Nanomedicine, Volume IIA: Biocompatibility
© 2003 Robert A. Freitas Jr. All Rights Reserved.
Robert A. Freitas Jr., Nanomedicine, Volume IIA: Biocompatibility, Landes Bioscience, Georgetown, TX, 2003
220.127.116.11 Biocompatibility with Neural Cells
Central nervous system (CNS) neurons, unlike those of the peripheral nervous system, do not spontaneously regenerate following injury, and it has been shown that in the developing CNS a combination of cell-adhesive and cell-repulsive cues guide growing axons to their targets [1528, 4961]. Neural cells respond to patterned surfaces [4962, 4963] (Section 18.104.22.168). For example, glass surfaces functionalized with spatially-precise patterns of cell-adhesive (peptide) regions and cell-repulsive (PEG) regions can control the direction of neuron cell adhesion and neurite outgrowth across the surface . Schwann cells have been cultured on and preferentially attach to micropatterned laminin-coated stripes separated by BSA, with cell orientation driven by the laminin-BSA interface . Lines of polylysine-conjugated laminin as narrow as 2.6 microns induce linear axonal guidance outgrowth and adherence of hippocampal neurons . Polyphosphoester polymers have high biocompatibility as nerve guide conduits . Self-assembling peptide scaffolds can serve as biologically compatible substrates for neurite outgrowth and synapse formation . Varying the mechanical , electrical [4969, 4970] and chemical  characteristics of the contact surface also influences the neurite outgrowth rate in neuronal contact guidance, and can even allow control of neuron shape .
Adhesion and patterning of cortical neurons has been investigated  on isolated islands of neuron-adhesive polyethylenimine (PEI) surrounded by a neuron-repellent fluorocarbon layer. The patterns consisted of PEI-coated wells (diameter 150 microns, depth 0.5 micron) etched in a fluorocarbon coating atop polyimide-coated glass. The separation distance between the PEI-coated wells was varied between 10-90 microns, resulting in highly compliant patterns of adhering cortical neurons after one day in vitro and interconnecting neurite fascicles between PEI-coated wells present on patterns with a separation distance of 10 microns after 8 days in vitro .
Immunoisolation of dopamine-secreting PC12 cells by microencapsulation in semi-permeable 75:25 2-hydroxyethyl methacrylate/methyl methacrylate (HEMA/MMA) copolymer membranes has been evaluated as a promising strategy for dopamine replacement for Parkinson’s disease . There was good biocompatibility between the HEMA/MMA copolymer and the host brain, as evidenced by the absence of gross tissue damage at the neuronal tissue/capsule interface and only a moderate inflammatory response by reactive astrocytes confined to the immediate vicinity of the injection tract , despite other work suggesting that pure MMA can be neurotoxic to human cortical neurons . In another experiment , neuronal and glial cells (Schwann cells and astrocytes) were immobilized within N-(2-hydroxypropyl) methacrylamide (HPMA) polymer hydrogels to produce cell-based polymer hybrid devices, with some cells exhibiting spreading or process outgrowth and secretion of laminin which offers a possible model for tissue replacement in the central nervous system using these cell-based polymer constructs. Similar constructs involving polycarbonate tubes filled with lens capsule-derived extracellular matrix coated with cultured neonatal Schwann cells are being studied for their ability to promote the regrowth of retinal ganglion cell (RGC) and other axons across surgically induced tissue defects in the CNS . Genetically engineered cells have been combined with biocompatible polymers to elicit axon regrowth across tissue defects in injured rat CNS , and the direct transplantation of neural tissue into the mammalian brain has been studied for a century .
Although early electrodes implanted in brain or peripheral nerve often left corrosion- or abrasion-related deposits , good long-term biocompatibility of various electrode materials has been demonstrated (1) at nerves [4980, 4981]; (2) in cochlear implants at scala tympani electrode arrays [4982, 4983] and potential CNS auditory prostheses ; (3) in retinal chip implants , semiconductor-based microphotodiode arrays designed to be placed under the neural retina in the subretinal space [4985-4987], and visual cortex microelectrode arrays ; (4) in other neural implants intended for mobilization of paraplegics, phrenic pacing, or cardiac assist ; and (5) for a variety of microwires  and electrode materials including silicon [4989-4991], platinum [4989, 4992], iridium [4989, 4993], polyesterimide-insulated gold wires , peptide-coated glassy carbon pins , carbon nanotubes , and polymer-based electrodes . Silicon nitride [4992, 5041] and silicon dioxide  are dielectrics used as an electrode passivation layer. Certain metals cannot be used in the brain without provoking necrosis and phagocytosis. For instance, copper induces active phagocytosis and silver yields inactive phagocytes after implantation for 37 days in rat brain . On the other hand, stainless steel and Nichrome (with varnish insulators such as Epoxylite or polyimide) can be implanted without producing any detectable damage beyond that of the initial trauma and brief phagocytosis limited to the edge of the electrode track . Larger electrodes create more tissue reaction at least up to 37 days . Other aspects of electrocompatibility are discussed in Section 22.214.171.124.
Many materials show good biocompatibility when implanted in the brain or CNS, including various gels [4998, 4999], biopolymers [5000-5003] and polymer capsules , hollow dialysis fibers , and other biomaterials [5006, 5007]. The overall neurobiocompatibility of diamond (Section 126.96.36.199) and diamond-like carbon , carbon nanotubes (Section 188.8.131.52) and functionalized fullerenes (e.g., Section 184.108.40.206(4)), carbon fiber , Nitinol (Section 220.127.116.11), and metal coatings such as tantalum, tungsten, platinum, gold, iridium, palladium, and brass (further altered to promote or inhibit cell growth and spreading by an additional overcoat of biological materials including ECM proteins, laminin, fibronectin, and collagen IV)  have also been examined. In one experiment , titanium microscrews and monofilament stainless steel wire were implanted into the parietal region of rabbits and produced no behavioral changes or neurological deficits suggestive of either systemic or localized toxicity from the implant materials. However, at 2 weeks the titanium had caused the largest inflammatory response in surrounding brain parenchyma based on analysis of markers for microglial proliferation, gliosis, and leukocyte infiltration. After 26 weeks the greatest leukocyte response was found with stainless steel implants, as compared to silicone elastomer which produced the least inflammation. Silicone elastomer has well-established brain biocompatibility and is commonly used as a neurosurgical implant material .
The neurobiocompatibility of bulk Teflon (Section 18.104.22.168(9)), Teflon implants (Section 22.214.171.124) and Teflon particles (Section 126.96.36.199) has already been briefly discussed. In general, Teflon is relatively inert with poor cell attachment when used as an implant in the central nervous system . For example, Proplast (a fluorocarbon polymer) shows no reaction with dura and brain , although this material was withdrawn from the market for other reasons (Section 188.8.131.52). As another example, a 12-micron thick Teflon film prevents adhesions between an implanted electrode array and the dura, in cat brains .
Special risks of particles in the brain should also be investigated further. For example, diffuse iron particles were found in the cortex of a patient who showed increasing frequency of seizures 12 years after a blunt head injury, which the researchers believed might have contributed to progressing traumatic epilepsy ; though strictly neurochemical alterations might be responsible for epileptogenesis or seizures . If seizures can be induced by particles of certain types in the cortex, this could have relevance for medical nanorobots navigating or residing in these spaces.
Motile nanorobots performing missions in brain tissue can be injected directly into nonvascular regions of brain tissue, thus entirely avoiding the blood-brain barrier (BBB) which serves as a formidable obstacle for traditional drug molecules, particularly peptides. According to one excellent brief summary , the BBB is found in all vertebrate brains and is formed around the endothelial cells of the brain capillaries (~640 km of vessels of total surface area ~9.3 m2). The endothelial cells comprising the tubular capillaries in brain are cemented together by intercellular tight junctions which eliminate a paracellular pathway of solute movement through the BBB, and the virtual absence of pinocytosis across brain capillary endothelium  eliminates transcellular bulk flow of circulating solute through the BBB. “Under these conditions, solute may gain access to brain interstitium via only one of two pathways: lipid mediation or catalyzed transport. Lipid-mediated transport is restricted to small molecules (with a molecular weight less than a threshold of approximately 700 Da) and is generally, but not always, proportional to the lipid solubility of the molecule. Catalyzed transport includes carrier-mediated or receptor-mediated processes. The BBB is actually composed of two membranes in series: the lumenal and the ablumenal membranes of the brain capillary endothelial cell, which are separated by approximately 300 nm of endothelial cytoplasm.” 
While there are direct routes for nanorobots into brain tissue that avoid the BBB (e.g., injection into the neuropil [6089, 6090], injection into cerebrospinal fluid , histonatation (Section 9.4.4), etc.), some mission scenarios might require bloodborne medical nanorobots to cross the blood-brain barrier. It has long been known that passive particles of colloidal size can receive special coatings that engage various naturally occurring endocytic and transcytic transport mechanisms  while causing no large-scale openings in the tight junctions of the brain endothelium . For example, polysorbate 80- or 85-coated biodegradable polybutylcyanoacrylate (PBCA) nanoparticles trigger phagocytic uptake by brain blood vessel endothelial cells  which allows particle-bound small molecules that normally do not cross the BBB to be transported across it. Overcoating with polysorbates apparently leads to the adsorption of apolipoproteins from blood plasma onto the nanoparticle surface [6095, 6096], whereupon the coated particles mimic low density lipoprotein (LDL) particles and can interact with the LDL receptor, leading to their uptake by the endothelial cells . Small cargo molecules that have been transported in this manner experimentally through the endothelium and thence into the neuropil include the Leu-enkephalin (analgesic) hexapeptide dalargin [6098-6100], the Met-enkephalin kyotorphin , the antitumor antibiotic doxorubicin , the NMDA receptor antagonist MRZ 2/576 , loperamide  and tubocurarine . The lipophilic antitumor drug camptothecin , the drug 3',5'-dioctanoyl-5-fluoro-2'-deoxyuridine (DO-FUdR) , tobramycin  and idarubicin  have been transported into the brain using ~200-nm solid lipid nanoparticles, and similarly the antitrypanosomal drug diminazenediaceturate has crossed the BBB using 364-442 nm lipid-drug conjugates . Some BBB penetration has even been shown by long-circulating pegylated nanoparticles .
However, nanorobots will most likely need to enter the neuropil themselves – not merely broadcast small-molecule effluents into it through the BBB from an extraendothelial location, or release cargo molecules from an intraendothelial waystation. In a cell culture model of the BBB using a co-culture of bovine brain capillary endothelial cells and rat astrocytes, lipid-coated ionically-charged nanoparticles 60-nm in diameter have been induced to cross the BBB by transcytosis without any degradation . More significantly, the BBB can be temporarily and reversibly opened to allow small-particle passage by osmotic disruption [6087-6089] via intracarotid infusion of hypertonic saccharide solution , e.g., mannitol or arabinose, which results in transient shrinkage of cerebrovascular endothelial cells with subsequent increased permeability of the tight junctions [6114, 6115]. This allows the passage of magnetite-dextran nanoparticles [6116-6118] (e.g., MION nanoparticle unimodal hydrodynamic diameter ~40 nm ), replication-defective adenovirus [6118-6120] (70-90 nm diameter ) particles, and herpes simplex virus (HSV) [6118, 6120, 6121] (150-200 nm diameter ) particles through the BBB endothelium and into the neuropil. The BBB can also be reversibly opened for some small molecules using the vasoactive peptide bradykinin analog Cereport (RMP-7, receptor mediated permeabilizer-7) , though apparently bradykinin itself is not as effective .
The BBB is also disrupted during diseases such as experimental allergic encephalomyelitis [6126, 6127], HIV encephalitis , and multiple sclerosis in which >1000-nm-size T cells and macrophages invade neural tissue through BBB tight junctions, and during experimental bacterial meningitis which produces focal pial venular leaks of in situ perfused 0.01% colloidal carbon black . Nanorobots could similarly locally manipulate the signaling pathways involved in BBB tight junction regulation , possibly commanding junctional gaps to open or close at need – e.g., ICAM-1-mediated signaling in brain endothelial cells is known to be a crucial regulatory step in the process of lymphocyte recruitment and migration through the BBB .
Fenart et al  notes that the customary drawback to methods that involve an increase in BBB permeability is that there is poor specificity, with circulating blood compounds such as albumin gaining indiscriminant and pathological access to the brain. However, in the case of medical nanorobots these methods could be applied on a highly localized basis, followed by rapid convoy formation entry (Section 184.108.40.206). A similar solution involves the protein transduction domains (PTDs) – naturally-occurring protein sequences that allow rapid crossing of cell membranes of all mammalian cell types without compromising membrane structure or function . PTDs have been demonstrated as suitable for in vivo delivery of “peptides, small proteins, full-length enzymes, DNA oligomers, peptide-nucleic acid oligomers, liposomes, and magnetic nanoparticles” across the blood brain barrier , and these “keys to the city” could in principle also be applied locally.
Additionally, the BBB is not a structurally perfect barrier. Gaps and imperfections of various sizes are naturally present. Nanorobots seeking entry to the neuropil from the bloodstream can search out and exploit these randomly-placed natural junctional gaps. BBB ultrastructure has been lightly studied  and 0.5-micron perijunctional gaps have been observed , but the author can find in the literature no precise estimate of the number density or distribution of micron-size gaps throughout the entire BBB network of the human brain. In one rat experiment  it was found that in control animals 0.4%-0.6% of circulating albumin appeared in the subendothelial space and in the basement membrane of control animals prior to osmotic disruption (rising to 56% 30 minutes after osmotic disruption), so many gaps of some size clearly exist. Another study  reports 0.5%-2.4% BBB penetration by various peptide molecules prior to BBB disruption. Certain brain regions (e.g., selected circumventricular organs including the pineal gland, neurohypophysis, and choroid plexus) are known to have particularly leaky BBB capillaries . The area postrema deserves mention as another possible site of circumventricular entry, as it is also the chemoreceptor trigger zone and is often the site that triggers nausea and vomiting in response to detection of toxic substances in the bloodstream. Hypertension can produce measurably leaky venules  and other leaks in the BBB . And while glycated albumin-gold colloid complexes injected into the common carotid artery do not significantly permeate the BBB, nevertheless “a few” gold particles are observed in the perivascular neuropil after 15 minutes .
If a population of Nbot nanorobots of (assumed cubical) volume Vbot transit in convoy (Section 220.127.116.11) at velocity vbot through randomly-placed >Vbot2/3-area holes in the BBB (i.e., large enough to admit one nanorobot at a time) of collective hole area Atotal with the objective of infusing the entire nanorobot population into the neuropil in tinfusion seconds, then vbot = Nbot Vbot / Atotal tinfusion. Taking Nbot = 109 nanorobots, Vbot = 1 µm3, and tinfusion = 100 sec, then even assuming a very conservative transit speed of vbot = 100 µm/sec, the total area of all ~micron-size holes need only be Atotal = 10-7 m2 or just ~0.000001% of the total BBB surface area. If the actual total area of micron-size holes Atotal is less than this 105 µm2, the transit velocity vbot or the infusion time tinfusion may be increased as required.
Even in the complete absence of large BBB junctional gaps as posited above, properly mission-designed active motile nanorobots could employ a combination of cytopenetration (Section 9.4.5), in cyto locomotion (Section 9.4.6) and histonatation (Section 9.4.4) through the BBB to achieve ready access to the neuropil.
Other aspects of nanorobotic neurobiocompatibility discussed elsewhere in this Volume include the hypothalamic induction of hypo- or hyperthermia analogously to Shapiro Syndrome (Section 15.2.7), the fate of microparticles placed in the brain (Section 18.104.22.168.1), neuronal chemorepellents (Section 22.214.171.124.1), neuronal exocytosis (Section 126.96.36.199.6), mechanical strain (Section 188.8.131.52) and membrane wounding (Sections 184.108.40.206.1 and 220.127.116.11.2) in neural cells, nanorobot-induced neural cytoskeleton disorganization (Section 18.104.22.168.1), motor neuron diseases (Section 22.214.171.124.2), intracellular shock wave damage to neurons (Section 126.96.36.199), the possibility of mechanically-induced neuron apoptosis (Section 188.8.131.52), and storage diseases in neurons (Section 184.108.40.206).
Last updated on 30 April 2004