The application of tumor cell-derived vesicles in oncology therapy

Tumor cell-derived vesicles are released by tumor cells, have a phospholipid bilayer, and are widely distributed in various biological fluids. In recent years, it has been found that tumor cell-derived vesicles contain proteins, metabolites and nucleic acids and can be delivered to recipient cells to perform their physiological functions, such as mediating specific intercellular communication, activating or inhibiting signaling pathways, participating in regulating the modulation of tumor microenvironment and influencing tumor development, which can be used for early detection and diagnosis of cancer. In addition, tumor cell-derived vesicles exhibit multiple properties in tumor therapeutic applications and may serve as a new class of delivery systems. In this review, we elaborate on the application of tumor cell-derived vesicles in oncology therapy (AU)

The application of tumor cell-derived vesicles in oncology therapy.

Tumor cell-derived vesicles are released by tumor cells, have a phospholipid bilayer, and are widely distributed in various biological fluids. In recent years, it has been found that tumor cell-derived vesicles contain proteins, metabolites and nucleic acids and can be delivered to recipient cells to perform their physiological functions, such as mediating specific intercellular communication, activating or inhibiting signaling pathways, participating in regulating the modulation of tumor microenvironment and influencing tumor development, which can be used for early detection and diagnosis of cancer. In addition, tumor cell-derived vesicles exhibit multiple properties in tumor therapeutic applications and may serve as a new class of delivery systems. In this review, we elaborate on the application of tumor cell-derived vesicles in oncology therapy.

Regulation of Hole Concentration and Mobility and First-Principle Analysis of Mg-Doping in InGaN Grown by MOCVD.

This work studied the regulation of hole concentration and mobility in p-InGaN layers grown by metalorganic chemical vapor deposition (MOCVD) under an N-rich environment. By adjusting the growth temperature, the hole concentration can be controlled between 6 × 1017/cm3 and 3 × 1019/cm3 with adjustable hole mobility from 3 to 16 cm2/V.s. These p-InGaN layers can meet different requirements of devices for hole concentration and mobility. First-principles defect calculations indicate that the p-type doping of InGaN at the N-rich limiting condition mainly originated from Mg substituting In (MgIn). In contrast with the compensation of nitrogen vacancy in p-type InGaN grown in a Ga-rich environment, the holes in p-type InGaN grown in an N-rich environment were mainly compensated by interstitial Mg (Mgi), which has very low formation energy.

Enzymatic synthesis of L-lactic acid from carbon dioxide and ethanol with an inherent cofactor regeneration cycle.

Efficient conversion of carbon dioxide is of great interests to today's endeavors in controlling greenhouse gas emission. A multienzyme catalytic system that uses carbon dioxide and ethanol to produce L-lactate was demonstrated in this work, thereby providing a novel reaction route to convert bio-based ethanol to an important building block for synthesis biodegradable polymers. The synthetic route has a unique internal cofactor regeneration cycle, eliminating the need of additional chemical or energy for cofactor regeneration. Lactate was successfully synthesized with 41% of ethanol converted in a batch reaction, while a turnover number of 2.2 day⁻¹ was reached for cofactor regeneration in a reaction with continuous feeding of ethanol. A kinetic model developed based on reaction kinetic parameters determined separately for each reaction step predicted well the reaction rates and yields of the multienzyme reaction system.

Enzymic thin film coatings for bioactive materials.

Bioactive materials have been explored for a broad range of applications including biocatalysts, biosensors, antifouling membranes and other functional and smart materials. We report herein a unique method for preparation of bioactive materials through a spin coating process. Specifically, we investigated the preparation of protease Subtilisin Carlsberg-coated plastic films and examined their activities for hydrolysis of chicken egg albumin (CEA). The process generated enzymic coatings with a typical loading of 13 microg/cm2, retaining 46% of the enzyme activity for hydrolysis of CEA in aqueous solutions. Interestingly, the surface-coated protease thin film not only catalyzed the hydrolysis of CEA in aqueous solutions, but also showed good activity for solid-state CEA that was coated on top of the enzyme thin film.

A novel high throughput immunomagnetic cell sorting system for potential clinical scale depletion of T cells for allogeneic stem cell transplantation.

OBJECTIVE: To develop an immunomagnetic cell separation system for allogeneic hematopoietic stem cell (HSC) transplantations, which can achieve a high level of T-cell depletion (at least 4.0 log(10)), high level of recovery of hematopoietic stem cells (>90%), with a high throughput (>10(6) cells/second). METHODS: Peripheral blood leukocytes (PBLs) from buffy coats were spiked with CD34-expressing cells (KG1a) to mimic a leukaphoresis product containing stimulated HSCs. T cells were labeled with anti-CD3(+) Dynabeads and separated in a quadrupole magnetic cell sorter (QMS). The performance of the system with respect to T-cell depletion and recovery of non-T cells and spiked KG1a was determined using four-color, flow cytometry analysis, with the aid of Trucount cell-concentration calibration beads. Limiting dilution assays were also performed to quantify the log(10) depletion of clonable T cells. RESULTS: While the general performance of the QMS system is governed by proven theoretical principles, significant system variability exist, not all of which can be explained by our current understanding. Consequently, a factorial design was employed, guided by JMP software, to optimize the labeling conditions and operation of the QMS focused on maximizing the depletion of T cell, and recovery of unlabeled cells including KG1a cells. From these studies, an optimized, no wash, immunomagnetic labeling protocol and optimized QMS operating conditions were developed. For an average initial cell concentration of 1.7 x 10(8) total cells, an average 3.96 +/- 0.33 log(10) depletion (range, 3.53-4.34) of CD3(+)CD45(+) cells with a mean 99.43% +/- 4.23% recovery of CD34(+)CD45(+) cells (range, 94.38-104.90%) was achieved at a sorting speed of 10(6) cells/s (n = 6). Limiting dilution assays on the T-cell depleted fractions, which gave a log(10) depletion of 3.51 for the clonable T cells. CONCLUSION: We suggest that this system will provide superior performance with respect to T-cell depletion and CD34(+) recovery for clinical allogeneic bone marrow transplants. Ongoing studies, on a clinical scale, are being conducted to demonstrate this claim.

Application of immunomagnetic cell enrichment in combination with RT-PCR for the detection of rare circulating head and neck tumor cells in human peripheral blood.

Detection of rare, circulating tumor cells (CTC's) in human peripheral blood is a potential indicator of prognosis and diagnosis in oncology. Typical methods to detect these CTC's are either by immunocytochemistry (ICCS) or RT-PCR. However without accurate, rapid, and reproducible enrichment processes, these detection techniques are labor intensive and/or unreliable. In this article, a repeatable enrichment process that included a flow-through immunomagnetic cell separation system, the quadrupole magnetic sorter (QMS) was optimized with the aid of a statistical analysis software package. The QMS was operated in a negative mode of operation by immunomagnetically targeting normal human peripheral blood lymphocytes (PBL) through the CD45 surface marker. Three head and neck squamous carcinoma cell lines (HNSCC), Detroit-562, SCC-4, and CAL-27, were used to determine the sensitivity of RT-PCR for the epidermal growth factor receptor (EGFR) in spiked PBL. The detection purity needed for detection was found to be one cell in 10(4), one cell in 10(3), and one cell in 10(5) for the Detroit-562, SCC-4, and CAL-27, respectively. The actual number of cancer cells needed for RT-PCR detection ranged from 30 to 1 cell. To mimic the potential concentration of rare CTC present in peripheral blood of cancer patients, the spiking concentration was chosen to be one cancer cell per 10(5) total leukocytes from healthy donors. Using a single step immunomagnetic labeling, the final, optimized enrichment process produced a 57.6 +/- 30.3-fold (n = 6) enrichment of the rare cancer cells with a final cancer cell recovery of (77.8 +/- 6.6)%.

Comparison of two immunomagnetic separation technologies to deplete T cells from human blood samples.

The objective of this study was to compare the performance of two immunomagnetic separation technologies to deplete T cells from buffy coats of human blood. Specifically, two versions of the commercial MACS(R) Technology: MiniMACS and SuperMACS, and a prototype, flow-through system, the QMS, were evaluated. Peripheral blood mononuclear leukocytes (PBL) were isolated from buffy coats and an immunomagnetic separation of CD3(+) cells was conducted using company and optimized labeling protocols. To mimic peripheral blood containing bone marrow purged hematopoietic stem cells, HSC, CD34 expressing-cells (KG1a) were spiked into PBL prior to T-cell depletion once optimized depletion conditions were determined. Once the labeling protocol was optimized, the MiniMACS system performed well by producing a highly enriched CD3(+) fraction, and a respectable level of depletion of T cells and recovery of KG1a cells in the depleted fraction; an average log(10) depletion of T cells of 2.88 +/- 0.17 and an average recovery of the KG1a cells of 60.8 +/- 5.94% (n = 14). The performance of the SuperMACS system was very similar with an average log(10) depletion of T cells of 2.89 +/- 0.22 and an average recovery of KG1a of 63.1 +/- 8.55% (n = 10). In contrast, the QMS system produced an average log(10) depletion of T cells of 3.98 +/- 0.33 (n = 16) with a corresponding average recovery of 57.9 +/- 16.6% of the spiked CD34+ cells. The aforementioned QMS performance values were obtained using sorting speeds ranging from 2.5 x 10(4) to 1.7 x 10(5) cells per second. It is suggested that the lack of a 100% recovery of the unlabeled KG1a cells is the result of a previously reported "drafting" phenomena which pulls unlabeled cells in the direction of the magnetically labeled cells thereby resulting in loss of the unlabeled cells.

Enrichment of rare cancer cells through depletion of normal cells using density and flow-through, immunomagnetic cell separation.

OBJECTIVE: To develop a reliable technique to enrich for rare cells in blood suspensions using only negative selection steps including a flow-through immunomagnetic cell separations system and by optimizing variables normally encountered during such enrichment processes. METHODS: A human breast cancer cell line was cultivated and spiked at a ratio of 1 cancer cell to 10(5) total leukocytes in buffy coat or 1 cancer cell to 10(8) total cells in whole blood samples. The final, optimized process consisted of: a red cell lysis step, immunomagnetically staining leukocytes with an anti-CD45 PE, anti- MACS sandwich, immunomagnetic sorting using a flow-through system (QMS), and a final cell analysis step using either an automated cell counter, filtration, and visual counting or a cytospin analysis. RESULTS: The final, optimized process produced a final enrichment of the rare cancer cells of 5.17 log(10) and an average, final recovery of 46%. It should be noted that a negative depletion protocol was used (i.e., no labeling of the rare cancer cells was used). CONCLUSIONS: To the authors' knowledge, no examples in the literature exist of a 5.17 log(10) enrichment of cancer cells in human blood using a negative depletion protocol. The closest example is a 4 log(10) enrichment in which two positive magnetic cell separation steps were used (none were used in this study). Ongoing studies are investigating further modifications of the precommercial, prototype flow-through immunmagnetic separation system to increase both the enrichment and recovery rate. However, even at current performance levels, the presented process could significantly improve visual and molecular analysis of rare cells in blood.

Application of magnetic agarose support in liquid magnetically stabilized fluidized bed for protein adsorption.

A novel magnetic agarose support (MAS) was fabricated for application in a liquid magnetically stabilized fluidized bed (MSFB). It was produced by water-in-oil emulsification method using a mixture of agarose solution and nanometer-sized superparamagnetic Fe(3)O(4) particles as the aqueous phase. The MAS showed good superparamagnetic responsiveness in a magnetic field. A reactive triazine dye, Cibacron blue 3GA (CB), was coupled to the gel to prepare a CB-modified magnetic agarose support (CB-MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption equilibrium and kinetic behavior of the CB-MAS. The dependence of bed expansion in the MSFB with a transverse magnetic field on liquid velocity and magnetic field intensity was investigated. Liquid-phase dispersion behavior in the MSFB was examined by measurements of residence time distributions and compared with that obtained in packed and expanded beds. Dynamic lysozyme adsorption in the MSFB was also compared with those in packed and expanded beds. The dynamic binding capacity at 10% breakthrough was estimated at 55.8 mg/mL in the MSFB, higher than that in the expanded bed (31.1 mg/mL) at a liquid velocity of 45 cm/h. The results indicate that the CB-MAS is promising for use in liquid MSFB for protein adsorption.

Expanded bed adsorption of protein with DEAE Spherodex M.

In this article, the bed expansion behavior and the hydrodynamic and protein adsorption properties of the DEAE Spherodex M in expanded bed with mobile phases of different viscosities have been studied. The axial liquid-phase dispersion coefficient is found to be on the order of 10(-6) m(2)/s, falling into the common range from 1.0 x 10(-6) to 1.0 x 10(-5) m(2)/s observed previously in expanded bed operation. Because of the small size of the adsorbent, the high pore diffusivity of protein and the favorable column efficiency (low dispersion coefficient), the dynamic binding capacity (DBC) of bovine serum albumin (BSA) at 5% breakthrough in the expanded bed reaches over 80% that of the equilibrium adsorption density (EAD). Moreover, a theoretical model with unadjustable model parameters is used for the prediction of the breakthrough curves. Computer simulations show that the model agrees well with the experimental results at breakthrough less than about 50%. It indicates that the model is promising in the prediction of protein breakthrough behavior because breakthrough profiles at 5-50% breakthrough points are more important in practical applications.

Particle size and density distributions of two dense matrices in an expanded bed system.

The size and density distributions of two commercial media, that is, Streamline particles and 6% agarose coated steel beads (6AS), in an expanded bed system has been studied with a glass column (26 mm I.D.) modified by side ports. The Streamline particles have a broad size distribution but a relatively uniform density, while the 6AS beads have both broad size and density distributions. The effect of liquid-phase flow velocity, liquid viscosity and settled bed height on the particle size and density distributions is investigated. It is found that the radial mean particle size and density of the two matrices are uniform, while axial classifications are obvious in the expanded beds. For the Streamline, the volume-weighted mean particle size decreases linearly with increasing expanded bed height. For the 6AS beads, however, the mean particle size is even in the axial direction, but the particle density decreases exponentially with the increase of bed height. Moreover, the mean particle size of the Streamline or the density of the 6AS beads is well expressed as a function of the normalized bed height (that is, the ratio of the distance from bed bottom to the expanded bed height). The liquid flow-rate, liquid viscosity and settled bed height influence the mean axial size or density distribution by affecting the expanded bed height.

Nd-Fe-B alloy-densified agarose gel for expanded bed adsorption of proteins.

Novel dense composite adsorbents for expanded bed adsorption of protein have been fabricated by coating 4% agarose gel onto Nd-Fe-B alloy powder by a water-in-oil emulsification method. Two composite matrices, namely Nd-Fe-B alloy-densified agarose (NFBA) gels with different size distributions and densities, NFBA-S (50-165 microm, 1.88 g/ml) and NFBA-L (140-300 microm, 2.04 g/ml), were produced. Lysozyme was used as a model protein to test the adsorption capacity and kinetics for the NFBA gels modified by Cibacron blue 3GA (CB-NFBA gels). Liquid-phase dispersion behavior in the expanded beds was examined by measurements of residence time distributions, and compared with that of Streamline SP (Amersham-Pharmacia Biotech, Sweden). The dependence of axial mixing in the expanded beds on flow velocity, bed expansion degree. settled bed height, and viscosity of liquid phase was investigated. Breakthrough curves of lysozyme in the expanded beds of the CB-NFBA gels were also examined. The dynamic binding capacity at 5% breakthrough was 23.3 mg/ml matrix for the CB-NFBA-S gels, and 16.7 mg/ml matrix for the CB-NFBA-L, at a flow velocity of 220 cm/h. The results indicate that the NFBA gels are promising for expanded bed adsorption of proteins.