3. 1. Assay validation
The calibration standards and quality controls for both pre-study and in-study validation were prepared as described in the experimental section. Measurement of assay precision (determined as percent coefficient variation (%CV)) and accuracy (determined as percent difference from theoretical (PDFT), with the theoretical value corresponding to the value of the standard curve) were calculated from seven and twelve standard curves for pre-study and in-study validation, respectively. The results of the first (pre-study) phase are presented in . The results of the second (in-study) phase are summarized in . In both phases we have observed high precision and accuracy, in that both the %CV and PDFT are less than 10%. The lowest calibration standard quantification was slightly less accurate (PDFT=-14.3%) in the second phase (). These values are within the limits suggested for analytical assays by the US FDA29
and those used in industry in support of pharmaceutical development27, 30
Assay precision and accuracy
To determine intra-assay (i.e. within one run) precision and accuracy, quality control hemoglobin standard samples were analyzed six times in one pre-study validation run. The results are presented in . Both precision and accuracy in this test were high, as %CV and PDFT were below 10%.
Inter-assay precision and accuracy were determined by analysis of three quality control hemoglobin standard samples, each analyzed the pre-study and in-study validation, as described above. The quality control samples were prepared from a hemoglobin standard of a known concentration of 80.0 mg/mL. Theoretical (calculated) concentrations of the quality control samples were as follows: 0.0625 (low), 0.125 (mid) and 0.625 (high) mg/mL. The results for pre-study validation are presented in . The results of in-study validation for quality controls are summarized in . The data demonstrated high precision and accuracy (%CV and PDFT <10%), with the exception of one low QC level in the in-study validation, for which variability between runs (%CV) was 12%. This low precision is still within the limits suggested by the FDA, and was for the lowest concentration calibration standard, which is most prone to variable absorbance readings.
This assay validation demonstrates that our 96-well format assay for quantitative determination of hemoglobin is reproducible and robust. Although there is no formal recommendation to the cut-off limit for precision and accuracy of the hemoglobin-specific assay, the obtained values are within requirements described for small molecules, i.e. within 15% 27, 29
3.2. Selection and Qualification of Positive and Negative Controls
An important aspect of an accurate evaluation of a material’s hemolytic properties is the use of relevant positive and negative controls. ASTM standard F-756-00 was developed for medical devices, and uses controls which are impractical for the evaluation of nanoparticles. Since the small size and unique physicochemical properties of nanoparticles may cause a variety of nanoparticle-specific interferences, initially, we aimed to include controls which were nanoparticles themselves. Polyethyleneglycol (PEG) solution was chosen as a negative control since this polymer is frequently attached to nanoparticles to increase compatibility with blood components1, 31
. Selection of the positive control was more challenging; originally we chose cationic polystyrene nanoparticles with nominal sizes of 20, 50 and 80 nm. Initially, high hemolytic activity was observed with each of these formulations (data not shown). However, when the surfactant/detergent Triton X-100 is removed from the polystyrene particle solution by dialysis prior to the assay, the particles adsorb plasma-free hemoglobin, aggregate and are removed along with undamaged cells during centrifugation. Triton X-100 is known to be hemolytic10
, and this was the reason for excluding polystyrene nanoparticles from positive control qualification. Another polymer which may produce a hemolytic response due to its high positive charge is Poly-L-lysine (PLL). Initially, this polymer, when tested in our assay, produced percent hemolysis greater than 8%, which according to the ASTM standard F756 qualifies this material as a positive control. Inter-assay performance of the positive (PLL) and negative (PEG) control samples was evaluated during pre-study validation. We compared the percent hemolysis calculated for each control sample from six validation runs. The results are presented in . To compare intra-assay performance, the positive and the negative control samples were analyzed six times in one validation run. The results are also presented in .
Qualification of positive and negative controls
Further studies of PLL in 96-well format assay revealed a high degree of inter-lot variability, depending on the lot of PLL and storage time, the percent hemolysis observed with this material varied from 5 to 70% (). Although precision and accuracy of PLL in pre-study validation conducted using the same lot of polymer met the acceptance limits (i.e. %CV and PDFT <15%, ), the high degree of inter-lot variability () disqualified this material from further use as positive control in this assay.
PLL disqualified as a positive control
The only material, which produced hemolysis consistently over the entire two year period was Triton-X 100 (). This is consistent with previously published studies12
, although other studies have used distilled water as a positive control14
. As with the calibration standards and quality controls, the variability in percent hemolysis (%CV) induced by Triton-X 100 and PEG in in-study validation has met the industrial requirement of being within 15%27, 30
Other parameters evaluated during assay validation included testing the effects of using freshly drawn blood versus stored blood, various blood incubation times with the test-materials, and different types of mixing during incubation (rotation versus shaking every 30 minutes). The results of these tests indicated that freshly-drawn blood can be stored up to 24 hours at 2-8°C with no appreciable effects on assay performance; prolonged storage (36 and 48 h) resulted in gradual increase in plasma free hemoglobin (PFH), which disqualified blood samples with PHF levels above 1mg/mL from the use in nanoparticle hemolysis test. The optimal time of sample incubation was 3 hours, and there was no significant difference in test results when rotation was used instead of mixing every 30 minutes (data not shown).
3.3. Nanoparticle interference with the assay and approaches to overcome it
The protocol described herein relies on the use of human blood anti-coagulated with Lithium-heparin. Other anti-coagulants are available commercially and may be used for this assay26
. The same applies to the assay buffer. All nanoparticles in this study were suspended and diluted in calcium and magnesium free PBS. If a test nanomaterial is prone to agglomeration in PBS, other erythrocyte-friendly buffers, e.g. saline, may be used instead. We used this protocol for analysis of a variety of nanoparticles: nanoliposomes, PAMAM dendrimers, triazine dendrimers, nanoemulsions, water-soluble fullerene derivatives, and polystyrene nanoparticles, and repeatedly observed nanoparticle interference with the assay. Several examples of this interference are summarized below.
Generation 6 (G6) amine-terminated PAMAM dendrimers tested in this assay at concentration of 125 μg/mL resulted in ~5% hemolysis (data not shown). This was consistent with previous studies of various nanomaterials demonstrating that cationic particles are more “reactive” than anionic or neutral particles and can damage cells (reviewed in ref.1
). When G5 amine terminated dendrimers were included in the analysis, no plasma-free hemoglobin was detected in supernatants (). When samples were analyzed during various steps of incubation, rapid (within first 15 minutes) coagulation of blood was observed (). Erythrocytes in the clot were protected from hemolysis, and blood clots were removed from the supernatants by centrifugation, giving a false-negative result. These findings were supported by a platelet aggregation study, in which G5 amine-terminated dendrimers caused approximately 80% platelet aggregation (). This data demonstrates that visual sample examination during all assay steps is important for the accurate interpretation of test-results. Another example of nanoparticle interference was observed when polystyrene nanoparticles were tested. shows the results of our assay for determination of the hemolytic properties of polystyrene nanoparticles. In this commonly used protocol, 20 and 50nm polysterene nanoparticle size standards were incubated in whole blood, the blood was centrifuged to remove undamaged erythrocytes and nanoparticles, and the percent hemolysis was determined by colorimetric detection of hemoglobin in the supernatant. Under these conditions, untreated (i.e. commercially supplied) particles with 20 and 50nm diameters were strongly hemolytic. In the case of the 50nm particles, spectroscopic analysis indicated a reduction in hemolysis following dialysis. Visual inspection of the microcentrifuge tubes (), however, showed the dialyzed 50 nm particles adsorb hemoglobin (compared to control tube), and the adsorbed hemoglobin precipitates with the particles upon centrifugation – yielding a false negative result. This phenomenon was not observed with polystyrene nanoparticle less than 50nm in size, but was evident for larger, 80nm polystyrene nanoparticles.
Nanoparticles interfere with the hemolysis assay
The most common mechanism of interference is due to the nanoparticle absorbance at or close to the assay wavelength (540 nm). This can be seen from , which shows the UV-Vis spectra of several nanoparticle samples. The third column of shows that the fullerene derivative, C3, causes hemolysis of almost 20%. However, when the same nanoparticles were used in a control sample without blood (i.e. sample containing all assay components except the blood is substituted with PBS), the absorbance of this sample at 540 nm was also very high and when extrapolated against the hemoglobin standard curve corresponded to a percent hemolysis of 18% ( 4th column). The same was true for citrated gold nanoparticles, some nanoemulsions, fullerene derivatives, and doxorubicin-loaded particles, all of which absorb near 540 nm (). For heavy particles (e.g. gold nanoparticles), an extra centrifugation step was used for removing the particles from the supernatant prior to evaluation on the plate reader. However, for the majority of nanoparticles, removal by centrifugation was not possible due to their small size and high solubility. For these particles, the results of the hemoglobin assay can be adjusted by subtraction of the absorbance of the no-blood control (i.e. sample containing all assay components except the blood is substituted by PBS). For example, for the C3 fullerene derivative, this adjusted result corresponds to a much lower percentage hemolysis ( last column). If the degree of interference is very high (i.e. OD value of nanoparticle sample is above that of the highest calibrator in the hemoglobin standard curve), then dilution of the nanoparticle sample is required prior to test in order to obtain accurate results after the adjustment procedure.
Nanoparticle optical properties as a source of interference with hemolysis assay
3.4. Relevance of in vitro assay to the in vivo testing
To evaluate the relevance of the described in vitro
assay for analysis of nanoparticle hemolytic properties, we have tested three nanotechnology-derived formulations approved by the US FDA for use in clinical applications. This analysis included, Doxil® (a liposomal formulation of doxorubicin), Abraxane® (albumin bound Paclitaxel nanoparticles) and, Propofol®. (a nanoemulsion-based anesthetic agent). Doxil® and Propofol® interfered with the assay when used at high concentrations. The results shown in are adjusted to account for this interference. A low percentage hemolysis was observed with both formulations in vitro
and it appeared to have no or weak relationship to the concentration of nanoparticle. This is in agreement with studies reporting low levels of erythrocyte damage by Doxil® in vivo
, and Propofol® in vitro32-35
. In both cases it was dose dependent, and minimized by using lower doses of formulations32-35
. It is important to notice that when both Doxil and Propofol were analyzed at high concentration (labeled as “stock” in ), the OD value of nanoparticle only control was above that of the assay highest calibrator. Subtraction procedure applied to adjust test result for the interference as described in the section above may not be accurate due to the high degree of nanoparticle interference with the assay. According to several studies in vitro
percent hemolysis is rated as “no concern’ when it varies from 5 to 25%36-39
. Our test results with Doxil® and Propofol® meet the criteria of some of these studies36
. Abraxane® did not induce any damage to red blood cells when tested in vitro
in our assay (), and there is no report in the literature on the hemolytic activity of this drug in vivo
Hemolysis assay applied to nanoparticulate pharmaceuticals approved fro clinical use
Thus, the assay described in our study demonstrated comparable negative test results for nanotechnology derived formulations tested in vivo. Although more comprehensive study is required to prove in vivo relevance of this in vitro method, the described assay can be used to exclude potentially harmful formulations from early preclinical testing (if in vitro result is >50% hemolysis) and to suggest potential complications to monitor during in vivo studies of nanomaterials.