Modeling and Optimizing High Pressure Liquid Chromatography (HPLC) Columns for the Separation of Biopharmaceuticals

One of the most critical steps in the production of pharmaceuticals is the separation of the desired compound from reaction byproducts and environmental contaminants. Among the most sensitive of these methods is High Pressure Liquid Chromatography (HPLC), in which an initial mixture of compounds is forced by high pressure fluid flow through a column packed with a porous solid medium. Size and charge interactions with the solid phase cause the compounds to elute at different times from the column. The performance of an HPLC column is highly dependent on properties such as the length, ambient temperature, inlet pressure, and solid medium porosity. The ideal parameters are conventionally determined by purchasing and physically testing a series of columns, which can be prohibitive in cost, time, and materials. Thus there currently exists a pressing need for computer models to simulate the separation of two or more compounds in order to expedite the onerous process of physical optimization. This study sought to simulate the physical phenomena that underlie the elution process in an HPLC column, and optimize the conditions such that species separation and purity are maximized. The computing software COMSOL was used to model the involved physics, which comprised the flow of a mobile phase through a porous matrix, modeled by the Navier-Stokes Brinkman equation; the diffusion and dispersion of two solutes in the matrix, modeled by the general mass transfer equation; and the effect of external heating on the materials’ behavior, modeled by the general heat equation. The geometry of the HPLC column consisted of an axisymmetric two-dimensional tube filled with a uniformly distributed porous matrix. This model column was evaluated by simulating the separation of creatine and creatinine, two closely-related molecules involved in muscle tissue energetics. Once the model was tailored to a high degree of accuracy in comparison with experimental data, the column and species parameters were optimized. The optimal geometry for the separation of creatine and creatinine by HPLC, was a column of diameter 1.05 mm and length 78.4 mm, with a packed bed of spherical particles 5 µm in diameter. The optimal column temperature for this particular situation was found to be lower, at 15℃, as this slightly increases peak resolution but also elution time. Though concentration plots derived from this model corroborated experimental elution absorbance plots with relatively high fidelity, lingering issues remain, including the unexpectedly small influence of temperature on elution characteristics. Future models may seek to correct this calculation error by including a less steep concentration gradient at the inlet at initial time points. Additionally, variations in column heating were found to have a very small effect on the diffusion of the solute bands, so the external temperature was excluded from the optimization process. The successful implementation of this model indicates that HPLC chromatography can be feasibly represented by computer modeling, and more specific models can reduce the time and material costs of extensive physical testing

Silica-Lipid Hybrid Microparticles as Efficient Vehicles for Enhanced Stability and Bioaccessibility of Curcumin

Kurkumin je aktivni sastojak koji ima višestruku ulogu, no njegova je uporaba ograničena zbog slabe topljivosti u vodi i stabilnosti, a time i slabe biološke raspoloživosti. Stoga je svrha ovoga rada bila osmisliti kako zaobići ta ograničenja. Postupkom emulgiranja dobivena je nanoemulzija s kurkuminom, a nakon toga sušenjem u vakuumu hibridne mikročestice nanoemulzije u silicijevom dioksidu. Udjel kurkumina u nanoemulziji bio je (0,30±0,02) %, a u mikročesticama (0,67±0,02) %. FTIR i XDR analizom utvrđeno je da je kurkumin u mikročesticama inkapsuliran u poroznom amorfnom silicijevom dioksidu. Antioksidacijska aktivnost kurkumina in vitro nije se smanjila nakon inkapsulacije. Simulacijom probave in vitro utvrđeno je da je biološka raspoloživost kurkumina u nanoemulziji i mikročesticama bila veća nego u kontrolnom uzorku. Stabilnost mikročestica ostala je ista tijekom 6 tjedana skladištenja u mraku pri temperaturama od 4, 25 i 40 °C. Osim toga, pokazalo se da su pri izlaganju svjetlosti, mikročestice imale bolju kemijsku stabilnost od nanoemulzije. Pri koncentraciji nanoemulzije manjoj od 45 μg/mL preživljavanje stanica bilo je veće od 80 %. Stoga možemo zaključiti da mikročestice mogu poslužiti kao nosači kurkumina te poboljšati njegovu topljivost, stabilnost pri izlaganju svjetlosti te biološku raspoloživost.Curcumin is an active ingredient with multiple functions, but its application is often restricted due to its poor water solubility, weak stability, and consequently low bioaccessibility. Based on this, the aim of this work is to develop a new vehicle to overcome these restrictions. Here we developed a curcumin-loaded nanoemulsion and then curcumin-loaded silica-lipid hybrid microparticles through emulsification and vacuum drying, respectively. The loading of curcumin in the nanoemulsion and microparticles was (0.30±0.02) and (0.67±0.02) %, respectively. FTIR and XRD analyses of microparticles revealed that curcumin was encapsulated in porous, amorphous silica. In vitro antioxidant activities showed that the encapsulation would not affect the antioxidant activity of curcumin. In vitro simulated digestion indicated that nanoemulsion and microparticles had higher curcumin bioaccessibility than the control group. The storage stability of microparticles remained the same during 6 weeks in the dark at 4, 25 and 40 °C. Moreover, the microparticles had a better chemical stability than nanoemulsion under the light. The cell viability was over 80 % when the concentration of nanocarriers was less than 45 μg/mL. Hence, the microparticles could be a promising means to load curcumin and improve its solubility, light stability and bioaccessibilit

Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide

Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-L-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications

Enhanced heating rate of black carbon above planetary boundary layer over megacities in summertime

The fast development of a secondary aerosol layer was observed over megacities in eastern Asia during summertime. Within three hours, from midday to early afternoon, the contribution of secondary aerosols above the planetary boundary layer (PBL) increased by a factor of 3-5, and the coatings on the black carbon (BC) also increased and enhanced its absorption efficiency by 50%. This tended to result from the intensive actinic flux received above the PBL which promoted the photochemical reactions. The absorption of BC could be further amplified by the strong reflection of solar radiation over the cloud top across the PBL. This enhanced heating effect of BC introduced by combined processes (intensive solar radiation, secondary formation and cloud reflection) may considerably increase the temperature inversion above the PBL. This mechanism should be considered when evaluating the radiative impact of BC, especially for the polluted regions receiving strong solar radiation

Engineering Patient-specific Liver Microtissues with Prolonged Phenotypic Maintenance and Disease Modeling Potential

The burden of liver diseases is increasing worldwide, accounting for two million deaths annually. In the past decade, tremendous progress has been made in the basic and translational research of liver tissue engineering, which seeks to build physiologically relevant liver models to better understand liver diseases, accelerate drug development, and advance regenerative medicine. Liver microtissues are small, three-dimensional (3D) hepatocyte cultures that recapitulate liver physiology and have been used in many biomedical applications. However, sourcing of high-quality human hepatocytes for microtissue fabrication poses a significant challenge. Since the inception of induced pluripotent stem cell (iPSC) technology, iPSC-derived hepatocyte-like cells (HLCs) have demonstrated significant improvement over other hepatocyte cell sources in many studies. Despite their promising potential, HLCs face certain challenges: they resemble fetal hepatocytes rather than adult hepatocytes; they undergo dedifferentiation quickly after reaching maturity; they are produced on a small scale; and they exhibit large donor-to-donor and batch-to-batch variability. This doctoral thesis focuses on engineering patient-specific liver microtissues with prolonged phenotypic maintenance and disease modeling potential. Chapter 1 provides a review of recent advances, challenges, and future directions in liver microtissue research. 3D microtissues can be generated by scaffold-free assembly or scaffold-assisted methods using macroencapsulation, droplet microfluidics, and bioprinting. Optimization of the hepatic microenvironment entails incorporating the appropriate cell composition for enhanced cell-cell interactions and niche-specific signals, and creating scaffolds with desired chemical, mechanical and physical properties. Perfusion-based culture systems such as bioreactors and microfluidic systems are used to achieve efficient exchange of nutrients and soluble factors in the microtissues. Chapter 2 describes our efforts in optimizing methods of generating human HLCs from the peripheral blood of selected donors. Peripheral blood mononuclear cells (PBMCs) were first reprogrammed to iPSCs using Sendai viruses carrying the four Yamanaka factors. We developed an optimized protocol for hepatocyte differentiation from iPSCs, and obtained HLCs that exhibited hepatocyte-specific phenotypes and functions that were comparable to other reports. We then demonstrated the one-step generation of homogeneous, microencapsulated liver microtissues in Chapter 3. Droplet microfluidics was used to produce double emulsion droplets that served as individual microenvironments where HLCs were encapsulated in methylated collagen and alginate. The cells self-assembled in 24 days, whereas 2D HLCs underwent dedifferentiation within 7 days of reaching maturity. The spheroids showed further maturation compared to the 2D HLCs at peak maturity. Co-culture of HLCs with human endothelial cells was also investigated in the 3D system, but no improvement was observed over monoculture spheroids with our current methods. To our knowledge, this is the first study to utilize droplet microfluidics to generate homogeneous, compartmentalized droplets that serve as optimized 3D microenvironments for HLC aggregation and maturation. It demonstrated the potential of using high-throughput droplet microfluidics to produce and encapsulate mature, functional human HLCs for long-term applications. In Chapter 4, we developed a TM6SF2 knockout and overexpression model in iPSCs to investigate its molecular function and potential role in nonalcoholic fatty liver disease (NAFLD). Transmembrane 6 superfamily member 2 (TM6SF2) is a protein of unknown function, and analysis from our model suggested that TM6SF2 dysregulation has a biphasic response. Our data showed that both knockout and overexpression can result in the upregulation of cholesterol biosynthesis and a defect in the proper processing of lipid droplets. Additionally, high expression of the TM6SF2 rs58542926 variant has an increased risk for cholesterol upregulation, compared to the major allele. Future works will focus on generating liver microtissues from the TM6SF2 knockout and transgene-expressing cells using droplet microfluidics, and validating our hypotheses with established biochemical and functional assays

Performance of Polydopamine Complex and Mechanisms in Wound Healing

Polydopamine (PDA) has been gradually applied in wound healing of various types in the last three years. Due to its rich phenol groups and unique structure, it can be combined with a variety of materials to form wound dressings that can be used for chronic infection, tissue repair in vivo and serious wound healing. PDA complex has excellent mechanical properties and self-healing properties, and it is a stable material that can be used for a long period of time. Unlike other dressings, PDA complexes can achieve both photothermal therapy and electro activity. In this paper, wound healing is divided into four stages: antibacterial, anti-inflammatory, cell adhesion and proliferation, and re-epithelialization. Photothermal therapy can improve the bacteriostatic rate and remove reactive oxygen species to inhibit inflammation. Electrical signals can stimulate cell proliferation and directional migration. With low reactive oxygen species (ROS) levels, inflammatory factors are down-regulated and growth factors are up-regulated, forming regular collagen fibers and accelerating wound healing. Finally, five potential development directions are proposed, including increasing drug loading capacity, optimization of drug delivery platforms, improvement of photothermal conversion efficiency, intelligent electroactive materials and combined 3D printing