at high temperatures and in presence of Ca+2 (5 mM) the optimum temperature shifted from 45°C to 55°C. The purified lipase displayed significant stability in the presence of several hydrophilic and hydrophobic organic solvents (25%, v/v) up to 168 h. The pure enzyme preparation exhibited significant stability and compatibility with oxidizing agents and commercial detergents as it retained 40-70% of its original activities. The values of Km and Vmax for p-nitrophenyl palmitate (p-NPP) under optimal conditions were determined to be 2.0 mg.mL-1 and 5000 μg.mL-1.min-1, respectively.

Computation of interactive effects and optimization of process parameters for alkaline lipase production by mutant strain of Pseudomonas aeruginosa using response surface methodology.

Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100). The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L(-1); starch, 15.0 g.L(-1); triton-X-100, 0.93 mL.L(-1); incubation temperature, 34.12 °C and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL(-1)). The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R(2)) value (0.9987). The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization.

Simultaneous production of alkaline lipase and protease by antibiotic and heavy metal tolerant Pseudomonas aeruginosa.

An efficient bacterial strain capable of simultaneous production of lipase and protease in a single production medium was isolated. Thirty six bacterial strains, isolated from diverse habitats, were screened for their lipolytic and proteolytic activity. Of these, only one bacterial strain was found to be lipase and protease producer. The 16S rDNA sequencing and phylogenetic analyses revealed that strain (NSD-09) was in close identity to Pseudomonas aeruginosa. The maximum lipase (221.4 U/ml) and protease (187.9 U/ml) activities were obtained after 28 and 24 h of incubation, respectively at pH 9.0 and 37 °C. Castor oil and wheat bran were found to be the best substrate for lipase and protease production, respectively. The strain also exhibited high tolerance to lead (1450 µg/ml) and chromium (1000 µg/ml) in agar plates. It also showed tolerance to other heavy metals, such as Co(+2) , Zn(+2) , Hg(+2) , Ni(+2) and Cd(+2) . Therefore, this strain has scope for tailing bioremediation. Presumably, this is the first attempt on P. aeruginosa to explore its potential for both industrial and environmental applications.

An oxidant and organic solvent tolerant alkaline lipase by P. aeruginosa mutant: downstream processing and biochemical characterization.

An extracellular alkaline lipase from Pseudomonas aeruginosa mutant has been purified to homogeneity using acetone precipitation followed by anion exchange and gel filtration chromatography and resulted in 27-fold purification with 19.6% final recovery. SDS-PAGE study suggested that the purified lipase has an apparent molecular mass of 67 kDa. The optimum temperature and pH for the purified lipase were 45 °C and 8.0, respectively. The enzyme showed considerable stability in pH range of 7.0-11.0 and temperature range 35-55 °C. The metal ions Ca(2+), Mg(2+) and Na(+) tend to increase the enzyme activity, whereas, Fe(2+) and Mn(2+) ions resulted in discreet decrease in the activity. Divalent cations Ca(+2) and Mg(+2) seemed to protect the enzyme against thermal denaturation at high temperatures and in presence of Ca(+2) (5 mM) the optimum temperature shifted from 45 °C to 55 °C. The purified lipase displayed significant stability in the presence of several hydrophilic and hydrophobic organic solvents (25%, v/v) up to 168 h. The pure enzyme preparation exhibited significant stability and compatibility with oxidizing agents and commercial detergents as it retained 40-70% of its original activities. The values of K(m) and Vmax for p-nitrophenyl palmitate (p-NPP) under optimal conditions were determined to be 2.0 mg.mL(-1) and 5000 µg.mL(-1).min(-1), respectively.

Computation of interactive effects and optimization of process parameters for alkaline lipase production by mutant strain of Pseudomonas aeruginosa using response surface methodology

Alkaline lipase production by mutant strain of Pseudomonas aeruginosa MTCC 10,055 was optimized in shake flask batch fermentation using response surface methodology. An empirical model was developed through Box-Behnken experimental design to describe the relationship among tested variables (pH, temperature, castor oil, starch and triton-X-100). The second-order quadratic model determined the optimum conditions as castor oil, 1.77 mL.L-1; starch, 15.0 g.L-1; triton-X-100, 0.93 mL.L-1; incubation temperature, 34.12 ºC and pH 8.1 resulting into maximum alkaline lipase production (3142.57 U.mL-1). The quadratic model was in satisfactory adjustment with the experimental data as evidenced by a high coefficient of determination (R²) value (0.9987). The RSM facilitated the analysis and interpretation of experimental data to ascertain the optimum conditions of the variables for the process and recognized the contribution of individual variables to assess the response under optimal conditions. Hence Box-Behnken approach could fruitfully be applied for process optimization.