ABSTRACT
BACKGROUND: Cancer remains a formidable global health challenge, currently affecting nearly 20 million individuals worldwide. Due to the absence of universally effective treatments, ongoing research explores diverse strategies to combat this disease. Recent efforts have concentrated on developing combined drug regimens and targeted therapeutic approaches. OBJECTIVE: This study aimed to investigate the anticancer efficacy of a conjugated drug system, consisting of doxorubicin and cisplatin (Dox-Cis), encapsulated within niosomes and modified with MUC-1 aptamers to enhance biocompatibility and target specific cancer cells. METHODS: The chemical structure of the Dox-Cis conjugate was characterized using Fourier Transform Infrared Spectroscopy (FTIR) and Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-Q-TOF/MS). The zeta potential and morphological parameters of the niosomal vesicles were determined through Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). In vitro assessments of cell viability and apoptosis were conducted on MUC-1 positive HeLa cells and MUC-1 negative U87 cells. RESULTS: The findings confirmed the successful conjugation of Dox and Cis within the niosomes. The Nio/Dox-Cis/MUC-1 formulation demonstrated enhanced efficacy compared to the individual drugs and their unencapsulated combination in both cell lines. Notably, the Nio/Dox-Cis/MUC-1 formulation exhibited greater effectiveness on HeLa cells (38.503 ± 1.407) than on U87 cells (46.653 ± 1.297). CONCLUSION: The study underscores the potential of the Dox-Cis conjugate as a promising strategy for cancer treatment, particularly through platforms that facilitate targeted drug delivery to cancer cells. This targeted approach could lead to more effective and personalized cancer therapies.
Subject(s)
Aptamers, Nucleotide , Cell Survival , Cisplatin , Doxorubicin , Liposomes , Mucin-1 , Humans , Doxorubicin/pharmacology , Doxorubicin/chemistry , Mucin-1/metabolism , Mucin-1/chemistry , Liposomes/chemistry , Cisplatin/pharmacology , Cisplatin/chemistry , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/pharmacology , Cell Survival/drug effects , Apoptosis/drug effects , Cell Line, Tumor , HeLa Cells , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Drug Compounding/methodsABSTRACT
BACKGROUND: Experimental coronary artery interventions are currently being performed on non-diseased blood vessels in healthy animals. To provide a more realistic pathoanatomical scenario for investigations on novel interventional and surgical therapies, we aimed to fabricate a stenotic lesion, mimicking the morphology and structure of a human atherosclerotic plaque. METHODS: In an interdisciplinary setting, we engineered a casting mold to create an atherosclerotic plaque with the dimensions to fit in a porcine coronary artery. Oscillatory rheology experiments took place along with long-term stability tests assessed by microscopic examination and weight monitoring. For the implantability in future in vivo setups, we performed a cytotoxicity assessment, inserted the plaque in resected pig hearts, and performed diagnostic imaging to visualize the plaque in its final position. RESULTS: The most promising composition consists of gelatin, cholesterol, phospholipids, hydroxyapatite, and fine-grained calcium carbonate. It can be inserted in the coronary artery of human-sized pig hearts, producing a local partial stenosis and interacting like the atherosclerotic plaque by stretching and shrinking with the vessel wall and surrounding tissue. CONCLUSION: This artificial atherosclerotic plaque model works as a simulating tool for future medical testing and could be crucial for further specified research on coronary artery disease and is going to help to provide information about the optimal interventional and surgical care of the disease.
ABSTRACT
We present the application of a photonic silicon chip-based optical sensor system for expeditious and phenotypic antifungal susceptibility testing. This label-free diagnostic assay optically monitors the growth of Candida auris at varying antifungal concentrations on a microwell-structured silicon chip in real-time, and antifungal susceptibility is detected within 6 h, four times faster than in the current gold standard method.
Subject(s)
Antifungal Agents , Candidiasis , Antifungal Agents/pharmacology , Candida , Candida auris , Silicon , Microbial Sensitivity TestsABSTRACT
Monoclonal antibodies are increasingly dominating the market for human therapeutic and diagnostic agents. For this reason, continuous methods-such as perfusion processes-are being explored and optimized in an ongoing effort to increase product yields. Unfortunately, many established cell retention devices-such as tangential flow filtration-rely on membranes that are prone to clogging, fouling, and undesirable product retention at high cell densities. To circumvent these problems, in this work, we have developed a 3D-printed microfluidic spiral separator for cell retention, which can readily be adapted and replaced according to process conditions (i.e., a plug-and-play system) due to the fast and flexible 3D printing technique. In addition, this system was also expanded to include automatic flushing, web-based control, and notification via a cellphone application. This set-up constitutes a proof of concept that was successful at inducing a stable process operation at a viable cell concentration of 10-17 × 106 cells/mL in a hybrid mode (with alternating cell retention and cell bleed phases) while significantly reducing both shear stress and channel blockage. In addition to increasing efficiency to nearly 100%, this microfluidic device also improved production conditions by successfully separating dead cells and cell debris and increasing cell viability within the bioreactor.
ABSTRACT
Quality by Design (QbD) is one of the most important tools for the implementation of Process Analytical Technology (PAT) in biopharmaceutical production. For optimal characterization of a monoclonal antibody (mAb) upstream process a stepwise approach was implemented. The upstream was divided into three process stages, namely inoculum expansion, production, and primary recovery, which were investigated individually. This approach enables analysis of process parameters and associated intermediate quality attributes as well as systematic knowledge transfer to subsequent process steps. Following previous research, this study focuses on the primary recovery of the mAb and thereby marks the final step toward a holistic characterization of the upstream process. Based on gained knowledge during the production process evaluation, the cell viability and density were determined as critical parameters for the primary recovery. Directed cell viability adjustment was achieved using cytotoxic camptothecin in a novel protocol. Additionally, the cell separation method was added to the Design of Experiments (DoE) as a qualitative factor and varied between filtration and centrifugation. To assess the quality attributes after cell separation, the bioactivity of the mAb was analyzed using a cell-based assay and the purity of the supernatant was evaluated by measurement of process related impurities (host cell protein proportion, residual DNA). Multivariate data analysis of the compiled data confirmed the hypothesis that the upstream process has no significant influence on the bioactivity of the mAb. Therefore, process control must be tuned towards high mAb titers and purity after the primary recovery, enabling optimal downstream processing of the product. To minimize amounts of host cell proteins and residual DNA the cell viability should be maintained above 85% and the cell density should be controlled around 15 × 106 cells/ml during the cell removal. Thereby, this study shows the importance of QbD for the characterization of the primary recovery of mAbs and highlights the useful implementation of the stepwise approach over subsequent process stages.
ABSTRACT
With antimicrobial resistance becoming a major threat to healthcare settings around the world, there is a paramount need for rapid point-of-care antimicrobial susceptibility testing (AST) diagnostics. Unfortunately, most currently available clinical AST tools are lengthy, laborious, or are simply inappropriate for point-of-care testing. Herein, we design a 3D-printed microfluidic gradient generator that automatically produces two-fold dilution series of clinically relevant antimicrobials. We first establish the compatibility of these generators for classical AST (i.e., broth microdilution) and then extend their application to include a complete on-chip label-free and phenotypic AST. This is accomplished by the integration of photonic silicon chips, which provide a preferential surface for microbial colonization and allow optical tracking of bacterial behavior and growth at a solid-liquid interface in real-time by phase shift reflectometric interference spectroscopic measurements (PRISM). Using Escherichia coli and ciprofloxacin as a model pathogen-drug combination, we successfully determine the minimum inhibitory concentration within less than 90 minutes. This gradient generator-based PRISM assay provides an integrated AST device that is viable for convenient point-of-care testing and offers a promising and most importantly, rapid alternative to current clinical practices, which extend to 8-24 h.
Subject(s)
Microfluidics , SiliconABSTRACT
Quality by Design principles are well described and widely used in biopharmaceutical industry. The characterization of a monoclonal antibody (mAb) production process is crucial for novel process development and control. Yet, the application throughout the entire upstream process was rarely demonstrated. Following previously published research, this study marks the second step toward a complete process characterization and is focused on the effect of critical process parameters on the antibody production efficiency and quality of the process. In order to conduct the complex Design of Experiments approach with optimal control and comparability, the ambr®15 micro bioreactor platform was used. Investigated parameters included the pH and dissolved oxygen set points, the initial viable cell density (iVCD) as well as the N-1 duration. Various quality attributes (e.g., growth rate, viability, mAb titer, and peak proportion) were monitored and analyzed using multivariate data analysis to evaluate the parameter effects. The pH set point and the initial VCD were identified as key process parameters with strong influence on the cell growth as well as the mAb production and its proportion to the total protein concentration. For optimization and improvement in robustness of these quality attributes the pH must be increased to 7.2, while the iVCD must be lowered to 0.2 × 106 cells/mL. Based on the defined design space, additional experiments verified the results and confirmed the intact bioactivity of the antibody. Thereby, process control strategies could be tuned toward high cell maintenance and mAb production, which enable optimal downstream processing.
ABSTRACT
In regenerative medicine, autologous peripheral blood derived endothelial colony forming cells (PB-derived ECFC) represent a promising source of endothelial cells (EC) for pre-endothelialization of arterial tissue engineered vascular grafts (TEVG) since they are readily attainable, can easily be isolated and possess a high proliferation potential. The aim of this study was to compare the phenotype of PB-derived ECFC with arterial and venous model cells such as human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) under dynamic cell culture conditions to find a suitable cell source of EC for pre-endothelialization. In this study PB-derived ECFC were cultivated over 24 h under a high pulsatile shear stress (20 dyn/cm2, 1 Hz) and subsequently analyzed. ECFC oriented and elongated in the direction of flow and expressed similar anti-thrombotic and endothelial differentiation markers compared to HAEC. There were significant differences observable in gene expression levels of CD31, CD34 and NOTCH4 between ECFC and HUVEC. These results therefore suggest an arterial phenotype for PB-derived ECFC both under static and flow conditions, and this was supported by NOTCH4 protein expression profiles. ECFC also significantly up-regulated gene expression levels of anti-thrombotic genes such as krueppel-like factor 2, endothelial nitric oxide synthase 3 and thrombomodulin under shear stress cultivation as compared to static conditions. Dynamically cultured PB-derived ECFC therefore may be a promising cell source for pre-endothelialization of arterial TEVGs.
Subject(s)
Arteries , Blood Vessel Prosthesis , Cell Culture Techniques , Cells, Cultured , Human Umbilical Vein Endothelial Cells/metabolism , HumansABSTRACT
Monoclonal antibodies (mAbs) are of great interest to the biopharmaceutical industry due to their widely used application as human therapeutic and diagnostic agents. As such, mAb require to exhibit human-like glycolization patterns. Therefore, recombinant Chinese hamster ovary (CHO) cells are the favored production organisms; many relevant biopharmaceuticals are already produced by this cell type. To optimize the mAb yield in CHO DG44 cells a corelation between stress-induced cell size expansion and increased specific productivity was investigated. CO2 and macronutrient supply of the cells during a 12-day fed-batch cultivation process were tested as stress factors. Shake flasks (500 mL) and a small-scale bioreactor system (15 mL) were used for the cultivation experiments and compared in terms of their effect on cell diameter, integral viable cell concentration (IVCC), and cell-specific productivity. The achieved stress-induced increase in cell-specific productivity of up to 94.94.9%-134.4% correlates to a cell diameter shift of up to 7.34 µm. The highest final product titer of 4 g/L was reached by glucose oversupply during the batch phase of the process.
ABSTRACT
The rapid development of nanotechnology and its applications in medicine has provided the perfect solution against a wide range of different microbes, especially antibiotic-resistant ones. In this study, a one-step approach was used in preparing silver nanoparticles (AgNPs) by mixing silver nitrate with hot Hypericum perforatum (St. John's wort) aqueous extract under high stirring to prevent agglomeration. The formation of silver nanoparticles was monitored by continuous measurement of the surface plasma resonance spectra (UV-VIS). The effect of St. John's wort aqueous extract on the formation of silver nanoparticles was evaluated and fully characterized by using different physicochemical techniques. The obtained silver nanoparticles were spherical, monodisperse, face-centered cubic (fcc) crystal structures, and the size ranges between 20 to 40 nm. They were covered with a capping layer of organic compounds considered as a nano dimension protective layer that prevents agglomeration and sedimentation. AgNPs revealed antibacterial activity against both tested Gram-positive and Gram-negative bacterial strains causing the formation of 13-32 mm inhibition zones with MIC 6.25-12.5 µg/mL; Escherichia coli strains were resistant to tested AgNPs. The specific growth rate of S. aureus was significantly reduced due to tested AgNPs at concentrations ≥½ MIC. AgNPs did not affect wound migration in fibroblast cell lines compared to control. Our results highlighted the potential use of AgNPs capped with plant extracts in the pharmaceutical and food industries to control bacterial pathogens' growth; however, further studies are required to confirm their wound healing capability and their health impact must be critically evaluated.
ABSTRACT
Hypericum perforatum Linn (St. John's wort) is a popular and widespread medicine in Syria, which is used for a wide range of conditions, including gastrointestinal diseases, heart disease, skin diseases, and psychological disorders. This widespread use prompted us to identify the main compounds of this plant from Syria that are responsible for its medicinal properties, especially since its components differ between countries according to the nature of the soil, climate, and altitude. This is, to the best of our knowledge, the first report in which St. John's wort, a plant native to Syria, is extracted using different solvents and its most important compounds are identified. In this study, the dried above-ground parts, i.e., leaves, stem, petals, and flowers, were extracted using different solvents (water, ethanol, methanol, and acetone) and extraction protocols. By increasing the polarity of the solvent, higher yields were obtained, indicating that mainly hydrophobic compounds were extracted. Therefore, we conclude that extraction using the tea method or using a mixture of water and organic solvents resulted in higher yields compared with pure organic solvents or continuous boiling with water for long periods. The obtained extracts were analyzed using high-performance liquid chromatography equipped with a diode array detector (HPLC-DAD), coupled with UV-visible spectrophotometry at a full spectrum (200-800 nm). The HPLC spectra of the extracts were almost identical at three wavelengths (260 nm for phloroglucinols (hyperforin and derivates), 590 nm for naphthodianthrones (hypericins), and 350 nm for other flavonols, flavones, and caffeoylquinic acids), with differences observed only in the intensity of the peaks. This indicates that the same compounds were obtained using different solvents, but in different amounts. Five standards (chlorogenic acid, quercetin, quercitrin hydrate, hyperoside, and hypericin) were used, and a comparison with retention times and ultraviolet (UV) spectra reported in the literature was performed to identify 10 compounds in these extracts: hyperforin, adhyperforin, hypericin, rutin, quercetin, quercitrin, quercitrin hydrate, hyperoside, biapigenin, and chlorogenic acid. Although the European Pharmacopoeia still describes ultraviolet spectroscopy as a method for determining the quantity of Hyperici herba, interference from other metabolites can occur. Combined HPLC-DAD and electrospray ionization-mass spectrometry (LC-ESI-MS) in the positive mode have therefore also been used to confirm the presence of these compounds in the extracts by correlating known masses with the identified masses or through characteristic fragmentation patterns. Total phenolic contents of the extracts were determined by the Folin-Ciocalteu assay, and antioxidant activity was evaluated as free radical scavenging capacity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The results indicate that the aqueous extracts prepared by the tea method gave the highest total phenols, while the pure organic solvents gave very low phenols. Also, the extracts that contain the largest amount of phenols gave lower IC50 values or higher antioxidant activity than that of others.
ABSTRACT
The past year has established the link between the COVID-19 pandemic and the global spread of severe fungal infections; thus, underscoring the critical need for rapid and realizable fungal disease diagnostics. While in recent years, health authorities, such as the Centers for Disease Control and Prevention, have reported the alarming emergence and spread of drug-resistant pathogenic fungi and warned against the devastating consequences, progress in the diagnosis and treatment of fungal infections is limited. Early diagnosis and patient-tailored therapy are established to be key in reducing morbidity and mortality associated with fungal (and cofungal) infections. As such, antifungal susceptibility testing (AFST) is crucial in revealing susceptibility or resistance of these pathogens and initiating correct antifungal therapy. Today, gold standard AFST methods require several days for completion, and thus this much delayed time for answer limits their clinical application. This review focuses on the advancements made in developing novel AFST techniques and discusses their implications in the context of the practiced clinical workflow. The aim of this work is to highlight the advantages and drawbacks of currently available methods and identify the main gaps hindering their progress toward clinical application.
Subject(s)
Antifungal Agents/therapeutic use , COVID-19/epidemiology , Mycoses/diagnosis , Mycoses/drug therapy , COVID-19/virology , Diagnostic Tests, Routine , Drug Resistance, Fungal , Humans , Microbial Sensitivity Tests , Mycoses/epidemiology , Mycoses/microbiology , Pandemics , SARS-CoV-2/isolation & purificationABSTRACT
The anterior gradient homologue-2 (AGR2) protein is an attractive biomarker for various types of cancer. In pancreatic cancer, it is secreted to the pancreatic juice by premalignant lesions, which would be an ideal stage for diagnosis. Thus, designing assays for the sensitive detection of AGR2 would be highly valuable for the potential early diagnosis of pancreatic and other types of cancer. Herein, we present a biosensor for label-free AGR2 detection and investigate approaches for enhancing the aptasensor sensitivity by accelerating the target mass transfer rate and reducing the system noise. The biosensor is based on a nanostructured porous silicon thin film that is decorated with anti-AGR2 aptamers, where real-time monitoring of the reflectance changes enables the detection and quantification of AGR2, as well as the study of the diffusion and target-aptamer binding kinetics. The aptasensor is highly selective for AGR2 and can detect the protein in simulated pancreatic juice, where its concentration is outnumbered by orders of magnitude by numerous proteins. The aptasensor's analytical performance is characterized with a linear detection range of 0.05-2 mg mL-1, an apparent dissociation constant of 21 ± 1 µM, and a limit of detection of 9.2 µg mL-1 (0.2 µM), which is attributed to mass transfer limitations. To improve the latter, we applied different strategies to increase the diffusion flux to and within the nanostructure, such as the application of isotachophoresis for the preconcentration of AGR2 on the aptasensor, mixing, or integration with microchannels. By combining these approaches with a new signal processing technique that employs Morlet wavelet filtering and phase analysis, we achieve a limit of detection of 15 nM without compromising the biosensor's selectivity and specificity.
ABSTRACT
The field of optogenetics is rapidly growing in relevance and number of developed tools. Among other things, the optogenetic repertoire includes light-responsive ion channels and methods for gene regulation. This review will be confined to the optogenetic control of gene expression in mammalian cells as suitable models for clinical applications. Here optogenetic gene regulation might offer an excellent method for spatially and timely regulated gene and protein expression in cell therapeutic approaches. Well-known systems for gene regulation, such as the LOV-, CRY2/CIB-, PhyB/PIF-systems, as well as other, in mammalian cells not yet fully established systems, will be described. Advantages and disadvantages with regard to clinical applications are outlined in detail. Among the many unanswered questions concerning the application of optogenetics, we discuss items such as the use of exogenous chromophores and their effects on the biology of the cells and methods for a gentle, but effective gene transfection method for optogenetic tools for in vivo applications.
Subject(s)
Gene Expression Regulation , Optogenetics , Animals , HumansABSTRACT
BACKGROUND AND AIMS: Anti-Ro52 antibody (Ab) reactivity is highly prevalent in autoimmune rheumatic diseases (ARDs), mainly Sjögren's syndrome (SjS) and systemic lupus erythematosus (SLE), but also in other inflammatory disorders. Thorough assessment of the prevalence, clinical significance and epitope specificity of Ro52-autoAbs in cancerous diseases is still lacking. MATERIAL AND METHODS: Anti-Ro52 Ab reactivity was tested in a large cohort of 490 patients with various malignant diseases. Ro52-autoAb epitope mapping by an in house line immunoassay was carried out using 5 recombinant Ro52 polypeptides spanning Ro52. RESULTS: Anti-Ro52 abs were significantly more prevalent in patients with ovarian cancer (30%) compared to patients with 6 other malignant diseases (median 8.1%, range 5.9-15.8%). The presence of anti-Ro52 abs in patients with ovarian cancer was strongly associated with better overall survival. Ro52 epitope mapping of patients with ovarian cancer was dissimilar to that of SLE and SjS ARDs, less frequently recognizing Ro52-1 and Ro52-4 fragments compared to patients with SLE and SjS. CONCLUSION: We demonstrate for first time an unexpectedly high frequency of anti-Ro52 abs in patients with ovarian cancer, their presence indicating better overall survival. Their distinguishing epitope profile may suggest a non-SLE or SjS-related stimulus for autoAb production.
Subject(s)
Lupus Erythematosus, Systemic , Ovarian Neoplasms , Sjogren's Syndrome , Autoantibodies , Autoantigens , Female , Humans , Lupus Erythematosus, Systemic/diagnosis , Ovarian Neoplasms/diagnosis , Prognosis , RibonucleoproteinsABSTRACT
Due to the increasing economic and social relevance of biotherapeutics, their production processes are continually being reconsidered and reoptimized in an effort to secure higher product concentrations and qualities. Monitoring the productivity of cultured cells is therefore a critically important part of the cultivation process. Traditionally, this is achieved by determining the overall product titer by high performance liquid chromatography (HPLC), and then calculating the specific cell productivity based on this titer and an associated viable cell density. Unfortunately, this process is typically time-consuming and laborious. In this study, the productivity of Chinese Hamster Ovary (CHO) cells expressing a monoclonal antibody was analyzed over the course of the cultivation process. In addition to calculating the specific cell productivity based on the traditional product titer determined by HPLC analysis, culture productivity of single cells was also analyzed via flow cytometry using a cold capture assay. The cold capture assay is a cell surface labelling technique described by Brezinsky et al., which allows for the visualization of a product on the surface of the producing cell. The cell productivity results obtained via HPLC and the results of cold capture assay remained in great accordance over the whole cultivation process. Accordingly, our study demonstrates that the cold capture assay offers an interesting, comparatively time-effective, and potentially cheaper alternative for monitoring the productivity of a cell culture.
ABSTRACT
The development of multifunctional nanoscale systems that can mediate efficient tumor targeting, together with high cellular internalization, is crucial for the diagnosis of glioma. The combination of imaging agents into one platform provides dual imaging and allows further surface modification with targeting ligands for specific glioma detection. Herein, transferrin (Tf)-decorated niosomes with integrated magnetic iron oxide nanoparticles (MIONs) and quantum dots (QDs) were formulated (PEGNIO/QDs/MIONs/Tf) for efficient imaging of glioma, supported by magnetic and active targeting. Transmission electron microscopy confirmed the complete co-encapsulation of MIONs and QDs in the niosomes. Flow cytometry analysis demonstrated enhanced cellular uptake of the niosomal formulation by glioma cells. In vitro imaging studies showed that PEGNIO/QDs/MIONs/Tf produces an obvious negative-contrast enhancement effect on glioma cells by magnetic resonance imaging (MRI) and also improved fluorescence intensity under fluorescence microscopy. This novel platform represents the first niosome-based system which combines magnetic nanoparticles and QDs, and has application potential in dual-targeted imaging of glioma.
Subject(s)
Glioma/diagnostic imaging , Liposomes/chemistry , Transferrin/chemistry , Animals , Cell Line, Tumor , Contrast Media , Ferric Compounds/chemistry , Glioma/genetics , Glioma/metabolism , Humans , Liposomes/metabolism , Magnetic Iron Oxide Nanoparticles/chemistry , Magnetic Resonance Imaging/methods , Magnetics , Microscopy, Electron, Transmission/methods , Nanoparticles , Polyethylene Glycols , Quantum Dots/chemistryABSTRACT
Even though the administration of chemotherapeutic agents such as erlotinib is clinically established for the treatment of breast cancer, its efficiency and the therapy outcome can be greatly improved using RNA interference (RNAi) mechanisms for a combinational therapy. However, the cellular uptake of bare small interfering RNA (siRNA) is insufficient and its fast degradation in the bloodstream leads to a lacking delivery and no suitable accumulation of siRNA inside the target tissues. To address these problems, non-ionic surfactant vesicles (niosomes) were used as a nanocarrier platform to encapsulate Lifeguard (LFG)-specific siRNA inside the hydrophilic core. A preceding entrapment of superparamagnetic iron-oxide nanoparticles (FexOy-NPs) inside the niosomal bilayer structure was achieved in order to enhance the cellular uptake via an external magnetic manipulation. After verifying a highly effective entrapment of the siRNA, the resulting hybrid niosomes were administered to BT-474 cells in a combinational therapy with either erlotinib or trastuzumab and monitored regarding the induced apoptosis. The obtained results demonstrated that the nanocarrier successfully caused a downregulation of the LFG gene in BT-474 cells, which led to an increased efficacy of the chemotherapeutics compared to plainly added siRNA. Especially the application of an external magnetic field enhanced the internalization of siRNA, therefore increasing the activation of apoptotic signaling pathways. Considering the improved therapy outcome as well as the high encapsulation efficiency, the formulated hybrid niosomes meet the requirements for a cost-effective commercialization and can be considered as a promising candidate for future siRNA delivery agents.
ABSTRACT
Smad8 is a transcriptional regulator that participates in the intracellular signaling pathway of the transforming growth factor-ß (TGF-ß) family. Full-length Smad8 is an inactive protein in the absence of ligand stimulation. The expression of a truncated version of the protein lacking the MH1 domain (cSmad8) revealed constitutive activity in genetically engineered mesenchymal stem cells and, in combination with BMP-2, exhibited a tendon cell-inducing potential. To further explore function and applicability of Smad8 in regenerative medicine recombinant production is required. Herein, we further engineered cSmad8 to include the transactivation signal (TAT) of the human immunodeficiency virus (HIV) to allow internalization into cells. TAT-hcSmad8 was produced in endotoxin-free ClearColi® BL21 (DE3), refolded from inclusion bodies (IBs) and purified by Heparin chromatography. Analysis of TAT-hcSmad8 by thermal shift assay revealed the formation of a hydrophobic core. The presence of mixed α-helixes and ß-sheets, in line with theoretical models, was proven by circular dichroism. TAT-hcSmad8 was successfully internalized by C3H10T1/2 cells, where it was mainly found in the cytoplasm and partially in the nucleus. Finally, it was shown that TAT-hcSmad8 exhibited biological activity in C3H10T1/2 cells after co-stimulation with BMP-2.
Subject(s)
Escherichia coli , Inclusion Bodies , Protein Refolding , Smad8 Protein , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Inclusion Bodies/chemistry , Inclusion Bodies/genetics , Inclusion Bodies/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Smad8 Protein/biosynthesis , Smad8 Protein/chemistry , Smad8 Protein/genetics , Smad8 Protein/isolation & purificationABSTRACT
The quality by design approach was introduced to the biopharmaceutical industry over 15 years ago. This principle is widely implemented in the characterization of monoclonal antibody production processes. Anyway, the early process phase, namely the inoculum expansion, was not yet investigated and characterized for most processes. In order to increase the understanding of early process parameter interactions and their influence on the later production process, a risk assessment followed by a design of experiments approach was conducted. The DoE included the critical parameters methotrexate (MTX) concentration, initial passage viable cell density and passage duration. Multivariate data analysis led to mathematical regression models and the establishment of a designated design space for the studied parameters. It was found that the passage duration as well as the initial viable cell density for each passage during the inoculum expansion have severe effects on the growth rate and viability of the early process phase. Furthermore, the variations during the inoculum expansion directly influenced the production process responses. This carry-over of factor effects highlights the crucial impact of early process failures and the importance of process analysis and control during the first part of mAb production processes.