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
126.96.36.199 Biocompatibility of Diamond Particles
In biomaterials research, it has been found [630, 631] that even though a bulk material may be well tolerated by the body, finely divided particles of the same material can often lead to severe and even carcinogenic complications in test animals. Differences in particle size influence histological reactions  and cytokine production . Many nanomedical applications will involve “particle” sized diamondoid objects (e.g., micron-scale individual medical nanorobots) so it is of great interest to review the experimental data relating to the reactions of specific cells to the presence of diamond particles. We already know that finely divided carbon particles are well tolerated by the body  – the passive nature of carbon in tissue has been known since ancient times, and both charcoal and lampblack (roughly spherical 10-20 nm particles) have been used for ornamental and official tattoos  (Section 188.8.131.52). Diamond particles are also well tolerated by cells:
(1) Neutrophils. A 1982 report of possible crystal-induced neutrophil activation [635-638] by 2- to 8-micron amorphous diamond crystals  was never confirmed. Indeed, to the contrary, diamond particles are traditionally regarded as biologically inert and noninflammatory . For example, Hedenborg and Klockars  used 4- to 8-micron diamond dust as an inert control in their experimental work, and found that diamond dust did not stimulate the production of reactive oxygen metabolite by polymorphonuclear (PMN) leukocytes – a proposed pathway for chronic inflammation and tissue injury of the lung (Section 15.1.2). Tse and Phelps  found that 3-micron diamond dust crystals in a 2 mg/cm3 concentration (~0.06% Nct; i.e., nanocrit, concentration in fluid, by volume, Section 184.108.40.206) were phagocytized by 21% of PMN cells (present at 7250 cells/mm3 concentration) after 45 minutes, but no chemotactic activity was generated. Higson and Jones  exposed horse and pig neutrophils to urate, hydroxyapatite, pyrophosphate and brushite crystals (all implicated in joint inflammation) – which induced superoxide and peroxide generation in a concentration- and temperature-dependent fashion – but exposing the neutrophils to diamond crystals at 37 oC produced no effect. Yet another experiment  tested the ability of various crystals to stimulate phagocytosis, degranulation, and secretion of cell movement (motility) factors (CMFs) from polymorphonuclear leukocytes. The experiment found that hydroxyapatite (HA) crystals stimulated some enzyme release and CMF generation, and monosodium urate monohydrate (MSUM) crystals much more so. But 4- to 8-micron diamond crystal fragments in suspension up to ~0.2% Nct in culture, while clearly interacting with PMN leukocytes, did not stimulate degranulation, CMF production, or cell death even at high crystal concentrations. MSUM and HA particles are generally regarded as having atomically “rough” surfaces with a negative surface charge or Zeta potential, whereas diamond particles are considered relatively “smooth” with little or no surface charge [633, 640].
(2) Monocytes and Macrophages. It has long been known that free carbon and diamond particles are ingested by cultured macrophages without harmful effects. For example, cells that have taken up large amounts of 2- to 4-micron diamond dust remain healthy for at least 30 hours, whereas cells succumb rapidly after ingesting silica . Phosphatase enzyme discharged into diamond-containing phagosomes by adherent lysosomes did not escape into the cytoplasm or nucleus , indicating that diamond does not damage these organelles (Section 220.127.116.11.4). In a more recent study , 2- to 15-micron particles of diamond, silicon carbide (SiC), hydroxyapatite (HA) and polymethylmethacrylate (PMMA) were suspended in serum-free cultures of human monocytes at a concentration of 0.5 mg/cm3 (~0.01% Nct in culture). All particles were phagocytosed, but while monocyte morphology changed after the ingestion of SiC and HA, there was no change after the ingestion of diamond, indicating no activation of the monocytes by the diamond. Interleukin-1beta production was indistinguishable for control and diamond cultures, but increased 30-fold in the HA cultures, 38-fold in the cultures exposed to SiC, and in a similar range to HA and SiC for the PMMA. The authors  concluded that diamond particles in serum-free monocyte culture are inert, despite being phagocytosed, unlike most other particles. They offered several possible explanations for this: differences in opsonization, surface charge, or intracellular ion release . Alternatively, different particles may be phagocytosed through different receptors on the monocyte surface. Macrophage responsiveness to diamond particles pre-exposed to protein-rich serum has not been extensively investigated, however.
(3) Fibroblasts. Early studies in the 1950s  and 1960s  found that micron-size diamond dust particles did not induce fibrogenic activity. Schmidt et al  note that diamond dust is nonfibrogenic in human monocyte-macrophages found in the lungs. In other words, fibroblasts are not recruited by macrophages in response to the presence of diamond dust. Diamond dust of sizes <0.5 micron and 1-2 microns did not induce the release of thymocyte proliferation factor or fibroblast proliferation factor at diamond particle concentrations up to ~0.1 mg/cm3 (~0.003% Nct in culture) . In another experiment , synthetic hydroxyapatite crystals at a concentration of 50 µg/cm3 in 1% and 10% serum stimulated 3H-thymidine uptake into quiescent canine synovial fibroblasts and human foreskin fibroblast cultures. Calcium pyrophosphate dihydrate crystals also stimulated uptake, as did calcium urate crystals markedly and sodium urate crystals more modestly. But 1- to 5-micron diamond crystals had no mitogenic effect on the fibroblasts at particle concentrations up to 0.4 mg/cm3 (~0.01% Nct in culture).
(4) Other Cells. The reactions of regenerating rabbit bone tissue to phagocytosable particles were studied  by dispersing various particles in hyaluronan and then introducing them into an implant-traversing canal, forming a bone harvest chamber. Tissue that entered the canal during the following 3 weeks was harvested. Particles of high density polyethylene, bone cement and chromium-cobalt injected in this fashion all provoked an inflammatory reaction in tissue entering the canal and caused a marked decrease in the amount of ingrown bone. But the phagocytosable 2- to 15-micron round-shaped diamond particles – introduced at a number density of ~60 million/cm3 (~0.7% Nct in culture) – produced no decrease in bone formation and appeared “comparatively harmless...there was no obvious cellular reaction to these particles.” Histologically, the diamond particles aggregated into clumps. Occasionally macrophages were seen nearby, but phagocytic cells remained few and dispersed, despite containing large amounts of ingested particulate diamond. There was no concentration of macrophages and giant cells such as is usually seen when PMMA or high-density polyethylene particles are implanted. Interestingly, 8- to 15-micron SiC particles also produced no inflammation or decrease in bone formation, even though the particles were “elongated splinters with sharp edges.” Finally, neurologist Stephen S. Flitman [personal communication, 1999] notes that diamond has never been shown to be neurotoxic.
(5) Inflammation. Tse and Phelps  found that 3-micron diamond crystals in a 10 mg/cm3 concentration (~0.3% Nct injection fluid) injected into canine knee joints produced little evidence of inflammation – intra-articular pressure remained low, along with the local cell count. Diamond particles are generally considered noninflammatory relative to the complement system  (Section 18.104.22.168), and produce no inflammation or edema in animal models [1848, 1849].
(6) Hemolysis. Dion et al  observed no detectable hemolysis in vitro by various ceramic powders tested, including diamond, graphite and alumina, after 60 minutes of exposure to a powder concentration of ~0.5 gm per cm3 of diluted blood (~14% Nct in vitro). The diamond powder in this experiment assayed ~1.25% impurities, mostly Zr.
(7) Other Biological Systems. Diamond particles have been found to have an “adjuvant” effect on one fungus-based insecticide against beetles , probably due to the abrasive properties of these particles.
(8) Adamantanes. Single-molecule units of diamond called adamantane (C10H16), when properly functionalized, possess useful pharmacological properties  including antiviral [5513-5521] (including anti-HIV [5522-5524]) activity, antiparkinsonian and antidementia activity [5525-5528], some anti-tumor activity [5529, 5530, 5565] though with toxicity problems at high dose , analgesic effects , and enhancement of immunotoxin activity . Interactions have been investigated between adamantanes and plasma proteins , cell adhesion proteins , enzymes [5534-5543, 5574], and receptors or channels [5558-5560]; with bacterial metabolism [5544, 5565, 5567] and viral assembly ; and with polynuclear [5545-5547], mononuclear [5548-5552], and peripheral blood  leukocytes. Lipid bilayer effects  and cellular uptake  of adamantane conjugates has been studied. Adamantane-based drugs such as amantadine [5554-5556] and tromantadine  are nearly completely excreted unchanged in the urine, and typically no metabolites having a hydroxylated adamantane ring system can be detected . Of course, the properties of crystalline diamond are due to its molecular structure in which each carbon is in sp3 hybridization and is bound to four other carbon atoms, and in n=1 adamantane, there is no single carbon that is bound to four other carbons. Polyadamantanes up to n=4 units have been chemically synthesized  and polyadamantanes up to n=11 units have been identified, extracted, purified and crystallized from natural petroleum [5841, 5842], although the biomedical properties of these nanometer-sized diamond molecules have yet to be investigated.
Last updated on 30 April 2004