Excellent Adsorption of Lead (II) and Chromium (VI) from Water Using Zwitterions (-NH3+ and -COO-) Functionalized Nano Lanthanum Oxide: Kinetic, Isotherm, Thermodynamic, and Surface Mechanism.

Zwitterion amino acid l-cysteine functionalized lanthanum oxide nanoparticles (l-Cyst-La2O3 NPs) have been synthesized for the first time with lanthanum acetate as the precursor, NH4OH as the base, and l-cysteine as the in situ functionalized mediator. The typical size of l-Cyst-La2O3 NPs was obtained in the range of 15-20 nm from the TEM technique. A cytotoxicity test of l-Cyst-La2O3 NPs was performed in Raw 264.7 cell lines, which were shown to be highly biocompatible. The point zero charge pH (pHPZC) of bare and l-Cyst functionalized La2O3 NPs was obtained at pH 6 and 2. The maximum uptake capacities of l-Cyst-La2O3 NPs at temperatures 25-45 °C were obtained as 137-282 mg/g for Pb2+ and 186-256 mg/g for Cr6+. All of these values are much higher than those reported in the literature with other nanomaterials. The presence of -SH, -NH2, and -COOH functional groups in zwitterion l-cysteine provides multiple binding sites leading to the high adsorption of Pb2+ and Cr6+. Five-cycle desorption studies were successfully performed to regenerate the spent l-Cyst-La2O3 NPs.

Maternal vitamin B12 in mice positively regulates bone, but not muscle mass and strength in post-weaning and mature offspring.

Vitamin B12 deficiency has been shown to affect bone mass in rodents and negatively impact bone formation in humans. In this study using mouse models, we define the effect of B12 supplementation in the wild-type mother and B12 deficiency in a mouse genetic model (Gif-/- mice) during gestation on bone and muscle architecture and mechanical properties in the offspring. Analysis of bones from 4-wk-old offspring of the wild-type mother following vehicle or B12 supplementation during gestation (from embryonic day 0.5 to 20.5) showed an increase in bone mass caused by an isolated increase in bone formation in the B12-supplemented group compared with vehicle controls. Analysis of the effect of B12 deficiency in the mother in a mouse genetic model (Gif-/- mice) on the long bone architecture of the offspring showed a compromised cortical and trabecular bone mass, which was completely prevented by a single injection of B12 in the B12-deficient Gif-/- mothers. Biomechanical analysis of long bones of the offspring born from B12-supplemented wild-type mothers showed an increase in bone strength, and conversely, offspring born from B12-deficient Gif-/- mothers revealed a compromised bone strength, which could be rescued by a single injection of B12 in the B12-deficient Gif-/- mother. Muscle structure and function analysis however revealed no significant effect on muscle mass, structure, and grip strength of B12 deficiency or supplementation in Gif-/- mice compared with littermate controls. Together, these results demonstrate the beneficial effect of maternally derived B12 in the regulation of bone structure and function in the offspring.

Evaluation of size, shape, and charge effect on the biological interaction and cellular uptake of cerium oxide nanostructures.

Cerium oxide (CeO2) at the nanoscale has prolifically attracted the immense interest of researchers due to its switchable oxidation states (Ce3+/Ce4+) that play a crucial role in many biological activities. The present work reports the evaluation of size, shape, and charge effect on the biological interaction with RAW 264.7 cells for three nanostructures of CeO2(CeO2NS) namely nanocubes (NCs), nanorods (NRs), and nanoparticles (NPs). These NS exhibits similar composition and have average diameter values in the order of NCs < NRs â‰… NPs. The values of zeta potential revealed the anionic nature of NS with surface charge in order of NCs < NPs < NRs. The cellular interaction of CeO2NS was analyzed for cytotoxicity, cellular uptake, and morphological studies. Quantitative determination of the uptake of CeO2NS exhibited concentration-dependent uptake in the order as NCs > NPs > NRs. The proposed possible mechanisms of cellular uptake revealed that different structures tended to use the various endocytosis pathways in different proportions.

Co-delivery of gemcitabine and simvastatin through PLGA polymeric nanoparticles for the treatment of pancreatic cancer: in-vitro characterization, cellular uptake, and pharmacokinetic studies.

Despite the ongoing extensive research, cancer therapeutics still remains an area with unmet needs which is hampered by shortfall in the development of newer medicines. The present study discusses a nano-based combinational approach for treating solid tumor. Dual-loaded nanoparticles encapsulating gemcitabine HCl (GM) and simvastatin (SV) were fabricated by double emulsion solvent evaporation method and optimized. Optimized nanoparticles showed a particle size of 258 ± 2.4 nm, polydispersity index of 0.32 ± 0.052, and zeta potential of -12.5 mV. The size and the morphology of the particles wee further confirmed by transmission electron microscopy (TEM) and scanning electron microscopy, respectively of the particles. The entrapment efficiency of GM and SV in the nanoparticles was 38.5 ± 4.5% and 72.2 ± 5.6%, respectively. The in vitro release profile was studied for 60 h and showed Higuchi release pattern. The cell toxicity was done using MTT assay and lower IC50 was obtained with the nanoparticles as compared to the pure drug. The bioavailability of GM and SV in PLGA nanoparticles was enhanced by 1.4-fold and 1.3-fold respectively, compared to drug solution. The results revealed that co-delivery of GM and SV could be used for its oral delivery for the effective treatment of pancreatic cancer.

Inhalable Particles for "Pincer Therapeutics" Targeting Nitazoxanide as Bactericidal and Host-Directed Agent to Macrophages in a Mouse Model of Tuberculosis.

Nitazoxanide (NTZ) has moderate mycobactericidal activity and is also an inducer of autophagy in mammalian cells. High-payload (40-50% w/w) inhalable particles containing NTZ alone or in combination with antituberculosis (TB) agents isoniazid (INH) and rifabutin (RFB) were prepared with high incorporation efficiency of 92%. In vitro drug release was corrected for drug degradation during the course of study and revealed first-order controlled release. Particles were efficiently taken up in vitro by macrophages and maintained intracellular drug concentrations at one order of magnitude higher than NTZ in solution for 6 h. Dose-dependent killing of Mtb and restoration of lung and spleen architecture were observed in experimentally infected mice treated with inhalations containing NTZ. Adjunct NTZ with INH and RFB cleared culturable bacteria from the lung and spleen and markedly healed tissue architecture. NTZ can be used in combination with INH-RFB to kill the pathogen and heal the host.

Purification of recombinant ovalbumin from inclusion bodies of Escherichia coli.

Recombinant ovalbumin expressed in bacterial host is essentially free from post-translational modifications and can be useful in understanding the structure-function relationship of the protein. In this study, ovalbumin was expressed in Escherichia coli in the form of inclusion bodies. Ovalbumin inclusion bodies were solubilized using urea and refolded by decreasing the urea concentration by dilution. Refolded protein was purified by anion exchange chromatography. Overall recovery of purified recombinant ovalbumin from inclusion bodies was about 30% with 98% purity. Purified recombinant ovalbumin was characterized by mass spectrometry, circular dichroism and fluorescence spectroscopy. Recombinant ovalbumin was shown to be resistant to trypsin using protease resistance assay. This indicated proper refolding of ovalbumin from inclusion bodies of E. coli. This method provides a simple way of producing ovalbumin free of post-translational modifications.

Recovery of bioactive protein from bacterial inclusion bodies using trifluoroethanol as solubilization agent.

BACKGROUND: Formation of inclusion bodies poses a major hurdle in recovery of bioactive recombinant protein from Escherichia coli. Urea and guanidine hydrochloride have routinely been used to solubilize inclusion body proteins, but many times result in poor recovery of bioactive protein. High pH buffers, detergents and organic solvents like n-propanol have been successfully used as mild solubilization agents for high throughput recovery of bioactive protein from bacterial inclusion bodies. These mild solubilization agents preserve native-like secondary structures of proteins in inclusion body aggregates and result in improved recovery of bioactive protein as compared to conventional solubilization agents. Here we demonstrate solubilization of human growth hormone inclusion body aggregates using 30% trifluoroethanol in presence of 3 M urea and its refolding into bioactive form. RESULTS: Human growth hormone was expressed in E. coli M15 (pREP) cells in the form of inclusion bodies. Different concentrations of trifluoroethanol with or without addition of low concentration (3 M) of urea were used for solubilization of inclusion body aggregates. Thirty percent trifluoroethanol in combination with 3 M urea was found to be suitable for efficient solubilization of human growth hormone inclusion bodies. Solubilized protein was refolded by dilution and purified by anion exchange and size exclusion chromatography. Purified protein was analyzed for secondary and tertiary structure using different spectroscopic tools and was found to be bioactive by cell proliferation assay. To understand the mechanism of action of trifluoroethanol, secondary and tertiary structure of human growth hormone in trifluoroethanol was compared to that in presence of other denaturants like urea and guanidine hydrochloride. Trifluoroethanol was found to be stabilizing the secondary structure and destabilizing the tertiary structure of protein. Finally, it was observed that trifluoroethanol can be used to solubilize inclusion bodies of a number of proteins. CONCLUSIONS: Trifluoroethanol was found to be a suitable mild solubilization agent for bacterial inclusion bodies. Fully functional, bioactive human growth hormone was recovered in high yield from inclusion bodies using trifluoroethanol based solubilization buffer. It was also observed that trifluoroethanol has potential to solubilize inclusion bodies of different proteins.

Preparation and Preclinical Evaluation of Inhalable Particles Containing Rapamycin and Anti-Tuberculosis Agents for Induction of Autophagy.

PURPOSE: Mycobacterium tuberculosis (Mtb) inhibits host defense mechanisms, including autophagy. We investigated particles containing rapamycin (RAP) alone or in combination with isoniazid (INH) and rifabutin (RFB) for: targeting lung macrophages on inhalation; inducing autophagy; and killing macrophage-resident Mtb and/or augmenting anti-tuberculosis (TB) drugs. METHODS: PLGA and drugs were spray-dried. Pharmacokinetics, partial biodistribution (LC-MS/MS) and efficacy (colony forming units, qPCR, acid fast staining, histopathology) in mice following dry powder inhalation were evaluated. RESULTS: Aerodynamic diameters of formulations were 0.7-4.7 µm. Inhaled particles reached deep lungs and were phagocytosed by alveolar macrophages, yielding AUC0-48 of 102 compared to 0.1 µg/ml × h obtained with equivalent intravenous dose. RAP particles induced more autophagy in Mtb-infected macrophages than solutions. Inhaled particles containing RAP alone in daily, alternate-day and weekly dosing regimens reduced bacterial burden in lungs and spleens, inducing autophagy and phagosome-lysosome fusion. Inhalation of particles containing RAP with INH and RFB cleared the lungs and spleens of culturable bacteria. CONCLUSIONS: Targeting a potent autophagy-inducing agent to airway and lung macrophages alone is feasible, but not sufficient to eliminate Mtb. Combination of macrophage-targeted inhaled RAP with classical anti-TB drugs contributes to restoring tissue architecture and killing Mtb.

Self-assembling tryptophan-based designer peptides as intracellular delivery vehicles.

A series of tryptophan-based peptides W1a, b-W4a, b, with diverse architectures were designed and synthesized. These tryptophan containing peptides can self-assemble to spherical particle. This self-assembled system was demonstrated to encapsulate rhodamine B and penetrate the cell membrane.

Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3).

Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with specific activity ∼3194IU/mg and ∼1493IU/mg were obtained from soluble and inclusion bodies (IBs) fraction with recovery of 56% and 74% in terms of activity, respectively. The recombinant enzyme was moderately thermostable (t1/2 60min at 50°C) and optimally active in wider alkaline pH range (7.0-10.5). Interaction of protein with its cofactor CaCl2 was found to stimulate the change in tertiary structure as revealed by near UV CD spectra. Intrinsic tryptophan fluorescence spectra indicated that tryptophan is involved in substrate binding and there might be independent binding of Ca(2+) and polygalacturonic acid to the active site. The recombinant enzyme was found to be capable of degrading pectin and polygalacturonic acid. The work reports novel conditions for refolding to obtain active recombinant pectate lyase from inclusion bodies and elucidates the effect of ligand and substrate binding on protein conformation by circular dichroism (CD) and fluorescence spectrofluorometry.

Development and characterization of itraconazole-loaded solid lipid nanoparticles for ocular delivery.

The purpose of this study was to investigate the feasibility of entrapping water-insoluble drug itraconazole into solid lipid nanoparticles (SLNs) for topical ocular delivery. The drug-loaded SLNs were prepared from stearic acid and palmitic acid using different concentrations of polyvinyl alcohol employed as emulsifier. SLNs were prepared by the melt-emulsion sonication and low temperature-solidification method and characterized for particle size, zeta potential, drug loading and drug entrapment efficiency. The mean particle size of SLNs prepared with stearic acid ranged from 139 to 199 nm, while the SLNs prepared with palmitic acid had particle size in the range of 126-160 nm. The SLNs were spherical in shape. Stearic acid-SLNs showed higher entrapment of drug compared with palmitic acid-SLNs. Differential scanning calorimetry (DSC) and X-ray diffraction measurements showed decrease in crystallinity of drug in the SLN formulations. The modified Franz-diffusion cell and freshly excised goat corneas were used to test drug corneal permeability. Permeation of itraconazole from stearic acid-SLNs was higher than that obtained with palmitic acid-SLNs. The SLNs showed clear zone of inhibition against Aspergillus flavus indicating antimicrobial efficacy of formulations.

Fusion and self-assembly of biodegradable polymer particles into scaffoldlike and membranelike structures at room temperature for regenerative medicine.

We report here a novel surfactant mediated fusion of polylactide particles into scaffoldlike structures at room temperature. In the presence of ethanol, evenly spread surfactant coated polylactide particles fused immediately into membranelike structures. Polymer scaffolds of the desired shape and size could be fabricated from polylactide particles using this fusion process. Desorption of surfactant molecules from the surface of the particles during ethanol treatment and the degree of solubility of the polymer in alcohol were found to be the main reasons for the fusion of particles into a scaffold at room temperature. TGA and DSC studies of the polylactide particles showed that the particles were stable at room temperature, and FTIR studies showed that there was no change in characteristics of the polymer after the fusion of particles into a scaffold-type structure. These scaffolds supported three-dimensional growth of animal cells in vitro and release model protein in a sustained manner for a long period of time. In an experimental animal wound model, the polylactide membranes showed faster wound closure, indicating its use as a passive dressing material. This polymer particle fusion process thus provides a novel method of scaffold fabrication for various biomedical applications.

Development of enteric submicron particles formulation of α-amylase for oral delivery.

Enteric submicron particles (SPs) formulations of α-amylase were prepared by w/o/w emulsion solvent evaporation using hydroxypropyl methylcellulose phthalate (HPMCP) and Eudragit L 100, to avoid gastric inactivation of α-amylase. Smaller internal and external aqueous phase volume provided maximum encapsulation efficiency (71.92-73.40%), least particle size (546.4-595.4 nm) and 23-26% loss of enzyme activity. Release studies in 0.1 N HCl confirmed the gastro-resistance of formulations. The anionic SPs aggregated in 0.1 N HCl (i.e. gastric pH 1.2), due to protonation of carboxylic groups of enteric polymer. The aggregates being < 500 µm size would not impede gastric emptying. However, at pH >5.0 (duodenal pH), SPs showed de-aggregation due to restoration of surface charge. HPMCP and Eudragit L 100 SPs facilitated almost complete release of α-amylase within 30 min at pH 6.0 and 6.8, respectively, following Higuchi kinetics. PXRD and DSC indicated amorphous character and scanning electron microscope showed spherical shape of SPs. In simulated gastro-intestinal pH condition, HPMCP and Eudragit L 100 SPs showed good digestion of cooked rice and could serve as potential carrier for oral enzyme delivery. Stability studies indicated the formulations as quite stable to ensure 2 years shelf life at room temperature.

Immunogenicity Evaluation of Thermostable Microparticles Entrapping Receptor Binding Domain of SARS-CoV-2 by Single Point Administration.

Receptor binding domain (RBD) of SARS-CoV-2 is a prime vaccine target against which neutralizing antibody responses are directed. Purified RBD as a vaccine candidate warrants administration of multiple doses along with adjuvants and use of delivery systems to improve its immunogenicity. The present investigation examines the immunogenicity of RBD delivered by biodegradable polymer particles from single dose administration. Mice upon single point immunization of RBD entrapped microparticles generated improved antibody response. The polymer microparticles showed better temperature stability and could be stored at 37 degrees for one month without any considerable loss of immunogenicity. Further, immunization with microparticles could elicit memory antibody response upon challenge after four months of single dose administration. Thus, using microparticles entrapping RBD as a vaccine candidate confer improved immunogenicity, temperature stability and recall response. These thermostable microparticles seem to be a potentially cost-effective approach which can help in dose reduction, provide a wider access of vaccines and accelerate the end of global pandemic.

Electrochemical Detection of Vibrio cholerae by Amine Functionalized Biocompatible Gadolinium Oxide Nanoparticles.

Herein, we report the biocompatible amine-functionalized gadolinium oxide nanoparticles (Gd2O3 NPs) for the possibility of electrochemical detection of Vibrio cholerae (Vc) cells. The microwave irradiation process is applied to synthesize Gd2O3 NPs. The amine (NH2) functionalization is carried out via overnight stirring with 3(Aminopropyl)triethoxysilane (APTES) at 55 °C. The size of NPs amine functionalized APETS@Gd2O3 NPs are determined by transmission electron microscopy (TEM). APETS@Gd2O3 NPs are further electrophoretically deposited onto indium tin oxide (ITO) coated glass substrate to obtain working electrode surface. The monoclonal antibodies (anti-CT) specific to cholera toxin associated to Vc cells are covalently immobilized onto the above electrodes using EDC-NHS chemistry and further BSA is added to obtain the BSA/anti-CT/APETS@Gd2O3/ITO immunoelectrode. Further, this immunoelectrode shows the response for cells in CFU range from 3.125 × 106 to 30 × 106 and is very selective with sensitivity and LOD 5.07 mA CFUs mL cm-2 and 0.9375 × 106 CFU respectively. To establish a future potential for APTES@Gd2O3 NPs in field of biomedical applications and cytosensing, the effect of APTES@Gd2O3 NPs on mammalian cells is also observed using in vitro cytotoxicity assay and cell cycle analysis.

Solubilization of inclusion body proteins using n-propanol and its refolding into bioactive form.

Inclusion bodies of recombinant human growth hormone (r-hGH) were isolated from Escherichia coli, enriched and solubilized in 100mM Tris buffer containing 6M n-propanol and 2M urea. Around 4 mg/ml of r-hGH from inclusion bodies were solubilized in 6M n-propanol-based buffer containing 2M urea. Existence of native-like secondary structure of r-hGH in 6M n-propanol solution was confirmed by CD and fluorescence spectra. Solubilized r-hGH was subsequently refolded by pulsatile dilution, purified to homogeneity and found to be functionally active. Tris buffer containing 6M n-propanol and 2M urea also effectively solubilized a number of proteins expressed as inclusion bodies in E. coli. Mild solubilization of inclusion body proteins, chaotropic effect of n-propanol at high concentration and kosmotropic effect at lower concentration helped in improved refolding of the solubilized protein. Around 40% of the r-hGH in the form of inclusion body aggregates was refolded into bioactive form while using n-propanol as solubilization agent. Solubilization with 6M n-propanol solution thus can be a viable alternative for achieving high throughput recovery of bioactive protein from inclusion bodies of E. coli.

Solubilization and Refolding of Inclusion Body Proteins.

Expression of heterologous proteins in E. coli often leads to the formation of protein aggregates known as inclusion bodies (IBs). Inclusion body aggregates pose a major hurdle in the recovery of bioactive proteins from E. coli. Usage of strong denaturing buffers for solubilization of bacterial IBs results in poor recovery of bioactive protein. Structure-function understanding of IBs in the last two decades have led to the development of several mild solubilization buffers, which improve the recovery of bioactive from IBs. Recently, combinatorial mild solubilization methods have paved the way for solubilization of wide range of inclusion bodies with appreciable refolding yield. Here, we describe a simple protocol for solubilization and refolding of an inclusion body protein with appreciable recovery.

Microparticles entrapping pneumococcal protein SP0845 show improved immunogenicity and temperature stability.

Protein based vaccines are the most safe and affordable strategy to combat pneumococcal disease circumventing the limitations of conventional polysaccharide-based vaccines like serotype dependence, high cost and inability to be administered to immunocompromised. SP0845 is a highly conserved vaccine candidate shown to provide protection against heterologous strains of Streptococcus pneumoniae, the primal cause of pneumonia. However, the associated poor immunogenicity warrants the need for adjuvants and multiple doses to mount desired responses. The present study relates to improve the immunogenicity of pneumococcal protein SP0845 by use of poly lactic acid biodegradable polymer microparticles. The immunization studies showed that microparticles elicited higher antibody response compared to alum adjuvanted protein and this immunopotentiation was achieved without the use of any additional adjuvant. They were also capable of eliciting secondary antibody response upon boosting after four months. Further, the particles upon storage at 25 and 37 °C for one month were still capable of mounting an immune response equivalent to those stored in cold chain. Thus, using microparticles entrapping SP0845 for immunization not only improve the immunogenicity but also offer better temperature stability. This can greatly reduce the cost and increase access of protein-based vaccine to resource limited settings.

Contraceptive efficacy of recombinant porcine zona proteins and fusion protein encompassing canine ZP3 fragment and GnRH in female beagle dogs.

PROBLEM: To manage population of dogs (Canis familiaris), the efficacy of recombinant proteins-based contraceptive vaccines to inhibit fertility has been evaluated in female beagle dogs. METHOD OF STUDY: Female beagle dogs (n = 4) were immunized with physical mixture of Escherichia coli-expressed recombinant porcine ZP3 with promiscuous T cell epitope of tetanus toxoid (TT-KK-pZP3) and porcine ZP4 with promiscuous T cell epitope of bovine RNase (bRNase-KK-pZP4), or with a fusion protein encompassing dog ZP3 fragment and two copies of GnRH with appropriate promiscuous T cell epitopes (dZP3-GnRH2 ); control animals received only alum, the adjuvant. The immunized animals were followed-up for antibody titres by ELISA as well as for fertility status subsequent to mating with male dogs. RESULTS: Active immunization of female dogs following a three injections schedule at 4-week intervals with a physical mixture of TT-KK-pZP3 + bRNase-KK-pZP4 as well as dZP3-GnRH2 , led to generation of significant antibody titres against respective recombinant proteins. Active immunization with dZP3-GnRH2 also led to generation of antibodies reactive with both dZP3 and GnRH. A booster dose on day 383 led to an increase in antibody titres and circulating antibodies against respective recombinant proteins could be observed on day 528. Antibodies in immune serum samples from dogs immunized with TT-KK-pZP3 + bRNase-KK-pZP4 or dZP3-GnRH2 reacted with native canine ZP as assessed by an indirect immunofluorescence assay. Mating studies revealed a reduced number of pregnancies as well as a significant reduction in the number of pups born in the female dogs immunized with dZP3-GnRH2 as compared to the adjuvanted control. Curtailment of pregnancy in dZP3-GnRH2 immunized group was associated with antibody titres against dZP3-GnRH2 . However, immunization with recombinant TT-KK-pZP3 + bRNase-KK-pZP4 did not significantly decrease the number of pups born as compared to the adjuvanted control. CONCLUSION: These studies revealed the potential of recombinant dZP3-GnRH2 -based contraceptive vaccine to curtail fertility in female dogs. Large scale studies to establish the efficacy and safety of this recombinant protein for the management of community dog population are thus warranted.