Complete Cell Culture Medium Research Articles

Chitosan-copper microparticles as doxorubicin microcarriers for bone tumor therapy

Doxorubicin (DOX) is a chemotherapeutic drug used in osteosarcoma treatments, usually administrated in very high dosages. This study proposes novel DOX microcarriers based on chitosan (CHT) physically crosslinked with copper(II) ions that will act synergically to inhibit tumor growth at lower drug dosage without affecting the healthy cells. Spherical CHT-Cu microparticles with a smooth surface and an average size of 30.1 ± 9.1 µm were obtained by emulsion. The release of Cu2+ ions from the CHT-Cu microparticles showed that 99.4 % of added cupric ions were released in 72 h of incubation in a complete cell culture medium (CCM). DOX entrapment in microparticles was conducted in a phosphate buffer solution (pH 6), utilizing the pH sensitivity of the polymer. The successful drug-loading process was confirmed by DOX emitting red fluorescence from drug-loaded microcarriers (DOX@CHT-Cu). The drug release in CCM showed an initial burst release, followed by sustained release. Biological assays indicated mild toxicity of CHT-Cu microparticles on the MG-63 osteosarcoma cell line, without affecting the viability of human mesenchymal stem cells (hMSCs). The DOX@CHT-Cu microparticles at concentration of 0.5 mg mL‒1 showed selective toxicity toward MG-63 cells.

DNA nanostructures prevent the formation of and convert toxic amyloid proteospecies into cytocompatible and biodegradable spherical complexes

AbstractThe deposition of insoluble proteinaceous aggregates in the form of amyloid fibrils within the extracellular space of tissues is associated with numerous diseases. The development of molecular approaches to arrest amyloid formation and prevent cellular degeneration remains very challenging due to the complexity of the process of protein aggregation, which encompasses an infinite array of conformations and quaternary structures. Polyanionic biopolymers, such as glycosaminoglycans and RNAs, have been shown to modulate the self‐assembly of amyloidogenic polypeptides and to reduce the toxicity induced by the formation of oligomeric and/or pre‐fibrillar proteospecies. This study evaluates the effects of double‐stranded DNA (dsDNA) nanostructures (1D, 2D, and 3D) on amyloid self‐assembly, fibril disaggregation, and the cytotoxicity associated with amyloidogenesis. Using the islet amyloid polypeptide (IAPP) whose pancreatic accumulation is the hallmark of type 2 diabetes, it was observed that dsDNA nanostructures inhibit amyloid formation by inducing the formation of spherical complexes in which the peptide adopts a random coil conformation. Interestingly, the DNA nanostructures showed a persistent ability to disassemble enzymatically and thermodynamically stable amyloid fibrils into nanoscale DNA/IAPP entities that are fully compatible with β‐pancreatic cells and are biodegradable by proteolysis. Notably, dsDNA nanostructures avidly trapped highly toxic soluble oligomeric species in complete cell culture media and converted them into non‐toxic binary complexes. Overall, these results expose the potent modulatory effects of dsDNA on amyloidogenic pathways, and these DNA nanoscaffolds could be used as a source of inspiration for the design of molecules to fight amyloid‐related disorders.

A new physiological medium uncovers biochemical and cellular alterations in Lesch-Nyhan disease fibroblasts

BackgroundLesch-Nyhan disease (LND) is a severe neurological disorder caused by the genetic deficiency of hypoxanthine–guanine phosphoribosyltransferase (HGprt), an enzyme involved in the salvage synthesis of purines. To compensate this deficiency, there is an acceleration of the de novo purine biosynthetic pathway. Most studies have failed to find any consistent abnormalities of purine nucleotides in cultured cells obtained from the patients. Recently, it has been shown that 5-aminoimidazole-4-carboxamide riboside 5ʹ-monophosphate (ZMP), an intermediate of the de novo pathway, accumulates in LND fibroblasts maintained with RPMI containing physiological levels (25 nM) of folic acid (FA), which strongly differs from FA levels of regular cell culture media (2200 nM). However, RPMI and other standard media contain non-physiological levels of many nutrients, having a great impact in cell metabolism that does not precisely recapitulate the in vivo behavior of cells.MethodsWe prepared a new culture medium containing physiological levels of all nutrients, including vitamins (Plasmax-PV), to study the potential alterations of LND fibroblasts that may have been masked by the usage of non-physiological media. We quantified ZMP accumulation under different culture conditions and evaluated the activity of two known ZMP-target proteins (AMPK and ADSL), the mRNA expression of the folate carrier SLC19A1, possible mitochondrial alterations and functional consequences in LND fibroblasts.ResultsLND fibroblasts maintained with Plasmax-PV show metabolic adaptations such a higher glycolytic capacity, increased expression of the folate carrier SCL19A1, and functional alterations such a decreased mitochondrial potential and reduced cell migration compared to controls. These alterations can be reverted with high levels of folic acid, suggesting that folic acid supplements might be a potential treatment for LND.ConclusionsA complete physiological cell culture medium reveals new alterations in Lesch-Nyhan disease. This work emphasizes the importance of using physiological cell culture conditions when studying a metabolic disorder.

Ulex Europaeus Agglutinin-I-Based Magnetic Isolation for the Efficient and Specific Capture of SW480 Circulating Colorectal Tumor Cells.

The efficient and specific capture of circulating tumor cells (CTCs) from patients’ peripheral blood is of significant value in precise cancer diagnosis and cancer therapy. As fine targeting molecules, lectins can recognize cancer cells specifically due to the abnormal glycosylation of molecules on the cancer cell membrane and the specific binding of lectin with glycoconjugates. Herein, a Ulex europaeus agglutinin-I (UEA-I)-based magnetic isolation strategy was developed to efficiently and specifically capture α-1,2-fucose overexpression CTCs from colorectal cancer (CRC) patients’ peripheral blood. Using UEA-I-modified Fe3O4 magnetic beads (termed MB-UEA-I), up to 94 and 89% of target cells (i.e., SW480 CRC cells) were captured from the cell spiking complete cell culture medium and whole blood, respectively. More than 90% of captured cells show good viability and proliferation ability without detaching from MB-UEA-I. In combination with three-color immunocytochemistry (ICC) identification, MB-UEA-I has been successfully used to capture CTCs from CRC patients’ peripheral blood. The experimental results indicate a correlation between CTC characterization and tumor metastasis. Specifically, MB-UEA-I can be applied to screen early CRC by capturing CTCs when served as a liquid biopsy. The presented work offers a new insight into developing cost-effective lectin-functionalized methods for biomedical applications.

Cytotoxic Effects of Rheum khorasanicum Extract on MCF-7 Human Breast Cancer Cell Line and ODC1 Gene Expression

Background: Polyamine metabolism has a critical role in cancer initiation and progression. The abnormal overexpression of the ornithine decarboxylase (ODC1) enzyme gene could be a characteristic of cancer cells. Objectives: The current study evaluated the cytotoxic effects of Rheum khorasanicum extract in three different solvents, including distilled water, ethanol, and n-hexane, on the MCF-7 breast cancer cell line and assessed the expression level of the ODC1 gene upon the abovementioned treatments. Methods: We cultured MCF-7 cancer cells in complete cell culture media and subsequently subjected them to increasing concentrations (0 - 200 μg/mL) of the extract. The cytotoxic activity of the extract was investigated using an MTT assay. The quantitative analysis of ODC1 gene expression was performed using real-time PCR. Results: The cell viability was significantly decreased in concentration- and time-dependent manners. The mRNA expression of the ODC1 gene declined gradually with the increasing concentrations of extracts. Conclusions: Our findings indicated that R. khorasanicum extract could potentially influence ODC1 gene expression in breast cancer cells and exert inhibitory effects on cancer cell proliferation.

Comparison of Hydrogen Peroxide Secretion From Living Cells Cultured in Different Formats Using Hydrogel-Based LSPR Substrates.

Reactive oxygen species (ROS), a number of reactive molecules and free radicals derived from molecular oxygen, are generated as by-products during mitochondrial electron transport within cells. Physiologically, cells are capable of metabolizing the ROS exploiting specific mechanisms. However, if excessive ROS accumulate inside the cells, it will cause the cells apoptosis or necrosis. Hydrogen peroxide (H2O2) is one of the essential ROS often participating in chemical reactions in organisms and regulating homeostasis in the body. Therefore, rapid and sensitive detection of H2O2 is a significant task in cell biology research. Furthermore, it has been found that cells cultured in different formats can result in different cellular responses and biological activities. In order to investigate the H2O2 secretion from the cells cultured in different formats, a hydrogel-based substrate is exploited to separate relatively large molecular (e.g., proteins) for direct measurement of H2O2 secreted from living cells in complete cell culture medium containing serum. The substrate takes advantage of the localized surface plasmon resonance (LSPR) method based on enzyme immunoprecipitation. In addition, the H2O2 secreted from the cells cultured in different dimensions (suspension of single cells and three-dimensional cell spheroids) treated with identical drugs is measured and compared. The spheroid samples can be prepared with ample amount using a designed microfluidic device with precise control of size. The results show that the H2O2 secretion from the cells are great affected by their culture formats.

Can the Self-Assembling of Dicarboxylate Pt(IV) Prodrugs Influence Their Cell Uptake?

The possibility of spontaneous self-assembly of dicarboxylato Pt(IV) prodrugs and the consequences on their uptake in cancer cells have been evaluated in different aqueous solutions. Four Pt(IV) complexes, namely, (OC-6-33)-diacetatodiamminedichloridoplatinum(IV), Ace, (OC-6-33)-diamminedibutanoatodichloridoplatinum(IV), But, (OC-6-33)-diamminedichloridodihexanoatoplatinum(IV), Hex, and (OC-6-33)-diamminedichloridodioctanoatoplatinum(IV), Oct, have been dispersed in i) milliQ water, ii) phosphate buffered saline, and iii) complete cell culture media (RPMI 1640 or DMEM) containing fetal bovine serum (FBS). The samples have been analyzed by dynamic light scattering (DLS) to measure the size and distribution of the nanoparticles possibly present. The zeta potential offered an indication of the stability of the resulting aggregates. In the case of the most lipophilic compounds of the series, namely, Oct and to a lesser extent Hex, the formation of nanosized aggregates has been observed, in particular at the highest concentration tested (10 μM). The cell culture media had the effect to disaggregate these nanoparticles, mainly by virtue of their albumin content, able to interact with the organic chains via noncovalent (hydrophobic) interactions. For Oct, at the highest concentration employed for the uptake tests (10 μM), the combination between passive diffusion and endocytosis of the self-assembled nanoparticles makes the cellular uptake higher than in the presence of passive diffusion only. During the study of cellular uptake on A2780 ovarian cancer cells pretreated with cytochalasin D, a statistically significant inhibition of endocytosis was observed for Oct. In these experimental conditions, the relationship between uptake and lipophilicity becomes almost linear instead of exponential. Since Oct anticancer prodrug is active at nanomolar concentrations, where the aggregation in culture media is almost abolished, this phenomenon should not significantly impact its antiproliferative activity.

Plasmonic gel films for time-lapse LSPR detection of hydrogen peroxide secreted from living cells

In this paper, we developed a low-cost and easy-to-fabricate hydrogel-based localized surface plasmon resonance (LSPR) substrate for sensitive, consistent, specific, and time-lapse detection of hydrogen peroxide (H2O2) secreted from living cells in cell culture medium. The uniform and reproducible LSPR substrate was made from agarose, gold nanorods (AuNRs), horseradish peroxidase (HRP), and 3-amino-9-ethylcarbazole (AEC). In H2O2 sensing, insoluble precipitates were generated in the gel film by an HRP-catalyzed reaction in the presence H2O2, and the precipitates near the AuNRs caused a LSPR shift of the substrate, which could be used for H2O2 quantification. Using the developed substrate, we showed that a H2O2 concentration as low as 0.5 μM could be detected in complete cell culture medium within 30 min with good reproducibility. In addition, we demonstrated time-lapse detection of H2O2 secreted from living cells after the cells were stimulated by ascorbic acid. The results indicated that the agarose gel of the substrate provided the filtration capability and the improved stability of the AuNRs allowing detection of H2O2 in cell culture medium without tedious sample preparation procedures. Moreover, the HRP-catalyzed reaction-based LSPR biosensing offered good selectivity for H2O2 detection and possessed great potential for various in vitro cell studies.

Self-Assembly of Solubilized Human Hair Keratins.

Human hair keratins have proven to be a viable biomaterial for diverse regenerative applications. However, the most significant characteristic of this material, the ability to self-assemble into nanoscale intermediate filaments, has not been exploited. Herein, we successfully demonstrated the induction of hair-extracted keratin self-assembly in vitro to form dense, homogeneous, and continuous nanofibrous networks. These networks remain hydrolytically stable in vitro for up to 5 days in complete cell culture media and are compatible with primary human dermal fibroblasts and keratinocytes. These results enhance the versatility of human hair keratins for applications where structured assembly is of benefit.

Colloidal stability and biological activity evaluation of microbial exopolysaccharide levan-capped gold nanoparticles

The main objectives of this study were to explore the suitability of the exopolysaccharide levan, biosynthesized by Bacillus subtilis, to aid in the formation of gold nanoparticles (AuNPs) and to investigate the colloidal stability and in vitro biological activity of this biopolymer-AuNPs complex. AuNPs (mainly spherical, 8-10 nm-sized, and monodispersed) were successfully synthesized in levan concentrations up to 0.5% w/v (L-AuNP0.5) while exposed to ultraviolet C (UVC) irradiation. The increase of levan quantity decreased the size of AuNPs according to Transmission Electron Microscopy (TEM) images and enhanced the colloidal stability significantly. The presence of L-AuNP0.5 at the highest treatment dose (1000 μg/mL) exhibited substantial cytotoxicity towards L-929 mouse fibroblasts for all incubation periods. Dose-dependent toxicity was observed for the first day while, after this threshold value, medium (100 μg/mL) and the lowest (10 μg/mL) treatment doses were non-cytotoxic during 7 days of incubation, implying dose and time-independent cell viabilities (> 95%) compared to the negative control (complete cell culture medium). There occurred a special surface interaction with cells and L-AuNP0.5, especially when the cells were subjected to deliberate starvation periods to increase L-AuNP0.5 internalization via passive and active endocytosis. Scanning Electron Microscopy (SEM) images showed high accumulation of L-AuNP0.5 around or inside the cell membrane after 7 days. Overall, this attribute (high uptake of L-AuNP0.5) could make them promising candidates for prospective cancer therapeutics or drug delivery systems by enabling the cell internalization of anticancer drugs.

Anticancer Activity of Liquid Treated with Microwave Plasma-Generated Gas through Macrophage Activation.

Reactive nitrogen species (RNS), including nitric oxide (NO·) has been known as one of the key regulatory molecules in the immune system. In this study, we generated RNS-containing water treated with microwave plasma-generated gas in which the major component was nitric oxide (PGNO), and the effect on the macrophage polarization was investigated. The RNS-containing water was diluted in complete cell culture media (PGNO-solution) into the concentration that did not induce cell death in RAW 264.7 murine macrophages. PGNO-solution upregulates M1-type macrophage activation and downregulates the characteristics of M2-type macrophage at the transcriptional level. In addition, the PGNO-solution-treated M2-like macrophages had higher potential in killing melanoma cells. The anticancer potential was also investigated in a syngeneic mouse model. Our results show that PGNO-solution has the potential to convert the fate of macrophages, suggesting PGNO-solution treatment as a supportive method for controlling the function of macrophages under the tumor microenvironment.

New approaches to the study of cell vital activity cultivated in different growing conditions with analysis of oxygen in the medium

Objectives - to develop new approaches to the study of morphofimctional state of chondroblasts, cultured at 37°C on a 3D carrier in different environments: in a CO2 incubator with 5% of CO2 and in a thermostat in an air-proof tube. Material and methods. The study cell culture - chondroblasts, isolated from the cartilage of the articular surfaces of the extra-fingers' phalanges. 3D carrier for cells - the demineralized lyophilized human spongiosa Lioplast®. The resulting tissue-engineered structures were grown in a complete cell culture medium at 37°С under different conditions: in a closed system in thermostat and in an open system in CO2 incubator (5% CO2). To assess the morphofunctional state of the cells on the surface of the 3D carrier, the picrosirius red staining, a LIVE/ DEAD® fluorescent dye kit, and scanning electron microscopy were used. The oxygen concentration in the culture medium was evaluated by the modified Winkler titration method. Results. The complex of morphological methods revealed the presence of living cells on the surface of human spongiosa within the 7-day period of cultivation. The cells either are fusiform or have a polygonal form and have a capacity to grow in 2 or more layers. The titrimetric analysis has demonstrated a decline in the concentration of dissolved oxygen in the medium with cellular tissue material in 7 days of cultivation. The concentration declined by 72.4% in a thermostat and by 63.5% in a CO2 incubator. In the tests tubes which contained only the medium and no cells, there was a similar decline in oxygen concentration by 47.3% in a thermostat and by 66.1% in a CO2 incubator. Conclusion. 1. A method of measuring the amount of dissolved oxygen in a culture medium, during the adhesive cell cultivation on a 3D carrier, was developed, based on the Winkler titration method. 2. A comparative analysis of the amount of dissolved oxygen in the medium in the process of chondroblast cultivation on a 3D human spongiosa carrier, both in a CO2 incubator and in a closed test tube, revealed an overall tendency to a decrease in the concentration of oxygen within 7 days of cultivation. 3. A decrease in oxygen concentration in the test tubes with human spongiosa samples (without cells), within the 7 days of cultivation, was registered. 4. An efficient and cost-saving method of graft manufacturing for the purposes of chondroplasty is the transfer of juvenile joint cartilage chondroblasts to 3D human spongiosa carriers and their further cultivation in air-proof test tubes copletely filled with medium within a period of 7 days.

Investigating the Interaction of Nanodiamonds with Human Serum Albumin and Induced Cytotoxicity

Nanodiamonds (NDs) have been recognized as emerging carbon-based delivery vehicles due to their biocompatibility. NDs were reported to be nontoxic and suited for biomedical applications in the complete cell culture medium. However, in this study, the cytotoxicity of NDs in serum-free medium was studied and indicated that serum proteins in cell culture medium played significant effect on the toxicity of NDs. Therefore, the interaction mechanism between NDs and a serum protein (human serum albumin, HSA) was first investigated by fluorescence quenching technique and circular dichroism (CD) spectrometry. The results suggest that HSA strongly bonds on the NDs surface to form “protein corona,” which not only prevents the aggregation of NDs and improves its stability but also inhibits the cytotoxicity of NDs. In serum-free medium, NDs exhibited obvious toxicity toward the human lung epithelial cell line (BEAS-2B) and showed concentration-dependent cytotoxicity. In the presence of bovine serum albumin (BSA), which shares structural homology and similar properties with HSA, toxicity of NDs was apparently inhibited. Therefore, the interaction between serum protein and NDs should be considered in the understanding of the biological effects of NDs exposure in biomedical applications.

Cytotoxicity of orthodontic temporary anchorage devices on human periodontal ligament fibroblasts in vitro.

ObjectivesThe objective of this study is to test cytotoxicity of four brands of commercially available orthodontic temporary anchorage devices (TADs).Setting and sample populationTwenty‐four (six for each brand, i.e., Aarhus [AO]; Dual top [RMO]; Vector TAS [ORMCO]; and Unitek TAD [3M UNITEK]) TADs were tested.Materials and methodsTwenty‐four (six for each brand, i.e., Aarhus [AO]; Dual top [RMO]; Vector TAS [ORMCO]; and Unitek TAD [3M UNITEK]) TADs were individually incubated in complete cell culture medium and shaken at a rate of 1.5 rpm at 37°C for 30 days to extract possible toxic substances in conditioned media (CM). To test cytotoxicity, human periodontal ligament fibroblasts were cultured and exposed to the CM for 24 hr, followed by the examinations of morphological changes, cell viability (MTT assay), and cell damage (lactate dehydrogenase [LDH] assay).ResultsNo morphological changes were observed in any of the four brands of TADs compared with the negative control. LDH assay showed that none of the four brands of TADs caused significant cell damage after CM treatment compared with the negative control (P > .05). No significant differences were found between any of the four brands of TADs (P > .05). MTT assay showed similar results as did the LDH assay, except for a statistically significant difference found in the TADs from 3M UNITEK compared with the negative control (P = .047).ConclusionsAccording to the International Standard Organization standards, except for the TAD from 3M, none of the other three brands of commercially available TADs (from AO, RMO, and ORMCO) exhibited significant cytotoxicity, suggesting their safe clinical applications.

Receptor-Mediated Field Effect Transistor Biosensor for Real-Time Monitoring of Glutamate Release from Primary Hippocampal Neurons.

Glutamate, one of the most important central excitatory neurotransmitters, plays crucial roles in nerve signal transduction and is implicated in several neurological disorders. However, no effective means has been developed for specific detection of glutamate released from primary cultured neurons. Here we present a reduced graphene oxide (RGO)-based field effect transistor (FET) biosensor functionalized with synthesized glutamate receptor for real-time monitoring of glutamate release from primary cultured rat hippocampus neurons. Metabotropic glutamate receptors (mGluR) was specifically synthesized and then immobilized on the RGO surface by 1-pyrenebutanoic acid succinimidyl ester (PASE) linker, after which target glutamate (pI = 3.22) could specifically bind to the synthesized mGluR in the neutral buffer, causing the charge density change. After the neurons were cultured on the sensing channel with a self-made liquid reservoir, the FET biosensor could discriminate glutamate in the femtomolar range in complete cell culture medium and generate encouraging results in real-time monitoring of glutamate release from primary rat hippocampus neurons. This work is the first report of specific and direct detection of glutamate molecules released from primary culture of differentiated central neurons, which may further help understand the nature of neuronal communication. Moreover, this work paves a way for the detection of electrochemically inactive small molecules released by cells.

3D printed Polylactid Acid based porous scaffold for bone tissue engineering: an in vitro study

The objective of this study was to fabricate PLA-based porous scaffold by 3D printing technology and to evaluate their cytotoxicity and biocompatibility under in vitro conditions in respect to bone tissue engineering. Pure PLA in filamentous form was processed via 3D printing technology of fused filament fabrication into porous scaffolds. The structure and porosity of scaffolds were measured by metrotomography. PLA scaffolds were pre-treated by human serum, foetal bovine serum and complete cell culture medium to enhance bio-attractivity of the scaffold's surface for the adherence of the cells. Cells were enzymatically isolated from the periosteum of the proximal tibia and then expanded in monolayer. Periosteum-derived osteoprogenitors (PDOs) were seeded on the pre-treated PLA scaffolds and subsequent cell proliferation was measured by commercially available cell proliferation assay. Adherence of PDOs on the PLA scaffold was confirmed by scanning electron microscopy (SEM). Prepared scaffolds had well-defined structure and were characterized by uniform distribution of pores. They were non-toxic and biocompatible with PDOs, however, PLA scaffold with the periosteum-derived progenitor cells was significantly better in the group of scaffolds pre-treated with normal human serum. The obtained PLA porous scaffolds favored attachment of periosteum derived progenitors and proliferation, furthermore, cells penetrated into the scaffold through the interstitial pores which was meaningful for cytocompatibility evaluation.

Stiffness and swelling characteristics of nanocellulose films in cell culture media

Nanocellulose is a material of interest for biomedical applications due to its morphological similarity with tissues’ own collagen. New cell culture substrates that mimic the human body tissue stiffness and extracellular matrix can be used to study cell behaviour in a way that is not possible for traditional plastic substrates. Five wood-based micro-and nanofibrillated cellulose films without additives were prepared to investigate the effects of nanocellulose charge density and fibril size on the mechanical properties of the films in liquids. Swelling behaviour of the films was studied in deionized water, in complete cell culture media (DMEM) and in CaCl2 solutions. Atomic force microscopy with a colloidal probe was used to measure Young’s modulus at the surface of the thin films submerged in the liquids. Cell culture media and CaCl2 solutions reduced the swelling of the films observed in deionized water, most probably due to a bridging effect by the calcium ions. The reduction was proportional to the charge of the particular nanocellulose. Young’s modulus in compression varied randomly on sample surfaces and appeared not to be directly related to topography of the films. In complete DMEM, the average Young’s modulus varied from 0.8 to 36.4 kPa, in the same stiffness range as two skin tissue layers, dermis and epidermis. Therefore, from mechanical properties point of view, these films seem suitable as cell culture substrates for skin tissue cells and could be used in skin tissue engineering.

Integration of a Copper-Containing Biohybrid (CuHARS) with Cellulose for Subsequent Degradation and Biomedical Control.

We previously described the novel synthesis of a copper high-aspect ratio structure (CuHARS) biohybrid material using cystine. While extremely stable in water, CuHARS is completely (but slowly) degradable in cellular media. Here, integration of the CuHARS into cellulose matrices was carried out to provide added control for CuHARS degradation. Synthesized CuHARS was concentrated by centrifugation and then dried. The weighed mass was re-suspended in water. CuHARS was stable in water for months without degradation. In contrast, 25 μg/mL of the CuHARS in complete cell culture media was completely degraded (slowly) in 18 days under physiological conditions. Stable integration of CuHARS into cellulose matrices was achieved through assembly by mixing cellulose micro- and nano-fibers and CuHARS in an aqueous (pulp mixture) phase, followed by drying. Additional materials were integrated to make the hybrids magnetically susceptible. The cellulose-CuHARS composite films could be transferred, weighed, and cut into usable pieces; they maintained their form after rehydration in water for at least 7 days and were compatible with cell culture studies using brain tumor (glioma) cells. These studies demonstrate utility of a CuHARS-cellulose biohybrid for applied applications including: (1) a platform for biomedical tracking and (2) integration into a 2D/3D matrix using natural products (cellulose).

Co-transfection of star-shaped PDMAEMAs enhance transfection efficiency of protamine/pDNA complexes in the presence of serum

In previous studies, we have found that un-complexed free cationic polymers greatly promote gene transfection efficiency of pDNA complexes. Dividing the gene transfection system into two parts, bound chains and free chains, we exploited mixing different biomaterials as the second component of our designed system to improve gene transfection outcome. In this study, we investigated the effectiveness of star-shaped poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) with different arm numbers as free chains (i.e., co-transfection agent) while using natural salmon protamine as bound chains. We explored the gene transfer ability of the system in both serum free and complete cell culture medium. Our results reveal that the star-shaped PDMAEMAs enhance gene transfection efficiency of protamine/pDNA complexes, in the same range and even better than the gold standard polyethyleneimine (PEI, branched type with 25 kg/mol molar mass) in serum-free and serum containing medium conditions, respectively. From 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Lactate Dehydrogenase (LDH) release data, it was found that the PDMAEMA stars have tolerable cytotoxicity but with significant membrane disruption level, especially when increasing the arm number. Flow cytometry experiments showed that PDMAEMA stars clearly improve the uptake rate as well as the number of protamine/pDNA complexes taken up by the target cells. Using Taqman Real-time Polymerase Chain Reaction (PCR), further exploration of the effect of PDMAEMAs during different gene transfection stages reflects that these un-bound star-shaped polymers mainly act upon the cellular internalization step. Our results indicate that the star-shaped PDMAEMAs are promising vectors for gene delivery.

Superoxide Dismutase Mimetic GC4419 Enhances the Oxidation of Pharmacological Ascorbate and Its Anticancer Effects in an H₂O₂-Dependent Manner.

Lung cancer, together with head and neck cancer, accounts for more than one-fourth of cancer deaths worldwide. New, non-toxic therapeutic approaches are needed. High-dose IV vitamin C (aka, pharmacological ascorbate; P-AscH−) represents a promising adjuvant to radiochemotherapy that exerts its anti-cancer effects via metal-catalyzed oxidation to form H2O2. Mn(III)-porphyrins possessing superoxide dismutase (SOD) mimetic activity have been shown to increase the rate of oxidation of AscH−, enhancing the anti-tumor effects of AscH− in several cancer types. The current study demonstrates that the Mn(II)-containing pentaazamacrocyclic selective SOD mimetic GC4419 may serve as an AscH−/O2•− oxidoreductase as evidenced by the increased rate of oxygen consumption, steady-state concentrations of ascorbate radical, and H2O2 production in complete cell culture media. GC4419, but not CuZnSOD, was shown to significantly enhance the toxicity of AscH− in H1299, SCC25, SQ20B, and Cal27 cancer cell lines. This enhanced cancer cell killing was dependent upon the catalytic activity of the SOD mimetic and the generation of H2O2, as determined using conditional overexpression of catalase in H1299T cells. GC4419 combined with AscH− was also capable of enhancing radiation-induced cancer cell killing. Currently, AscH− and GC4419 are each being tested separately in clinical trials in combination with radiation therapy. Data presented here support the hypothesis that the combination of GC4419 and AscH− may provide an effective means by which to further enhance radiation therapy responses.