Phagocytosis Of Microspheres Research Articles

The expression and activation of protease-activated receptor-2 correlate with skin color.

Skin color results from the production and distribution of melanin in the epidermis. The protease-activated receptor-2 (PAR-2), expressed on keratinocytes but not on melanocytes, is involved in melanosome uptake via phagocytosis, and modulation of PAR-2 activation affects skin color. The pattern of melanosome distribution within the epidermis is skin color-dependent. In vitro, this distribution pattern is regulated by the ethnic origin of the keratinocytes, not the melanocytes. Therefore, we hypothesized that PAR-2 may play a role in the modulation of pigmentation in a skin type-dependent manner. We examined the expression of PAR-2 and its activator, trypsin, in human skins with different pigmentary levels. Here we show that PAR-2 and trypsin are expressed in higher levels, and are differentially localized in highly pigmented, relative to lightly pigmented skins. Moreover, highly pigmented skins exhibit an increase in PAR-2-specific protease cleavage ability. Microsphere phagocytosis was more efficient in keratinocytes from highly pigmented skins, and PAR-2 induced phagocytosis resulted in more efficient microsphere ingestion and more compacted microsphere organization in dark skin-derived keratinocytes. These results demonstrate that PAR-2 expression and activity correlate with skin color, suggesting the involvement of PAR-2 in ethnic skin color phenotypes.

Investigation of in vitro interactions between different polymeric surfaces and blood proteins via phagocytosis phenomena

The effect of various polymeric materials on blood components and their in vitro phagocytosis was the object of the present research. Polystyrene- (PS) and polymethylmetacrylate- (PMMA) based microspheres were produced by phase-inversion polymerization and chemically modified to obtain different surface hydrophilicities. The interactions between blood proteins and chemically- and biologically-modified surfaces were investigated and compared to plain microspheres. Adsorption properties of albumin, fibrinogen and total immunoglobulines on microspheres were tested. Hydrophilic surfaces have high ability for human serum albumin (HSA) adsorption, which also leads to less phagocytosis of microspheres in vitro. In the case of activated PMMAPVA microspheres, both protein adsorption and phagocytosis were significant. Interaction of blood proteins with microspheres did not cause any change in phagocytosis by leukocytes and monocytes. BSA adsorption on microspheres with different hydrophilicities showed the same blood protein adsorption results and phagocytosis was not detected. On the other hand, the highest level of phagocytosis was found with fibronectin-modified microspheres. The changes occurring in intrinsic and extrinsic coagulation mechanisms were determined by measuring the activated partial tromboplastin time (APTT) and the prothrombin time (PT). PT values of blood samples did not increase by treatment with microspheres, except for PS/HEMA, while chemical modification caused important prolongation in APTT.

Contribution of bone marrow cells to liver regeneration after partial hepatectomy in mice

Background/Aims : We examined whether bone marrow (BM) cells can commit to liver-consisting cells during liver regeneration after partial hepatectomy, using mice transplanted with green fluorescent protein (GFP) positive BM from GFP transgenic mice. Methods : Partial hepatectomy or sham operation was performed. Lineage marker analysis of GFP positive liver cells was by immunostaining and flow cytometry. DiI-labeled acetylated low-density lipoprotein uptake or microsphere phagocytosis was examined in vitro. Lineage marker expression in BM and peripheral blood (PB) cells, and the vascular endothelial growth factor (VEGF) concentration in the liver were also examined. Results : In hepatectomized mice, significantly more GFP positive cells participated in liver sinusoid than in sham-operated mice, expressing CD31 but not albumin. The percentage of cells that incorporated acetylated low-density lipoprotein but not microspheres was 69.5±3.4%, while 28.3±2.6% incorporated both, revealing sinusoidal endothelial and Kupffer cells, respectively. Increased expression of the CD31 and CD16/CD32 on GFP positive liver cells was also detected. The elevation of the VEGF concentration during liver regeneration and the increase in the CD34 and Flk-1 expression in the liver, BM, and PB cells suggested endothelial progenitor cell mobilization. Conclusions : GFP cell-marking provided direct evidence of the BM cells participation in liver regeneration after hepatectomy, where the majority was committed to sinusoidal endothelial cells probably through endothelial progenitor cell mobilization.

Microcalorimetry does not predict the cellular phagocytosis of latex microspheres

Current literature highlights the potential suitability of microcalorimetry for the investigation of cell–drug interactions. Previous work using bacteria or antigens derived from infectious organisms yielded conclusions that heat production is a quantitative means of measuring phagocytosis. In this study we evaluated the potential of flow-through microcalorimetry as a method of quantifying the phagocytosis of microsphere particulates. The technique avoids the need to incorporate radioactive or fluorescent markers into the particulate formulation, and would be widely applicable in biopharmaceutical research. Using the monocyte cell line Mono Mac 6 a power output of 9.00 μW per million cells was increased significantly on addition of zymosan, lipopolysaccaride (LPS) and phorbol myristate acetate but not following exposure to FITC labelled latex microspheres (LM). TNFα production increased on exposure to zymosan, LPS and LPS–phorbol myristate acetate, though not on exposure to LB. An assay was developed which allowed the quantification of internalised particulates in phagocytic cells using fluorescent activated cell sorting (FACS). In contrast to the microcalorimetric and TNFα data FACS revealed that 20% of the MM6 population phagocytosed a mean of 1.35 LM. Microcalorimetry and measurements of TNFα production are assays of cellular activation a phenomenon not necessarily associated with phagocytosis. FACS, however, serves as a specific and quantitative measure of phagocytosis. Microcalorimetry may not be a suitable technique for the quantitative assessment of the phagocytosis of drug delivery particulates.

The development of embolizing materials for chemo-embolization therapy of hepatocellular carcinoma

We developed chitin-containing cisplatin (CDDP) albumin microspheres (MS). We also investigated physical properties of MS in vitro, anti-tumor effect of MS in VX2 tumor model rabbits in vivo and clinical evaluation of MS. The CDDP contents, specific surface areas and yield of MS increased with an increase in the concentration of chitin. These findings suggest that accumulation of chitin on the surface of microsphere was the cause of expansion of the coated area as the concentration of chitin increased. In vitro CDDP releases decreased as the concentration of chitin increased. The initial burst effect of the drug was to be controlled by the increase of concentration of chitin. The chitin-containing CDDP microspheres showed similar CDDP release patterns irrespective of the concentration of chitin between 1 and 6 h after the administration of CDDP microspheres. After 6 h, the blood Pt levels increased with an increase in the concentration of CDDP microspheres suggesting a significant effect of the concentration of chitin on microsphere decomposability in vivo. Tumor growth rate was increasingly suppressed as the concentration of chitin increased. Clinical findings indicated that antitumor activity of chitin-containing CDDP albumin microspheres would have a chemoembolic effect by embolizing the hepatic artery. In the tumor and necrosis portions phagocytosis of microspheres by macrophage-like giant cell was shown. Consequently, the biocompatibility and decomposable properties of chitin-containing CDDP albumin microspheres were demonstrated.

Characterization of mannose receptor-dependent phagocytosis mediated by Mycobacterium tuberculosis lipoarabinomannan.

The macrophage mannose receptor (MR) along with complement receptors mediates phagocytosis of the M. tuberculosis virulent strains Erdman and H37Rv. We have determined that the terminal mannosyl units of the M. tuberculosis surface lipoglycan, lipoarabinomannan (LAM), from the Erdman strain serve as ligands for the MR. The biology of the MR (receptor binding and trafficking) in response to phagocytic stimuli is not well characterized. This study analyzes the MR-dependent phagocytosis mediated by Erdman LAM presented on a 1-micron-diameter phagocytic particle. Erdman LAM microspheres exhibited a time- and dose-dependent rapid increase in attachment and internalization by human monocyte-derived macrophages (MDMs). In contrast, internalization of LAM microspheres by monocytes was minimal. Microsphere internalization by MDMs was visualized and quantitated by immunofluorescence and confocal and electron microscopy and resembled conventional phagocytosis. Phagocytosis of LAM microspheres by MDMs was energy, cytoskeleton, and calcium dependent and was mannan inhibitable. Trypsin treatment of MDMs at 37 degrees C, which depleted surface and recycling intracellular pools of the MR, reduced the subsequent attachment of LAM microspheres. Trypsin treatment at 4 degrees C allowed for subsequent recovery of LAM microsphere phagocytosis at 37 degrees C by recycled MRs. Pretreatment of MDMs with cycloheximide influenced LAM microsphere phagocytosis to only a small extent, indicating that MR-dependent phagocytosis of the microspheres was occurring primarily by preformed recycled receptors. This study characterizes the requirements for macrophage phagocytosis of a LAM-coated particle mediated by the MR. This model will be useful in further characterization of the intracellular pathway taken by phagocytic particles coated with different LAM types in macrophages following ingestion.

Relating the phagocytosis of microparticles by alveolar macrophages to surface chemistry: the effect of 1,2-dipalmitoylphosphatidylcholine

This study examines the potential of 1,2-dipalmitoylphosphatidylcholine (DPPC), a major component of lung surfactant, to reduce the phagocytosis of microspheres by altering the cellular interactions occurring in the alveoli. These microspheres could be designed to act as a controlled delivery system for small molecules, peptides or proteins for pulmonary administration. Microspheres were prepared using poly(lactic-co-glycolic acid) (PLGA, 50/50) and encapsulated peroxidase as a model protein. DPPC was included in some formulations. The interaction of PLGA and DPPC-PLGA microspheres with phagocytic cells was evaluated using lung macrophages in culture. X-ray Photoelectron Spectra (XPS) results indicate that the inclusion of DPPC in the microspheres alters the microsphere surface chemistry, with the DPPC covering a large portion of the microsphere surface. The dominance of DPPC on the microsphere surface is highly beneficial in moderating the interactions occurring between the microspheres and phagocytic cells in the lung. Fluorescent confocal microscopy indicates that only 25% of cells internalized DPPC-coated particles, whereas 70% of those cells exposed to particles without the DPPC coating internalized particles after one hour of incubation.

Phagocytosis of microspheres with modified surfaces

A series of hydrogel microspheres having different polystyrene (PSt) contents was prepared by the precipitation polymerization of acrylamide (AAm) and comonomers followed by the seeded polymerization of styrene (St). The resulting AAm-St microspheres had different morphologies and surface properties dependent on the amount of St charged in the seeded polymerization. These AAm-St microspheres were contacted with granulocytes in phosphatebuffered saline (PBS) or plasma at 37 °C to clarify the effect of surface structure of the microspheres and of blood components on phagocytosis. Phagocytosis of the microspheres by granulocytes in PBS was enhanced with increasing PSt content in the microspheres. This suggested that the hydrophobicity of the microspheres is one of the major factors deciding the extent of phagocytosis in PBS. On the other hand, the extent of phagocytosis in plasma depended on the PSt content in the microspheres in an unexpected manner. The microspheres with a small content of PSt were less susceptible to phagocytosis than the microspheres containing no St. Phagocytosis in inactivated serum was slight, regardless of the type of microspheres. These results suggested that complement and the complement-immunogloblin G complex are responsible for the enhancement of phagocytosis of hydrophilic and hydrophobic microspheres, respectively.

In vivo and electron microscopic studies of rat liver after intravenous injection of polyamino acid microspheres.

In vivo and electron microscopy were used to study the hepatocellular responses of rat livers to intravenously injected polymeric microspheres. Two microsphere preparations with different surface characteristics and degradability were used in this study. In vivo microscopy revealed that both poly(benzyl L-glutamate) (PBLG) and poly(hydroxypropyl L-glutamine) (PHPG) microspheres caused disturbance in the microcirculation of rat liver up to 2 months after injection. The observed changes included stagnant flow and adherence of white blood cells to the endothelial lining of venules an sinusoids. Kupffer cell (KC) activation following phagocytosis of microspheres was evidenced by the enlargement of KCs and increased number of KCs taking up fluorescent latex particles. Electron microscopy of rat livers revealed a wide range of hepatocellular injury associated with the administration of PBLG and PHPG microspheres. These results indicate that a small amount of remaining microspheres is sufficient to induce continuous disturbance to hepatic microcirculation and that particulate drug carriers should be designed to be rapidly degraded so that the return to normal liver function is possible.

In vitro phagocytosis of polylactide microspheres by retinal pigment epithelial cells and intracellular drug release.

We investigated phagocytosis of biodegradable microspheres containing a drug by retinal pigment epithelial (RPE) cells and drug release within the cells to evaluate the potential usefulness of microspheres for intracellular drug delivery. The biodegradable polymers used were L-lactic acid, and DL-lactic acid with different molecular weights or the copolymers of different monomer compositions. The microspheres containing a non-bioactive fluorescent dye (rhodamine 6GX) as a model drug, were prepared by a solvent evaporation method. The in vitro release of the dye from the microspheres was examined. Phagocytosis of the microspheres by RPE cells was conducted to evaluate the extent of phagocytosis by phase-contrast microscopy and transmission electron microscopy. The RPE cells ingesting the microspheres at different stages were examined by fluorescent microscopy to estimate the intracellular release of the dye. The dye was released with time from every microsphere and the release was controlled by changing the type of polymers constituting microspheres. The microspheres containing the dye were phagocytosed by RPE cells and the dye was released intracellularly with time. The present study indicates that the drug incorporated in the microspheres was delivered into RPE cells by way of phagocytosis and released within the cells. It is concluded that this microsphere system is a promising delivery form capable of drug targeting to RPE cells.

Effects of chemotactic peptide f-met-leu-phe (FMLP) on C3b receptor (CR1) expression and phagocytosis of microspheres by human neutrophils

FMLP caused maximal upregulation of CR1 on neutrophils at a concentration of 10(-8) M but caused maximal enhancement of CR1-dependent phagocytosis of C3b.IgG-coated microspheres only at a concentration of 10(-6) M. There were positive correlations between FMLP-mediated upregulation of CR1 and FMLP-mediated enhancement of phagocytosis (correlation coefficient = 0.73, slope = 2.2) and between FMLP-mediated upregulation of CR1 and FMLP-mediated increase in total cell-associated microspheres (correlation coefficient = 0.88, slope = 1.3). The phagocytic capacity of both untreated and 10(-6) M FMLP-treated neutrophils was completely inhibited by fluid phase C3b and partially inhibited by aggregated IgG. The data suggest that CR1 upregulation is required but is not sufficient for maximal phagocytosis by the leukocytes. The data also suggest that FMLP at the higher concentrations may impart a phagocytic function to CR1, activate other phagocytic receptors, elicit phagocytosis-inducing mediators or may elicit a separate mechanism of phagocytosis. During the study, it was observed that there was considerable individual variation among different neutrophil preparations with respect to CR1 expression and binding and phagocytic capacity.

Effects of hydroxyurea on the phagocytosis of microspheres in V79 cells.

Phagocytosis of fluorescent microspheres in V79 cells arrested in the S phase by hydroxyurea (HU) was investigated by flow cytometry. The phagocytic activity increased with the exposure time of HU. No dependency on the HU concentration was observed in the concentration used. Cell size (projection area) and membrane fluidity, two factors effected by HU treatment, were examined in relation to phagocytic activity, and were found to be significantly increased. The elevation in phagocytic activity could be interpreted in terms of the alteration in the two physiological factors.

Macrophage phagocytosis of biodegradable microspheres composed of L-lactic acid/glycolic acid homo- and copolymers.

A variety of biodegradable microspheres were prepared from L-lactic acid, DL-lactic acid, or glycolic acid homopolymers and copolymers of different molecular weights and monomer compositions. Phagocytosis of the microspheres by mouse peritoneal macrophages was studied in cell culture system using scanning electron microscopy as well as light microscopy. The diameter of microspheres prepared was less than 2 microns, regardless of the starting polymers. No dependence of the chemical nature of starting polymers was observed on the extent of phagocytosis of the microspheres by macrophages. Precoating the microspheres with water-soluble macromolecules such as proteins had great influence on phagocytosis by macrophages. It was demonstrated that precoating with bovine serum albumin and non-proteinaceous macromolecules reduced the phagocytosis of microspheres, while bovine gamma-globulin, human fibronectin, bovine tuftsin, and gelatin precoating enhanced the phagocytosis. This trend was not influenced by the presence of serum. Only in the case of gelatin precoating, the phagocytosis was greatly enhanced by the presence of serum as compared to precoating with other proteins. Microscopic observation clearly indicated that the phagocytosed microspheres were gradually degraded in the macrophage interior with the incubation time, leading to release of a fluorescent dye encapsulated in the microspheres. The rate of microsphere degradation in cells could be controlled by changing the molecular weight and the monomer composition of the copolymers comprising the microspheres.

Evaluation of the phagocytosis of microspheres in V79 cells by flow cytometry.

Phagocytosis of microspheres in V79 Chinese hamster lung cells was investigated by flow cytometry. Fluorescent microspheres (1.8 micron diameter) were used as the ingesta. Change in the number of V79 cells containing fluorescent microspheres was measured as an index of phagocytic activity. With time there was a sigmoidal increase in cells containing microspheres. The phagocytosis of microspheres is partially explained by two parameters introduced to describe the sigmoidal curves.

Phagocytosis of Bioactive Microspheres

The influence of several proteins on the uptake of microspheres was investigated using mouse peritoneal macrophages. Thioglycollate-stimulated macrophages were cultivated for 3 h with protein-grafted and protein-coated cellulose microspheres smaller than 2 μm in the presence and the absence of serum. Bovine serum albumin reduced the phagocytosis of microspheres, while y-globulin, human fibronectin, bovine tuftsin, and gelatin enhanced the phagocytosis. This trend was not influenced substantially by the presence of serum and the mode of surface binding of the proteins; that is, covalent grafting or physical adsorption (coating). However, in the case of gelatin binding, phagocytosis was greatly enhanced by the presence of serum as compared with the other proteins.

Simultaneous analysis of cell surface antigens and cell morphology using monoclonal antibodies conjugated to fluorescent microspheres

A new method for simultaneous analysis of cell surface antigens and cell morphology using monoclonal antibodies conjugated to fluorescent microspheres (‘immunospheres’) is described. Wright's staining was performed on cells after reaction with immunospheres, and a direct correlation of cell surface antigen expression and cell morphology was made. Mild formalin fixation of cells inhibited phagocytosis of microspheres, which is a potential source of confusion in the analysis of cell surface binding. Rapid, accurate analysis of cell surface antigen expression in single cell suspensions of heterogeneous human hematopoietic and lymphoid cell populations was facilitated by this method.

Phagocytosis of microspheres containing an anticancer agent by tumor cells [formula omitted

The uptake of suspended human albumin microspheres containing 6-mercaptopurine-8- 14C by several tumor cell lines has been followed in cell culture. In a preliminary study, HeLa, KB, and human glioblastoma cells have all been shown to phagocytize the drug-containing spheres. We suggest that if studies now in progress confirm the ability of the ingested anticancer agent to survive lysosomal digestion of the albumin spheres, phagocytosis of such spheres may prove to be a method of delivering drug to malignant cells while preventing these agents from producing toxic effects in nonphagocytic tissues.