Cell intracellular barriers

Additional cell membrane barriers (plasma membrane, and possibly organelle membranes) will have to be overcome if the drug target is located intracellularly. It thus turns out... 

Figure 6 Intracellular barriers to gene delivery include cellular uptake, intracellular transport, endosome escape, vector unpacking, and nuclear uptake. The gene carrier illustrated here has targeting moieties on the vector surface that are specific for cell surface receptors. The dotted arrow represents the prerequisite step of bypassing physiological barriers of the lung, such as the mucosal layer, and reaching the target cell surface.

Genes can be delivered using either ex vivo or in vivo strategy. The ex vivo strategy encounters intracellular barriers presented by the cell that include the cell, endosome, and nuclear membranes (Fig. 6.38). 

Once the cell has been targeted, the vector particle has to be internahzed by the cellular uptake machinery via endocytotic or phagocytotic uptake processes. This step can be taken rather easily by polyplexes, as appropriate receptor-binding ligands and/or cationic charges may enhance intracellular uptake of particles into endosomal vesicles. Several intracellular barriers then have to be overcome for successful transgene expression. Endosomal release was found to be a major bottleneck for many non-viral vectors [151,167]. The vector particle needs to survive and escape from the endosomal vesicular compartment, traffick the cytoplasmic environment, target the nucleus, enter the nucleus, and expose the carried nucleic acid to the cellular transcription machinery. 

Fig. 2 Cell entry and intracellular barriers to nucleic acid drug delivery...

Although iron deficiency is a common problem, about 10% of the population are genetically at risk of iron overload (hemochromatosis), and elemental iron can lead to nonen2ymic generation of free radicals. Absorption of iron is stricdy regulated. Inorganic iron is accumulated in intestinal mucosal cells bound to an intracellular protein, ferritin. Once the ferritin in the cell is saturated with iron, no more can enter. Iron can only leave the mucosal cell if there is transferrin in plasma to bind to. Once transferrin is saturated with iron, any that has accumulated in the mucosal cells will be lost when the cells are shed. As a result of this mucosal barrier, only about 10% of dietary iron is normally absorbed and only 1-5% from many plant foods. 

Decreased cerebral blood flow, resulting from acute arterial occlusion, reduces oxygen and glucose delivery to brain tissue with subsequent lactic acid production, blood-brain barrier breakdown, inflammation, sodium and calcium pump dysfunction, glutamate release, intracellular calcium influx, free-radical generation, and finally membrane and nucleic acid breakdown and cell death. The degree of cerebral blood flow reduction following arterial occlusion is not uniform. Tissue at the... 

One should realize that the intracellular compartment as depicted in Figure 2 represents multiple cell types, whereas in vitro studies normally utilize a single cell type pertinent to characterizing specific attributes of drug transport in that cell system. The method of Shah et al. [51] would be of great benefit to investigating blood-brain barrier transport, consistent with a vascular-extravascular subcompartment brain model. 

While the lactate-H+ symporter and the K+/H+ exchanger are involved in acidification of the cell, the Na+/H+ exchanger present in the basal cells exports protons out of the cell in exchange for Na+ [139]. It was observed that removal of Na+ from the Ringer s solution decreased intracellular pH by 0.5 unit in basal cells, possibly due to inhibition of the Na+/H+ exchanger. As the basal cells are the precursors for the superficial cells of the corneal epithelium, it is quite likely that similar exchange processes are also present in the superficial layer, the principal barrier to ion and drug transport [99,103],... 

Each cell is surrounded by a plasma membrane that separates the cytoplasmic contents of the cell, or the intracellular fluid, from the fluid outside the cell, the extracellular fluid. An important homeostatic function of this plasma membrane is to serve as a permeability barrier that insulates or protects the cytoplasm from immediate changes in the surrounding environment. Furthermore, it allows the cell to maintain a cytoplasmic composition very different from that of the extracellular fluid the functions of neurons and muscle cells depend on this difference. The plasma membrane also contains many enzymes and other components such as antigens and receptors that allow cells to interact with other cells, neurotransmitters, blood-borne substances such as hormones, and various other chemical substances, such as drugs. 

As outlined in previous sections, escape of polyplexes from endosomes to the cytosol can be a major bottleneck in delivery. Membrane-active polymer domains or other conjugated molecules can help to overcome this barrier (see Sect. 2.3), but they may trigger cytotoxicity when acting extracellularly or at the cell surface. Therefore membrane-crossing agents either have to be inherently specific for endo-somal compartments (for example by pH-specificity), or they have to be modified to be activated in endosomes. For example, the reducing stimulus of intracellular vesicles has been used to activate formulations containing less active disulfide precursors of LLO [163] or Mel [170]. 

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