scholarly journals Optimization of layering technique and secondary structure analysis during the formulation of nanoparticles containing lysozyme by quality by design approach

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260603
Author(s):  
Katalin Kristó ◽  
Reihaneh Manteghi ◽  
Yousif H-E. Y. Ibrahim ◽  
Ditta Ungor ◽  
Edit Csapó ◽  
...  
Keyword(s):  
Particle Size ◽  
Enzyme Activity ◽  
Secondary Structure ◽  
Process Parameters ◽  
Encapsulation Efficiency ◽  
Quality By Design ◽  
Mixing Time ◽  
Shell Nanoparticles ◽  
Optimization Parameters ◽  
Α Helix

In our study, core-shell nanoparticles containing lysozyme were formulated with precipitation and layering self-assembly. Factorial design (DoE) was applied by setting the process parameters during the preparation with Quality by Design (QbD) approach. The factors were the concentration of lysozyme and sodium alginate, and pH. Our aim was to understand the effect of process parameters through the determination of mathematical equations, based on which the optimization parameters can be predicted under different process parameters. The optimization parameters were encapsulation efficiency, particle size, enzyme activity and the amount of α-helix structure. The nanoparticles were analysed with transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD) spectroscopy. Based on our results, we found that pH was the most important factor and pH 10 was recommended during the formulation. Enzyme activity and α-helix content correlated with each other very well, and particle size and encapsulation efficiency also showed very good correlation with each other. The results of the α-helix content of FTIR and CD measurements were very similar for the precipitated lysozyme due to the solid state of lysozyme. The mixing time had the best influence on the encapsulation efficiency and the particle size, which leads to the conclusion that a mixing time of 1 h is recommended. The novelty in our study is the presentation of a mathematical model with which the secondary structure of the protein and other optimization parameters can be controlled in the future during development of nanoparticle based on the process parameters.

Systematic Development and Optimization of an in-situ Gelling System for Moxifloxacin Ocular Nanosuspension using High-pressure Homogenization with an Improved Encapsulation Efficiency

2018 ◽  
Vol 24 (13) ◽  
pp. 1434-1445 ◽  
Author(s):  
Lalit Kumar Khurana ◽  
Romi Singh ◽  
Harinder Singh ◽  
Manju Sharma
Keyword(s):  
High Pressure ◽  
Process Parameters ◽  
Encapsulation Efficiency ◽  
Quality By Design ◽  
Polynomial Equation ◽  
Single Type ◽  
Application Site ◽  
High Pressure Homogenization ◽  
In Situ Gelling

Background: The objective of this study was to apply Quality by Design (QbD) principles on process parameter optimization for the development of hybrid delivery system (combination of (SLNs) and In-situ gelling system) for hydrophilic drug Moxifloxacin Hydrochloride (MOX) to achieve its controlled delivery, which otherwise may not be possible through single type of technology. Methods: Risk assessment studies were carried out to identify probable risks influencing CQAs on the product. In design of experiments (DoE), the process parameters (independent variables) i.e., chiller temperature X1, High Pressure Homogenization (HPH) pressure X2, and HPH cycles X3 were optimized using a three-factor two level face-centered central composite design to streamline the influence on three responses, namely encapsulation efficiency Y1, particle size Y2 and outlet temperature Y3. Independent and dependent variables were analyzed to establish a full-model second-order polynomial equation. F value is used to confirm the omission of insignificant parameters/interactions to derive a reduced-model polynomial equation to predict the Y1, Y2 and Y3 for optimized moxifloxacin in situ gelled nanosuspension. Results: Desirability plots showed the effects of X1, X2, and X3 on Y1, Y2 and Y3, respectively. The design space is generated to obtain optimized process parameters viz. chiller temperature (-5°C), HPH pressure 800 – 900 bar and 8 cycles that resulted in nanosuspension with ≈ 500 nm size, encapsulation efficiency >65% and final formulation temperature <23°C that were necessary to maintain the formulation in a liquid state. Conclusion: Quality by Design (QbD) approach is recently been encouraged by regulatory bodies to improve the quality of the finished product. This approach proved to be a useful tool in the development of robust nanosuspension of highly hydrophilic drugs with improved efficiency. Results indicate that such hybrid gel systems can be used to control the release of SLNs from application site and prolong their action in a sustained manner.

scholarly journals Caffeic Acid Phenethyl Ester Loaded PLGA Nanoparticles: Effect of Various Process Parameters on Reaction Yield, Encapsulation Efficiency, and Particle Size

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Serap Derman
Keyword(s):  
Particle Size ◽  
Process Parameters ◽  
Encapsulation Efficiency ◽  
Spherical Shape ◽  
Processing Parameters ◽  
Caffeic Acid Phenethyl Ester ◽  
Plga Nanoparticles ◽  
Reaction Yield ◽  
Uniform Size

CAPE loaded PLGA nanoparticles were prepared using the oil in water (o/w) single emulsion solvent evaporation methods. Five different processing parameters including initial CAPE amount, initial PLGA amount, PVA concentration in aqueous phase, PVA volume, and solvent type were screened systematically to improve encapsulation of hydrophobic CAPE molecule, simultaneously minimize particle size, and raise the reaction yield. Obtained results showed that the encapsulation efficiency of the nanoparticles significantly increased with the increase of the initial CAPE amount (p<0.05) and particle size (p<0.05). Furthermore, the particle size is significantly influenced by initial polymer amount (p<0.05) and surfactant concentration (p<0.05). By the optimization of process parameters, the nanoparticles produced70±6% reaction yield,89±3% encapsulation efficiency,-34.4±2.5 mV zeta potential, and163±2 nm particle size with low polydispersity index0.119±0.002. The particle size and surface morphology of optimized nanoparticles were studied and analyses showed that the nanoparticles have uniform size distribution, smooth surface, and spherical shape. Lyophilized nanoparticles with different CAPE and PLGA concentration in formulation were examined forin vitrorelease at physiological pH. Interestingly, the optimized nanoparticles showed a high (83.08%) and sustained CAPE release (lasting for 16 days) compared to nonoptimized nanoparticle.

Effect of Temperature and pH on the Secondary Structure and Denaturation Process of Jumbo Squid Hepatopancreas Cathepsin D.

2019 ◽  
Vol 26 (7) ◽  
pp. 532-541 ◽  
Author(s):  
Cadena-Cadena Francisco ◽  
Cárdenas-López José Luis ◽  
Ezquerra-Brauer Josafat Marina ◽  
Cinco-Moroyoqui Francisco Javier ◽  
López-Zavala Alonso Alexis ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Secondary Structure ◽  
Cathepsin D ◽  
Thermal Denaturation ◽  
Protein Denaturation ◽  
Marine Organisms ◽  
Effect Of Temperature ◽  
Jumbo Squid ◽  
Α Helix ◽  
Circular Dichroïsm

Background: Cathepsin D is a lysosomal enzyme that is found in all organisms acting in protein turnover, in humans it is present in some types of carcinomas, and it has a high activity in Parkinson's disease and a low activity in Alzheimer disease. In marine organisms, most of the research has been limited to corroborate the presence of this enzyme. It is known that cathepsin D of some marine organisms has a low thermostability and that it has the ability to have activity at very acidic pH. Cathepsin D of the Jumbo squid (Dosidicus gigas) hepatopancreas was purified and partially characterized. The secondary structure of these enzymes is highly conserved so the role of temperature and pH in the secondary structure and in protein denaturation is of great importance in the study of enzymes. The secondary structure of cathepsin D from jumbo squid hepatopancreas was determined by means of circular dichroism spectroscopy. Objective: In this article, our purpose was to determine the secondary structure of the enzyme and how it is affected by subjecting it to different temperature and pH conditions. Methods: Circular dichroism technique was used to measure the modifications of the secondary structure of cathepsin D when subjected to different treatments. The methodology consisted in dissecting the hepatopancreas of squid and freeze drying it. Then a crude extract was prepared by mixing 1: 1 hepatopancreas with assay buffer, the purification was in two steps; the first step consisted of using an ultrafiltration membrane with a molecular cut of 50 kDa, and the second step, a pepstatin agarose resin was used to purification the enzyme. Once the enzyme was purified, the purity was corroborated with SDS PAGE electrophoresis, isoelectric point and zymogram. Circular dichroism is carried out by placing the sample with a concentration of 0.125 mg / mL in a 3 mL quartz cell. The results were obtained in mdeg (millidegrees) and transformed to mean ellipticity per residue, using 111 g/mol molecular weight/residue as average. Secondary-structure estimation from the far-UV CD spectra was calculated using K2D Dichroweb software. Results: It was found that α helix decreases at temperatures above 50 °C and above pH 4. Heating the enzyme above 70°C maintains a low percentage of α helix and increases β sheet. Far-UV CD measurements of cathepsin D showed irreversible thermal denaturation. The process was strongly dependent on the heating rate, accompanied by a process of oligomerization of the protein that appears when the sample is heated, and maintained a certain time at this temperature. An amount typically between 3 and 4% α helix of their secondary structure remains unchanged. It is consistent with an unfolding process kinetically controlled due to the presence of an irreversible reaction. The secondary structure depends on pH, and a pH above 4 causes α helix structures to be modified. Conclusion: In conclusion, cathepsin D from jumbo squid hepatopancreas showed retaining up to 4% α helix at 80°C. The thermal denaturation of cathepsin D at pH 3.5 is under kinetic control and follows an irreversible model.

Optimization and Characterization of Aqueous Micellar Formulations for Ocular Delivery of an Antifungal Drug, Posaconazole

2020 ◽  
Vol 26 (14) ◽  
pp. 1543-1555 ◽  
Author(s):  
Meltem E. Durgun ◽  
Emine Kahraman ◽  
Sevgi Güngör ◽  
Yıldız Özsoy
Keyword(s):  
Particle Size ◽  
Zeta Potential ◽  
Size Distribution ◽  
Encapsulation Efficiency ◽  
Fungal Infections ◽  
In Vitro Release ◽  
Pluronic F127 ◽  
Glycol Succinate ◽  
Vitro Release

Background: Topical therapy is preferred for the management of ocular fungal infections due to its superiorities which include overcoming potential systemic side effects risk of drugs, and targeting of drugs to the site of disease. However, the optimization of effective ocular formulations has always been a major challenge due to restrictions of ocular barriers and physiological conditions. Posaconazole, an antifungal and highly lipophilic agent with broad-spectrum, has been used topically as off-label in the treatment of ocular fungal infections due to its highly lipophilic character. Micellar carriers have the potential to improve the solubility of lipophilic drugs and, overcome ocular barriers. Objective: In the current study, it was aimed optimization of posaconazole loaded micellar formulations to improve aqueous solubility of posaconazole and to characterize the formulations and to investigate the physical stability of these formulations at room temperature (25°C, 60% RH), and accelerated stability (40°C, 75% RH) conditions. Method: Micelles were prepared using a thin-film hydration method. Pre-formulation studies were firstly performed to optimize polymer/surfactant type and to determine their concentration in the formulations. Then, particle size, size distribution, and zeta potential of the micellar formulations were measured by ZetaSizer Nano-ZS. The drug encapsulation efficiency of the micelles was quantified by HPLC. The morphology of the micelles was depicted by AFM. The stability of optimized micelles was evaluated in terms of particle size, size distribution, zeta potential, drug amount and pH for 180 days. In vitro release studies were performed using Franz diffusion cells. Results: Pre-formulation studies indicated that single D-ɑ-tocopheryl polyethylene glycol succinate (TPGS), a combination of it and Pluronic F127/Pluronic F68 are capable of formation of posaconazole loaded micelles at specific concentrations. Optimized micelles with high encapsulation efficiency were less than 20 nm, approximately neutral, stable, and in aspherical shape. Additionally, in vitro release data showed that the release of posaconazole from the micelles was higher than that of suspension. Conclusion: The results revealed that the optimized micellar formulation of posaconazole offers a potential approach for topical ocular administration.

scholarly journals The Study of Carbon Recovery from Electrolysis Aluminum Carbon Dust by Froth Flotation

Metals ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 145
Author(s):  
Hesong Li ◽  
Jiaoru Wang ◽  
Wenyuan Hou ◽  
Mao Li ◽  
Benjun Cheng ◽  
...  
Keyword(s):  
Particle Size ◽  
Particle Size Distribution ◽  
Size Distribution ◽  
Mixing Time ◽  
Froth Flotation ◽  
Aluminum Electrolysis ◽  
Carbon Powder ◽  
Molten Salt Electrolysis ◽  
Carbon Recovery ◽  
Aluminum Smelting

A large amount of carbon dust is generated in the process of aluminum smelting by molten salt electrolysis. The carbon dust is solid hazardous waste but contains a large quantity of recyclable components such as carbon and fluoride. How to recycle carbon dust more effectively is a challenge in the aluminum electrolysis field. In this study, X-ray diffraction, scanning electron microscope, and other methods were used to analyze the phase composition of electrolytic aluminum carbon dust. The effects of particle size distribution of carbon dust, impeller speed, reagent addition, mixing time, and flotation time on the flotation recovery of carbon dust were studied. The optimal flotation conditions were obtained and the flotation products were analyzed. The results show that the optimal particle size distribution is 70% of particles below 200 mesh, corresponding to a grinding time of 11 min. The optimum speed of the flotation machine was to be between 1600 and 1800 r/min with the best slurry concentration of 20–30% and 5 min mixing time, and the collector kerosene was suitable for adding in batches. Under the above conditions, the recovered carbon powder with a carbon content of 75.6% was obtained, and the carbon recovery rate was 86.9%.

scholarly journals Synthesis and Advanced Characterization of Polymer-Protein Core-Shell Nanoparticles

Catalysts ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 730
Author(s):  
Erik Sarnello ◽  
Tao Li
Keyword(s):  
Particle Size ◽  
Small Angle ◽  
Core Shell ◽  
Assembly Process ◽  
Shell Nanoparticles ◽  
X Ray ◽  
X Ray Scattering ◽  
Wide Range ◽  
Core Shell Nanoparticles ◽  
Ray Scattering

Enzyme immobilization techniques are widely researched due to their wide range of applications. Polymer–protein core–shell nanoparticles (CSNPs) have emerged as a promising technique for enzyme/protein immobilization via a self-assembly process. Based on the desired application, different sizes and distribution of the polymer–protein CSNPs may be required. This work systematically studies the assembly process of poly(4-vinyl pyridine) and bovine serum albumin CSNPs. Average particle size was controlled by varying the concentrations of each reagent. Particle size and size distributions were monitored by dynamic light scattering, ultra-small-angle X-ray scattering, small-angle X-ray scattering and transmission electron microscopy. Results showed a wide range of CSNPs could be assembled ranging from an average radius as small as 52.3 nm, to particles above 1 µm by adjusting reagent concentrations. In situ X-ray scattering techniques monitored particle assembly as a function of time showing the initial particle growth followed by a decrease in particle size as they reach equilibrium. The results outline a general strategy that can be applied to other CSNP systems to better control particle size and distribution for various applications.

scholarly journals 708 Application of a novel mSENS drug delivery technology for mRNA therapeutics

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A750-A750
Author(s):  
Sojin Lee ◽  
Joon Young Park ◽  
Goo-Young Kim ◽  
Sang Woo Jo ◽  
Minhyuk Yun ◽  
...  
Keyword(s):  
Particle Size ◽  
Protein Expression ◽  
Cancer Vaccine ◽  
Encapsulation Efficiency ◽  
Tumor Model ◽  
Cell Responses ◽  
Delivery Platform ◽  
Mrna Therapeutics

BackgroundSuccessful clinical translation of mRNA therapeutics requires an appropriate delivery strategy to overcome instability of mRNA and facilitate cellular uptake into the cells.1 Several lipid based nanoparticle approaches that encapsulate mRNA, notably lipid nanoparticle (LNP), have been developed, but their efficiency for delivery to certain target tissues and toxicity profiles still have room for improvement. The application of a novel polymer based nanoparticle technology platform, so called Stability Enhanced Nano Shells (SENS) for mRNA (mSENS) as a mRNA delivery platform for a cancer vaccine was demonstrated.MethodsThe physicochemical properties of mSENS formulation, particle size and encapsulation efficiency, were characterized using dynamic light scattering (DLS) and gel retardation assay. Using luciferase-encoding mRNA, the protein expression levels in vitro and in vivo were evaluated by luciferase assay or bioluminescence imaging (BLI), respectively. For cancer vaccine studies, antigen (tyrosinase-related protein 2 (Trp-2))-specific T cell responses were assessed by immunophenotyping mouse splenocytes using flow cytometry and by the enzyme-linked immunosorbent spot (ELISPOT) assay. The anti-tumor efficacy was studied in B16F10 lung tumor model in C57BL/6 mice. Liver and systemic toxicity of mSENS treated mice was evaluated through blood chemistry and complete blood count (CBC) tests.ResultsA library of mSENS formulations complexed with luciferase-encoding mRNA, were characterized for their particle size, surface charge, encapsulation efficiency, colloidal stability, and in vitro and in vivo luciferase protein expression level. Upon systemic administration in mice, varying biodistribution profiles were observed, implicating the potential for tailored delivery to target tissues. Particularly, cancer vaccine application was further developed leveraging the formulation with preferential spleen delivery. Following vaccination with Trp-2 mRNA encapsulated with mSENS (Trp-2 mRNA-mSENS) in B16F10 tumor bearing mice, strong Trp-2 antigen-specific IFN-γ T-cell responses were observed. Generated anti-tumor immunity also marked suppression of B16F10 lung tumors were observed in Trp-2-mSENS immunized mice compared to non-immunized controls, demonstrating the potential of mSENS as a mRNA delivery platform for the application for vaccine.ConclusionsProprietary biodegradable polymer based-mSENS platform offers an attractive delivery strategy for mRNA by tailoring to specific therapeutic applications. Depending on the application, whether it’s a vaccine or protein replacement, a rationally designed mSENS formulation can efficiently distribute mRNA to specific tissues. In particular, application of a splenic mSENS formulation for a cancer vaccine has been demonstrated in murine tumor model. In summary, mRNA delivery through mSENS platform is expected to provide significant opportunities in clinical development for mRNA therapeutics.Ethics ApprovalThe study was approved by Samyang Biopharmaceuticals’ IACUC (Institutional Animal Care and Use Committee), approval number SYAU-2027.ReferencePiotr S. Kowalski, Arnab Rudra, Lei Miao, and Daniel G. Anderson, delivering the messenger: advances in technologies for therapeutic mRNA delivery. Molecular Therapy Vol. 27 No 4 April 2019.

scholarly journals Liposomes Loaded with Unsaponifiable Matter from Amaranthus hypochondriacus as a Source of Squalene and Carrying Soybean Lunasin Inhibited Melanoma Cells

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1960
Author(s):  
Erick Damian Castañeda-Reyes ◽  
Elvira Gonzalez de Mejia ◽  
Fred Joseph Eller ◽  
Mark A. Berhow ◽  
María de Jesús Perea-Flores ◽  
...  
Keyword(s):  
Particle Size ◽  
Unsaponifiable Matter ◽  
Encapsulation Efficiency ◽  
Inhibitory Concentration ◽  
Melanoma Cells ◽  
Amaranthus Hypochondriacus ◽  
Liposome Formulation ◽  
Reported Health ◽  
Amaranth Oil ◽  
Melanoma Cell Lines

Amaranthus hypochondriacus is a source of molecules with reported health benefits such as antioxidant activity and cancer prevention. The objective of this research was to optimize the conditions for preparing a liposome formulation using amaranth unsaponifiable matter as a source of squalene in order to minimize the particle size and to maximize the encapsulation efficiency of liposomes for carrying and delivering soybean lunasin into melanoma cell lines. Amaranth oil was extracted using supercritical dioxide carbon extraction (55.2 MPa pressure, 80 °C temperature, solvent (CO2)-to-feed (oil) ratio of 20). The extracted oil from amaranth was used to obtain the unsaponifiable enriched content of squalene, which was incorporated into liposomes. A Box–Behnken response surface methodology design was used to optimize the liposome formulation containing the unsaponifiable matter, once liposomes were optimized. Soybean lunasin was loaded into the liposomes and tested on A-375 and B16-F10 melanoma cells. The squalene concentration in the extracted oil was 36.64 ± 0.64 g/ 100 g of oil. The particle size in liposomes was between 115.8 and 163.1 nm; the squalene encapsulation efficiency ranged from 33.14% to 76.08%. The optimized liposome formulation contained 15.27 mg of phospholipids and 1.1 mg of unsaponifiable matter. Cell viability was affected by the liposome formulation with a half-maximum inhibitory concentration (IC50) equivalent to 225 μM in B16-F10 and 215 μM in A-375. The liposomes formulated with lunasin achieved 82.14 ± 3.34% lunasin encapsulation efficiency and improved efficacy by decreasing lunasin IC50 by 31.81% in B16-F10 and by 41.89% in A-375 compared with unencapsulated lunasin.

scholarly journals Development and Stability Studies of Novel Liposomal Vancomycin Formulations

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Krishna Muppidi ◽  
Andrew S. Pumerantz ◽  
Jeffrey Wang ◽  
Guru Betageri
Keyword(s):  
Particle Size ◽  
Encapsulation Efficiency ◽  
Antimicrobial Agents ◽  
Preparation Method ◽  
Physical Stability ◽  
Liposomal Formulations ◽  
Promising Strategy ◽  
Activity Variable ◽  
Distribution Studies

A promising strategy to improve the therapeutic efficiency of antimicrobial agents is targeted therapy. Although vancomycin has been considered a gold standard for the therapy of MRSA pneumonia, clinical failure rates have also been reported owing to its slow, time-dependent bactericidal activity, variable lung tissue penetration and poor intracellular penetration into macrophages. Liposomal encapsulation has been established as an alternative for antimicrobial delivery to infected tissue macrophages and offers enhanced pharmacodynamics, pharmacokinetics and decreased toxicity compared to standard preparations. The aim of the present work is to prepare vancomycin in two different liposomal formulations, conventional and PEGylated liposomes using different methods. The prepared formulations were optimized for their particle size, encapsulation efficiency and physical stability. The dehydration-rehydration was found to be the best preparation method. Both the conventional and PEGylated liposomal formulations were successfully formulated with a narrow particle size and size distribution and % encapsulation efficiency of and , respectively. Both the formulations were stable at C for 3 months. These formulations were successfully used to evaluate for their intracellular killing of MRSA and in vivo pharmacokinetic and bio-distribution studies.

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