Blood Contact Properties

One important aspect of nanoparticle used for medical applications is the assurance that they will not cause toxicity to blood elements when injected into a patient.

Hemolysis (i.e., damage to red blood cells) can lead to life-threatening conditions such as anemia, hypertension, arrhythmia, and renal failure. In our laboratory we have developed a protocol to evaluate hemolytic properties of nanoparticles based on the existing ASTM International standard used to characterize other materials.147,156 We have identified several problems when applying existing protocol for nanoparticles characterization. For example, colloidal gold nanoparticles with size 5-50 nm have absorbance at 535 nm, which overlaps with the assay wavelength of 540 nm. Removal of these particles by centrifugation was required prior to sample evaluation for the presence of plasma-free hemoglobin to avoid false-positive results. Though it worked well for gold particles with size 10-50 nm, centrifugation may be problematic for other nanoparticles. For example, small colloidal gold particles with a size of 5 nm require higher centrifugation force to be removed from the supernatant. Hemoglobin has a size of 5 nm; therefore, one cannot exclude the possibility that ultracentrifugation of supernatant may pellet hemoglobin along with the gold particles and thus result in a false-negative result. Ultracentrifugation is not feasible for fullerenes or dendrimer particles. Analysis of polystyrene particles of 20, 50, and 80 nm revealed another complication. We found that particle preparation damages red blood cells; the damage is caused by the surfactant used during particle manufacturing, and is detected for 20- and 50-nm particles. For 80-nm particles, the hemolysis assay showed false-negative results due to the adsorption of hemoglobin by the particles. When we applied dialysis to remove surfactant, 50-nm particles revealed same phenomenon as 80-nm particles, i.e., they caused hemolysis, but adsorbed hemoglobin resulting in a false-negative result. Another potential problem with the application of standard hemolysis protocol for nanoparticles characterization is that metal-containing particles may oxidize hemoglobin and result in a change in assay OD responses. Therefore, assay procedures may require slight modifications depending on the particle type. In general, inclusion of particle test samples without blood allowed for quick assessment of the potential particle interference with the assay. In some instances, deduction of the result generated for blood-free particle control from that obtained for blood-plus particle sample was possible and allowed estimation of particle potential to cause damage to erythrocytes.

Blood coagulation. Blood coagulation may be affected by nanomaterials. For example, modification of surface chemistry has been shown to improve immunological compatibility at the particle-blood interface: application of polyvinyl chloride resin particles resulted in 19 + 4% decrease in platelet count, indicating platelet adhesion/aggregation and increased blood coagulation time; the same particle preparation coated with PEG affected neither the platelet count nor elements of coagulation cascade.157 Similarly, folate-coated and PEG-coated Gd nanoparticles did not aggregate platelets or activate neutrophils.158 The evaluation of nanoparticle effects on blood coagulation includes studies on platelet function and coagulation factors. The in vitro cascade includes platelet aggregation assay and four coagulation assays measuring prothrombin time, activated partial thromboplastin time, thrombin time, and reptilase time.

Interaction with plasma proteins. High-resolution, 2D PAGE may be the method of choice to investigate plasma protein adsorption by nanoparticles. 2D PAGE has been used in several labs to isolate and identify plasma proteins adsorbed on the surface of stealth polycyanoacrylate particles8, liposomes159,160, solid lipid161, and iron oxide nanoparticles.162 Proteins commonly identified on several types of nanomaterials include antithrombin, C3 component of complement, alpha-2-macroglobulin, haptoglobin, plasminogen, immunoglobulins, albumin, fibrinogen, apolipoprotein, and transthyretin; albumin, immunoglobulins, and fibrinogen are the most abundant. Studies using this approach revealed that surface chemistry is important for protein adsorption. For example, Peracchia et al. demonstrated that coating with PEG results in approximately a fourfold reduction in protein binding by polycyanoacrylate particles.163 Gessner et al. prepared polystyrene latex model nanoparticles with different surface charges. This study demonstrated that increasing surface charge density results in a quantitative increase in plasma protein adsorption, but did not show significant differences in the qualitative composition of the absorbed protein mixture.164

One of the most important step in this procedure is the separation of particles from plasma after incubation is complete. Ultracentrifugation was shown to be successful for isolation of iron oxide, solid lipid particles, and some polymer-based particles.8,160,162 Gel filtration was applicable to liposomes,159 solid lipid, and iron oxide nanoparticles. Thode and colleagues compared four methods for isolation of iron oxide particles, i.e., ultracentrifugation, static filtration, magnetic separation, and gel filtration. Depending on the method used for particle separation from bulk plasma, different quantities of the same proteins and different species of proteins were identified on the particles of the same size and surface chemistry.162 For example, albumin was the predominant protein if static filtration and gel filtration were employed, while small quantities of this protein were found after ultracentrifugation; it was almost undetectable when magnetic separation was used. There was no difference in isolation of fibrinogen among the four methods. Comparable quantities of IgG gamma-chain were isolated using ultracentrifugation, static filtration, and magnetic separation, while gel filtration appeared to be inefficient in isolation of this protein.162 Attention has to be paid to the sample preparation to avoid artificial protein adsorption due to desorption during the separation, for example. Other critical steps are the number of washes to remove an excess of bulk plasma, the type of wash buffer, and a buffer to dislodge protein from the particle surface. In our lab we also found that using polypropylene low-retention tubes and pipette tips is crucial for isolation of particle-specific proteins (unpublished data).

Complement activation and phagocytosis. Following intravenous administration, nanoscale drug carriers may suffer a drawback in that they may be taken up by cells of the mononuclear phagocytic system. Consequently, such uptake facilitates clearance of nanoparticulate carriers and associated drugs, thus leading to a decrease in drug efficacy.165 The initial adsorption of plasma proteins such as components of complement and immunoglobulins promotes nanoparticles clear-ance.166,167 Therefore, the investigation of a nanoparticle's ability to interact with and activate a complement and uptake by mononuclear cells seems to be one of the key assays in the preclinical characterization cascade. Classical immunoassays used to evaluate complement activation, such as the total hemolytic complement assay (CH50) and the alternative pathway (rabbit CH50 or APCH50), are based on the hemolysis of rabbit erythrocytes. These hemolytic assays can be used to measure functional activity of specific components of either pathway. The main challenge in applying these assays for nanomaterial characterization is the ability of nanoparticles per se to lyse RBCs, thus generating false-positive results. To overcome this limitation, the approach for evaluation of complement activation includes two techniques. One is a qualitative yes or no rapid screen for the presence of C3 cleavage products using western blot. The second assay is a quantitative evaluation of samples found positive at an initial screen for the presence of C4a, C3a, and C5a components of complement using a flow cytometry-based multiplex array.

The difficulties with application of standard phagocytosis assay are: (1) light microscopy used in traditional phagocytosis assay168 is not applicable to nanoparticles due to their smaller size; (2) when light microscopy is substituted with TEM, visualization of particle may be complicated since their size is similar to that of cell organelles resulting in ambiguous interpretation of TEM data, thus TEM is limited to electron dense metal containing particles (see Figure 7.4); (3) labeling of nanoparticles with fluorescent tags9,169 is a superior approach for visualizing internalized particles, but should be avoided in preclinical tests as chemical attachment of fluorophore may create a new molecular entity with properties widely divergent from those of the original particle; (4) application of luminol to detect phagocytosed particles8 provides an exceptional technique which can overcome all limitations listed above; however, it is not free of applicability reservations as well (e.g., nanoparticles may interfere with activation of luminol once it is internalized, etc.).

CFU-GM assays. CFU-GM assays allow for the evaluation of potential nanoparticles interference with growth and differentiation of bone marrow stem cells into granulocyte and macrophages. This assay may provide valuable information for development of anti-cancer nano-technology platforms. Myelosuppression is a very common dose-limiting toxicity associated with the use of oncology cytotoxic drugs. Incorporation of such drugs into nanoparticle carriers targeted to specific cancer cells may help to reduce toxicity to normal tissues, including bone marrow, and should be considered during initial characterization of nanocarriers.

FIGURE 7.4 Study of internalization of 30-nm colloidal gold nanoparticles by murine macrophage cell line RAW 264.7 by TEM. (a) Analysis of single cell. (b) Zoom-in analysis of the selected area in the same cell.
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