AbstractMacromolecules can be introduced into cultured mammalian cells by fusing recipients with red cells, loaded by hypotonic hemolysis with the macromolecule of interest. This technique is called r
The object of our work is to define the possible role of hypotonically loaded erythrocytes as carriers to target drugs to the reticuloendothelial system. We have examined choices of drugs for loading into the erythrocytes and have considered methods of altering potentially useful agents so that they will load. We have demonstrated that the delivery of bleomycin to the reticuloendothelial system of mice, inside erythrocyte carriers, potentiates the effect of this drug on phagocytosis. We speculate, that this targeted delivery of bleomycin to phagocytes could be beneficial in the treatment of diseases characterized by an important phagocytic component.
The phospholipids of the red cell membrane are asymmetrically distributed across the bilayer. Loss of this asymmetry by certain normal and pathological red cells is correlated with their increased adherence both in vitro and in vivo. Red cells loaded under conditions which result in loss of asymmetry adhere to cultured endothelial cells and are phagocytized in vitro by macrophages more readily than red cells loaded under conditions in which asymmetry is maintained. These results suggest that preserving phospholipid asymmetry during loading may prevent undesired in vivo interactions by reinfused red cell carriers.
In iron overload resulting from repeated blood transfusion, phagocytic reticuloendothelial cells are the primary and major sites of iron accumulation. Removal of this iron by the iron chelator deferoxamine can be enhanced by targeted delivery of this agent to reticuloendothelial cells. We have used resealed erythrocyte ghosts as a model system for delivery of deferoxamine to reticuloendothelial cells and have demonstrated a several-fold enhancement of urine iron excretion in children and adults with congenital or acquired iron-loading anemias. Although presently not cost-effective, this approach provides a useful, practical and safe model for [1] iron chelator targeting to reticuloendothelial cells, and [2] the clinical use of resealed erythrocyte ghosts for targeting pharmacologic agents to reticuloendothelial cells.
Preparation, characteristics and properties of the three types of erythrocyte ghost are compared. These include light scattering properties, morphology, retention of cellular components and biochemical function including membrane transport. In vivo studies in the BALB/c mouse and Beagle dog have allowed the potential to be assessed of these erythrocyte ghosts as drug and enzyme cellular carriers in the treatment of human diseases. The development of the carrier erythrocyte with restored cellular ATP content and properties, including in vivo survival, that are closely similar to those of the normal erythrocyte is also described and its potential discussed.
G6PD-deficient erythrocytes (Mediterranean type of this enzyme disorder) were loaded with a) normal G6PD purified to homogeneity from human erythrocytes, b) G6PD Mediterranean purified from deficient granulocytes. The first set of experiments led to complete normalization of biochemical properties of erythrocytes, as assessed by evaluating their metabolic competence under steady state conditions and under oxidative stress as well. The second type of experiment allowed us to conclude that mutant G6PD is not appreciably destroyed within the affected erythrocytes.
Two different dialysis methods were used to G6PD load the erythrocytes of G6PD deficient subjects. With both methods the yield of entrapment was about 30-40% of the added G6PD. The functional behaviour of these G6PD loaded erythrocytes was evaluated by HMS either in resting conditions or under oxidative stimulation. An increase of the metabolic activity of the G6PD loaded erythrocytes was found. This model provides an useful approach in studying the hemolytic activity of a variety of agents.
Electric pulses in the range of 1 to 5 kV/cm and of durations 1 to 200 microseconds have been used to open up pores of limited size in various cell types. In the case of erythrocytes, these pores were shown to admit molecules as large as tetrasaccharides. Loading of clinically active drugs has also been attempted. Erythrocytes loaded with drugs can be resealed without loss of the hemoglobin content. With mouse erythrocytes, we have demonstrated the feasibility of using erythrocytes (not hemoglobin depleted ghosts) as drug carriers for prolonging the drug level in the circulation. Other possible applications of the method are also discussed.
Radiolabeled DNA fragments or nuclear proteins were encapsulated within human erythrocytes, and the erythrocytes were then fused with cultured mammalian cells using Sendai virus. Autoradiography revealed that 125I-labeled DNA fragments remained dispersed in the cytoplasm and disappeared with a half-life of 24 hours. In contrast, the nuclear proteins, HMG1, HMG2, HMG17 and histone H1, rapidly localized within HeLa nuclei and exhibited half lives greater than 80 hours. Several biochemical criteria indicate that the association of the injected nuclear proteins with chromatin faithfully mimics the behavior of their endogenous counterparts.
This chapter reviews the biochemical and physiological effects of the incorporation of allosteric effectors of hemoglobin in red blood cells. The amount of O2 that can be released in a particular organ depends on the critical O2 partial pressure characteristic for the organ and on the architecture of the microvasculature. This O2 release capacity is controlled by the microcirculation and molecular parameters of the intracellular Hb. The O2 release capacity can be enhanced by an increase in the co-operability of the Hb molecule; and/or by right shifting of the entire O2-binding curve toward higher O2 partial pressures. An abnormally high affinity of hemoglobin for oxygen shifts the O2-binding curve to the left and the P50 (O2 partial pressure at which 50% saturation of hemoglobin occurs) decreases causing a lower oxygen release to the tissues. In human RBC, the right-shift is controlled by several allosteric mechanisms, such as Bohr effect, 2, 3-bis-phosphoglycerate (DPG), and CO2-binding.
Autologous red blood cells were loaded with L-asparaginase and injected intravenously into monkeys. A single dose of 1850 IU/Kg suppressed plasma asparagine for 19 days compared to 10 days for the same dose injected free in solution. Rabbit antisera to asparaginase was used to passively immunize guinea-pigs. These animals were then challenged with RBC-entrapped asparaginase versus asparaginase free in solution. RBC entrapment allowed a ten fold greater dose of asparaginase before anaphylaxis became a problem. RBC entrapment prolongs the duration of action of asparaginase and offers protection against anaphylaxis. Clinical trial in patients with acute lymphoblastic leukemia is recommended.
Red cell-mediated microinjection facilitates the rapid introduction of macromolecules into large numbers of culture cells. Efficient injection of cells in monolayer using PEG as fusogen can be accomplished using any of three alternative agglutinating agents (PHA, Con A, or polylysine) to attach loaded red cells to recipient cells. These three method yield similar efficiencies of microinjection and postfusion viability, as shown in Table I. Either of the methods, after being tailored to fit particular needs, can be expected to produce results similar to those in Table I with some variability due to differences in cell types. One drawback when PHA is used as agglutinin is the inability to remove adherent, nonfused red cells from cultures after fusion. Reversible attachment of red cells is possible using Con A or polylysine. For each of the latter agglutinins, reversal of binding detaches the great majority of the red cells; virtually all nonfused red cells may be eliminated by performing an additional simple step in either case.
