The Neuromuscular Origins of Kinematic Variability during Perturbed Walking OPEN
Published: xx xx xxxx

Heather E Stokes
Joint Department of Biomedical Engineering
University of North Carolina at Chapel Hill and North Carolina State University
Chapel HillNCUSA

Jessica D Thompson
Joint Department of Biomedical Engineering
University of North Carolina at Chapel Hill and North Carolina State University
Chapel HillNCUSA

Jason R Franz firstname.lastname@example.com
Joint Department of Biomedical Engineering
University of North Carolina at Chapel Hill and North Carolina State University
Chapel HillNCUSA

The Neuromuscular Origins of Kinematic Variability during Perturbed Walking OPEN
Published: xx xx xxxx10.1038/s41598-017-00942-xReceived: 21 October 2016 Accepted: 20 March 20171 Scientific RepoRts | 7: 808 |. Correspondence and requests for materials should be addressed to J.R.F. (email:
We investigated the neuromuscular contributions to kinematic variability and thus step to step adjustments in posture and foot placement across a range of walking speeds in response to optical flow perturbations of different amplitudes using a custom virtual environment. We found that perturbations significantly increased step width, decreased step length, and elicited larger trunk sway compared to normal walking. However, perturbation-induced effects on the corresponding variabilities of these measurements were much more profound. Consistent with our hypotheses, we found that: (1) perturbations increased EMG activity of the gluteus medius and postural control muscles during leg swing, and increased antagonist leg muscle coactivation during limb loading in early stance, and (2) changes in the magnitude of step to step adjustments in postural sway and lateral foot placement positively correlated with those of postural control and gluteus medius muscle activities, respectively, in response to perturbations. However, (3) interactions between walking speed and susceptibility to perturbations, when present, were more complex than anticipated. Our study provides important mechanistic neuromuscular insight into walking balance control and important reference values for the emergence of balance impairment.Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 Scientific RepoRts | 7: 808 |

Walking balance control involves coordinated adjustments in posture (i.e., head and trunk stabilization) and foot placement from step to step [1][2][3][4] . Particularly in unpredictable and challenging environmental conditions, these adjustments depend on the integration of reliable sensory feedback and the planning and execution of appropriate motor responses 5,6 . Accordingly, sensory and mechanical perturbations are increasingly used to study corrective motor responses in walking as well as the onset and progression of balance deficits 4, 6-12 . Kinematic variability is frequently used to quantify the magnitude and efficacy of balance corrections in the presence of perturbations 6,[8][9][10] . However, few studies have investigated the neuromuscular mechanisms underlying kinematic variability and thus step to step adjustments in posture and foot placement during walking. This is especially notable for responses to visual (i.e., optical flow) perturbations 6,[8][9][10][11] , which elicit profound increases in kinematic variability only through the visual perception of imbalance.

Electromyographic (EMG) recordings of muscle activity during perturbed walking can provide insight into the extent to which dynamic balance corrections are neurally mediated via muscular actions. For example, by imposing lateral mechanical perturbations of the swing leg, Rankin et al. 4 revealed the important role of swing phase gluteus medius activity in adjusting foot placement laterally with each step, thereby regulating mediolateral translation of the trunk relative to the stance foot 4 . However, the neuromuscular adjustments underlying corrections in response to optical flow perturbations during walking may have unique biomechanical origins. Specifically, we recently proposed that optical flow perturbations elicit postural disturbances in walking via visuomotor entrainment, wherein head and trunk kinematics synchronize to the perturbation via postural adjustments to instinctively unify visual with somatosensory and vestibular feedback 10 . We suspect that these postural disturbances subsequently elicit step to step adjustments in foot placement, particularly in the mediolateral direction, to preserve whole-body balance. Thus, our observations allude to potentially coordinated changes in postural control muscle activities to orchestrate postural adjustments with those of gluteus medius activity to regulate lateral foot placement from step to step. However, kinematic measurements alone are unable to discern passive mechanics from active control in these postural and foot placement adjustments. Indeed, we also observe robust entrainment of foot trajectories to even the most complex optical flow perturbations 10 , which is unlikely to be explained by leg muscle adjustments alone.

In addition to the step to step kinematic adjustments used to accommodate balance perturbations during walking, which we suspect occur predominantly during leg swing, those perturbations may also require increased leg joint stability, particularly during limb loading in early stance. The concurrent activation of antagonist muscles is a well-documented phenomenon in motor control thought to increase joint stiffness to improve joint stability, thereby mitigating the effects of unexpected environmental demands or perturbations [13][14][15] . For example, we and others have shown that older adults walk with greater lower leg (i.e., ankle flexor-extensor) and upper leg (i.e., knee flexor-extensor) antagonist muscle coactivation than young adults 13,15,16 . Moreover, at least in arm reaching tasks, antagonist muscle coactivation and limb stiffness increase in the presence of external force perturbations 14 . However, the neuromuscular response of antagonist leg muscles, and thus leg joint stability, when accommodating sensory perturbations during walking remains unclear.

Finally, walking speed may also systematically influence the relation between kinematic variability and muscle activity in the presence of balance perturbations. During normal, unperturbed walking, Dingwell and Marin 17 simultaneously analyzed dynamic stability and kinematic variability and suggested that walking slower improves one's resilience to perturbations, despite increasing their movement variability. However, dynamic stability quantifies resilience to small perturbations arising normally during walking, and may not reflect the response to larger balance disturbances. Intuitively, compared to walking faster, walking slower would provide more time to plan and execute appropriate kinematic adjustments to maintain balance in the presence of perturbations. Accordingly, balance perturbations should elicit smaller increases in kinematic variability and muscle activity when walking at slower speeds. However, Goodworth et al. 7 recently found that postural sway was less sensitive to surface perturbations when applied at a speed modestly faster than preferred 7 . Thus, this premise remains equivocal.

Therefore, the purpose of this study was to investigate the association between step to step kinematic adjustments and specific changes in muscle activity across a range of walking speeds using optical flow perturbations of different amplitudes. First, we hypothesized that perturbations would increase EMG activity of the gluteus medius and postural control muscles during leg swing, and increase the coactivation of antagonistic leg muscles during limb loading in early stance. Second, we hypothesized that changes in the magnitude of step to step adjustments in postural sway and lateral foot placement would be positively correlated with those of postural control and gluteus medius muscle activities, respectively, in response to perturbations. Finally, we hypothesized that perturbation-induced changes in kinematic variability and EMG activity would be smaller at slower walking speeds.

Methods

Subjects and Experimental Protocol. Twelve healthy young adults (7 females, mean ± standard deviation, age: 25 ± 4.6 yrs, height: 1.70 ± 0.10 m, mass: 65.3 ± 10.8 kg) participated in this study. The experimental protocol was approved by and performed in accordance with the relevant guidelines and regulations of the University of North Carolina Internal Review Board, and subjects provided written informed consent prior to participating. Subjects also completed a health questionnaire prior to participating to ensure they had no known neuromuscular, cardiovascular, or orthopedic diseases.

All testing was completed on a dual-belt, force-measuring treadmill (Bertec, Inc., Columbus, OH) surrounded by an immersive, semi-circular curved screen measuring 2.24 m height and 2.83 m wide (Fig. 1A). Subjects started by walking on the treadmill at a comfortable speed for 5 minutes to acclimate to the walking environment and allow their movement patterns to stabilize. Subjects were then fit to an overhead harness which they wore for the duration of the experiment. In fully randomized order, subjects then completed a series of twelve 2-minute walking trials while watching a speed-matched virtual hallway projected onto the curved screen. During some trials, motion of the virtual hallway included continuous mediolateral (ML) optical flow perturbations consisting of a sum of three sinusoids, such that the full amplitude was applied at 0.250 Hz and half that amplitude was applied at 0.125 Hz and 0.442 Hz. The perturbations were consistent with the visual feedback associated with head movements, such that the hallway's end moved very little compared to that in the foreground, thereby inducing balance corrections rather than heading corrections. At each of three speeds (0.75, 1.25, and 1.75 m/s), subjects walked both normally (i.e., unperturbed) and with each of three perturbation amplitudes (20,35, and 50 cm). We opted to analyze the measurements outlined below during the second minute of each two-minute trial to allow some time for subjects to reach a steady state neuromuscular response to the ongoing perturbations. Finally, we used a photocell timing system (Brower Timing, Draper, UT) to measure subjects' preferred overground walking speed from the average of three times taken to traverse the middle 2 m of a 10 m walkway at their normal, comfortable speed (i.e., 1.29 ± 0.18 m·s −1 ).

Measurements and Data Analysis. A 14-camera motion capture system (Motion Analysis, CA) tracked 30 retro-reflective fmarkers placed on the subject's pelvis, right and left legs, and cervical spine (C7). Three-dimensional marker trajectories were collected at 100 Hz and then low-pass filtered using a 4th order Butterworth filter and a cut-off frequency of 8 Hz. Heel strikes were identified from peaks in the fore-aft position of the heel markers relative to the sacral marker using published procedures 18 . From this data, we computed step widths (SW) from consecutive mediolateral heel positions which were averaged over a period from 12-25% of each stride corresponding to mid-stance prior to heel rise 19 .

Step lengths (SL) were calculated as the relative anterior-posterior positions of consecutive heel markers at 20% of each stride plus the treadmill belt translation during that step 8 . Using time series of SW and SL, we computed their respective variabilities (SWV and SLV) as the standard deviation over all steps taken during each trial. Finally, from the kinematic data, we quantified postural sway as the average and standard deviation of peak-to-peak mediolateral (ML) trunk motion, defined using the trajectory of a marker placed over the C7 vertebra.

In addition, after preparing the shaved skin with alcohol, we collected electromyographic (EMG) recordings of right leg and postural control muscles using wireless single differential electrodes (Trigno, Delsys, Inc., Boston, MA). Following the guidelines of Cram and Kasman 20 , we collected muscle activities at 1000 Hz of the following ten muscles: medial gastrocnemius (MG), soleus (SOL), tibialis anterior (TA), peroneous longus (PL), vastus lateralis (VL), medial hamstring (MH), gluteus medius (GM), external oblique (EO), and erector spinae (ES). We visually inspected the EMG signals while subjects contracted each instrumented muscle to ensure signal quality and electrode placement. A custom MATLAB script processed all of the EMG data (Mathworks, Inc., Natick, MA). EMG activity, bandpass filtered (20-450 Hz), rectified, and normalized to the mean value during unperturbed walking at 1.25 m/s, was averaged within six gait cycle phases: early (0-10%), midstance (10-30%), terminal stance (30-60%), initial swing (60-73%), midswing (73-87%), and terminal swing (87-100%) 19 .

Statistical Analysis. We calculated mean values of step kinematics, postural sway variability, and EMG amplitudes across all strides for each subject. First, a two-way repeated measures analysis of variance (rmANOVA) tested for main effects of and interactions between walking speed (3 levels: 0.75, 1.25, and 1.75 m/s) and perturbation amplitude (4 levels: 0, 20, 35, and 50 cm). When a significant main effect or interaction was found, we performed post-hoc pairwise comparisons versus unperturbed walking using Tukey's Honest Significant Difference to correct for multiple comparisons. We then used a series of correlations between specific changes in kinematic variability and those in EMG activity from unperturbed walking, pooled across all walking speeds and perturbation amplitudes, to test our third hypothesis. Specifically, we first calculated partial Pearson's correlation coefficients between: (i) changes in step width variability and changes in GM activity, and (ii) changes in postural sway variability and changes in GM, EO, and ES activity, while controlling for walking speed as a covariate. We focused these correlation analyses to include GM, EO, and ES activity only within those gait cycle phases identified as having a significant main effect of perturbation amplitude in the rmANOVA. In our final planned comparison, for gait cycle phases identified as having concurrent significant main effects of perturbation amplitude among these three muscles, we calculated Pearson's correlation coefficients between changes in GM activity and changes in EO and ES activity. For all comparisons, we defined significance using an alpha level of 0.05.

Results

Perturbation effects on kinematic variability and EMG activity. In response to perturbations, subjects took wider, shorter, and more variable steps and increased their postural sway variability compared to walking normally (perturbation main effect, p's < 0.001) (Fig. 1B). For example, post-hoc comparisons revealed that, on average across the range of speeds tested, subjects took 14% wider (p < 0.001) and 3% shorter steps (p < 0.001), accompanied by 73% larger SWV (p < 0.001), 36% larger SLV (p < 0.001), and 52% larger postural sway variability (p < 0.001) in response to the largest amplitude perturbation (i.e., 50 cm). Accompanying these kinematic changes, EMG activity of all but one lower leg (Fig. 2), thigh (Fig. 3), and postural control muscle (Fig. 4) increased in the presence of optical flow perturbations. Moreover, these increases were phase-dependent. Lower leg muscles increased significantly during early to midstance in a manner consistent with larger antagonist coactivations, averaging a 72% increase in the TA (p = 0.026; Fig. 2A), a 19% increase in the MG and SOL (p's < 0.025; Fig. 2B,C), and a 57% increase in the PL (p = 0.046; Fig. 2D) in response to the largest amplitude perturbation. A hip abductor, GM activity also increased by an average of up to 15% during early to midstance (p's < 0.024), but also by an average of up to 29% during terminal swing (p = 0.001; Fig. 3A), concurrent with significant increases in MH activity (p = 0.017; Fig. 3B). Also during terminal swing, EO activity increased by an average of up to 62% in response to perturbations (p = 0.049; Fig. 4A).

Correlations between kinematic variability and EMG activity in the presence of perturbations. We found moderate and significant positive correlations between perturbation-induced increases in SWV and those in GM activity during terminal swing (R 2 = 0.30; Fig. 5A). We also found significant yet more modest positive correlations between these outcome measures during early to midstance (Fig. 5B,C). Perturbation-induced increases in postural sway variability correlated significantly with EO (R 2 = 0.23; Fig. 6A) and GM (R 2 = 0.16; Fig. 6B) activity during terminal swing. Finally, during the terminal swing phase, significant increases in EO activity in response to perturbations strongly and significantly correlated with concurrent increases in GM activity (R 2 = 0.49; Fig. 6C).

Effects of walking speed on response to perturbations. Walking faster increased SWV and all

EMG amplitudes but decreased SW, SLV, and ML postural sway amplitude and variability (speed main effect, p's < 0.029) (Fig. 1B,C). For example, during unperturbed walking, SWV increased by 12% and SLV decreased by 35% across the range of walking speeds tested (Fig. 1C). Post-hoc comparisons for the kinematic outcome measures revealed that walking faster muted the response to perturbations for SL, SLV, and SW (Fig. 1B,C).     Fig. 1C). In addition, of the instrumented muscles, only the modest PL activity during midswing exhibited a significant speed by amplitude interaction, with larger perturbation effects at 1.75 m/s compared to the slower speeds (p = 0.01; Fig. 2D).

Discussion

The scientific premise of this work was motivated by the potential for sensory perturbations to reveal fundamental insight into human walking balance control and translational insight into the onset and progression of balance deficits. We found that perturbations significantly increased step width, decreased step length, and elicited larger trunk sway compared to normal, unperturbed walking. However, perturbation-induced effects on the corresponding variabilities of these measurements, indicative of step to step kinematic adjustments, were much more profound. Kinematic variability is often used as a marker of balance control and greater variability is often ascribed to people at greater risk of falls 21,22 . Thus, our primary purpose was to investigate the neuromuscular mechanisms governing observed increases in step to step kinematic adjustments in response to optical flow perturbations and their dependence on walking speed.

Compared to those during unperturbed walking, the activities of eight of nine instrumented trunk and leg muscles increased significantly in response to perturbations. But, these increases were phase-dependent in a manner that we interpret based on their functional relevance to walking balance control. First, as hypothesized, optical flow perturbations increased gluteus medius, external oblique, and erector spinae activities during ipsilateral leg swing. Growing evidence implicates gluteus medius activity during terminal swing in adjusting lateral foot placement to preserve walking balance 4,23 . Indeed, a recent neuromechanical analysis combined EMG with perturbations of swing leg trajectories and concluded that gluteus medius activity during leg swing regulates the mediolateral translation of the trunk relative to the stance foot 4 . Here, we found that perturbation-induced increases in GM activity also progressed into the early to midstance phase. However, consistent with its purported role in governing lateral step placement, and also as hypothesized, changes in terminal swing GM activity exhibited a far stronger positive correlation with step to step adjustments in lateral foot placement. In addition, we suspect that concurrent swing phase increases in erector spinae and external oblique activity serve to meet the demands of trunk lateral rotation and stability in the presence of perturbations. As hypothesized, changes in the magnitude of step to step adjustments in postural sway positively correlated with those of external oblique EMG in response to perturbations. We recently found evidence for a high degree of coordination between kinematic adjustments in posture and foot placement during walking that may be governed by neuromechanical mechanisms unique to perturbations of optical flow 10 . Specifically, we posit that optical flow perturbations elicit postural disturbances via visuomotor entrainment of head and trunk kinematics, unifying visual with somatosensory and vestibular feedback but subsequently eliciting step to step adjustments in lateral foot placement to preserve whole-body balance. Together, our current findings imply that trunk muscles such as the external oblique may actively orchestrate postural adjustments elicited by perturbations, while the gluteus medius may subsequently respond by orchestrating adjustments in lateral foot placement to preserve whole-body balance. In support of this coordination between posture and foot placement, we found that increases in GM activity and EO activity were not only simultaneous during the terminal swing phase, but were also significantly and strongly correlated (i.e., R 2 = 0.49). Future neuromechanical modeling studies may provide the definitive insight needed to substantiate these empirical results.

We also hypothesized that perturbations would increase the coactivation of antagonistic leg muscles to improve joint stability, particularly with limb loading during the early stance phase. Antagonist leg muscle coactivation, often present in people with balance deficits, is considered a feedforward control strategy to increase leg joint stiffness and thereby aid in responding when balance is challenged 16,24,25 . In support of our hypothesis, we observed simultaneous perturbation-induced increases in TA, SOL, and MG activities, antagonistic muscles spanning the ankle, only during the early to midstance phase. In addition, we found concurrent increases in PL activity, a muscle also thought to play a role in ankle joint stability 26 . Together, these lower leg muscle responses suggest that the step to step kinematic adjustments used to accommodate balance perturbations during leg swing also increase the need for ankle joint stability during early to midstance. Our results are thus well aligned with the broader motor control literature, for example that from reaching tasks, during which antagonist muscle coactivation and limb stiffness increase in proportion to the magnitude of external force perturbations 14 .

Finally, some previous studies have found that walking slower improves metrics of dynamic stability, thought to characterize one's resilience to perturbations, despite increasing kinematic variability 17 . Most of our outcome measures were speed-dependent and, in partial agreement with prior work [27][28][29] , we found that step length variability and postural sway variability increased with slower walking speed. However, interactions between walking speed and susceptibility to optical flow perturbations, when present, were more complex than anticipated. Of the speed by amplitude interactions examined across the kinematic measurements, only three emerged as statistically significant; one (i.e., step width) alluded to a larger response to perturbations at 0.75 m/s, one (i.e., step width variability) to those at 1.25 m/s, and one (i.e., postural sway variability) to those at 1.75 m/s. Compared to walking normally, subjects increased their step width in response to perturbations most at the slowest speed, 0.75 m/s. In contrast, step to step adjustments in step width (i.e., SWV) and postural sway variability increased least on average in response to perturbations applied at 0.75 m/s, a finding more consistent with the conceptual premise of Dingwell and Marin (2006). For example, perturbation-induced increases in postural sway variability were, on average, up to 81% larger at 1.75 m/s than at 0.75 m/s. Together, these findings suggest an increased dependence on, or at least utilization of, wider steps to withstand perturbations if walking balance is challenged at slower speeds, a general anticipatory strategy 30 that may alleviate the need for more reactive step to step kinematic adjustments. Though, interestingly, we did not see nearly the same extent of speed by amplitude interactions for the EMG-based measurements. In fact, only perturbation-induced increases in peroneus longus activity were speed-dependent; these increases were significantly larger at the fast walking speed. This implies an increased demand for lateral ankle stability when balance is challenged at fast walking speeds. Ultimately, given the complex nature of interactions between walking speed and susceptibility to optical flow perturbations, we can only partially accept our second hypothesis that walking slower would improve one's resilience to perturbations.

There are several limitations of this study that should be considered when interpreting our results. First, we only considered continuous, mediolateral optical flow perturbations that may not reflect kinematic and muscular adjustments, or correlations between them, when responding to a more discrete sense of imbalance. Given the continuous nature of the perturbations, we correlated subject-average increases in EMG activity with those in kinematic variability. However, there may be important and presumably complex step by step associations between muscle activity and kinematic variability that were not accounted for in this study, but could be perhaps through the use of more acute perturbations. In addition, we used complex perturbations that we presumed would be difficult for subjects to anticipate or predict. However, although unlikely, we cannot exclude the possibility that subjects learned to anticipate the perturbation dynamics. This is particularly relevant given our choice to analyze the second minute of walking data following the onset of perturbations, prior to which subjects could have exhibited some neuromuscular adaptation. While our intent was to allow some time for subjects to reach a steady state response, the reported perturbation-induced effects may be muted compared to those immediately following perturbation onset. In addition, although we studied a broad range of walking speeds, these speeds were fixed and constant on the treadmill. Subjects were thus unable to modulate their walking speed when responding to perturbations. Ultimately, it is not clear how these components of our experimental design may have influenced the results of this study.

Conclusion

In conclusion, our findings point to specific muscular contributions to orchestrating the kinematic adjustments elicited by balance perturbations, in particular those of posture and lateral foot placement, in order to preserve whole-body balance. First, we found that perturbations increased EMG activity of the gluteus medius and postural control muscles in a coordinated manner during leg swing and the coactivation of antagonist lower leg muscles during limb loading in early stance. Second, we found that perturbation-induced increases in gluteus medius and postural control muscle activities positively correlated with step to step adjustments in lateral foot placement and postural sway, respectively. Finally, we found that interactions between walking speed and susceptibility to perturbations, when present, were more complex than anticipated. Ultimately, this study provides mechanistic insight into walking balance control and important reference values for the emergence of balance deficits due to aging or disease.

Figure 1 .
1(A) Subjects walked in an immersive virtual environment while watching a hallway with and without continuous mediolateral optical flow perturbations. Group average (±standard deviation) foot placement and trunk kinematic (B) magnitudes and (C) variabilities across the three walking speeds during normal and visually perturbed walking. Asterisks (*) indicate significantly different from normal, unperturbed walking in Tukey's post-hoc pairwise comparisons (p < 0.05). SW: Step width; SL: Step length; SWV: Step width variability; SLV: Step length variability.

Figure 2 .
2Perturbation-induced changes in lower leg muscle activities. Group average electromyographic (EMG) recordings from the lower leg muscles during normal (±standard error) and visually perturbed walking as a function of the gait cycle. Amplitudes are normalized to the mean values over a complete stride walking normally at 1.25 m/s. Solid horizontal lines with numerical indicators represent gait cycle phases with significant main effects of perturbation amplitude (p < 0.05) (i.e., 1: early stance; 2: midstance; 5: midswing). Each of these identified phases is also accompanied by bar graphs illustrating the group average (±standard error) muscle activity for each condition, pooled across walking speeds. Asterisks (*) indicate significantly different from normal, unperturbed walking in Tukey's post-hoc pairwise comparisons (p < 0.05). (A) TA: Tibalis anterior; (B) MG: Medial gastrocnemius; (C) SOL: Soleus; (D) PL: Peroneus longus.

Figure 3 .
3Perturbation-induced changes in upper leg muscle activities. Group average electromyographic (EMG) recordings from the thigh muscles during normal (±standard error) and visually perturbed walking as a function of the gait cycle. Amplitudes are normalized to the mean values over a complete stride walking normally at 1.25 m/s. Solid horizontal lines with numerical indicators represent gait cycle phases with significant main effects of perturbation amplitude (p < 0.05) (i.e., 1: early stance; 2: midstance; 5: midswing; 6: terminal swing). Each of these identified phases is also accompanied by bar graphs illustrating the group average (±standard error) muscle activity for each condition, pooled across walking speeds. Asterisks (*) indicate significantly different from normal, unperturbed walking in Tukey's post-hoc pairwise comparisons (p < 0.05).(A) GM: Gluteus medius; (B) MH: Medial hamstring; (C) VL: vastus lateralis.

Figure 4 .
4Perturbation-induced changes in postural control muscle activities. Group average electromyographic (EMG) recordings from postural control muscles during normal (±standard error) and visually perturbed walking as a function of the gait cycle. Amplitudes are normalized to the mean values over a complete stride walking normally at 1.25 m/s. Solid horizontal lines with numerical indicators represent gait cycle phases with significant main effects of perturbation amplitude (p < 0.05) (i.e., 5: midswing; 6: terminal swing). Each of these identified phases is also accompanied by bar graphs illustrating the group average (±standard error) muscle activity for each condition, pooled across walking speeds. Asterisks (*) indicate significantly different from normal, unperturbed walking in Tukey's post-hoc pairwise comparisons (p < 0.05). (A) EO: External oblique; (B) ES: Erector spinae.

Based on significant speed by amplitude interactions, perturbation-induced effects were largest at 0.75 m/s for SW (p = 0.040;Fig. 1B), largest at 1.25 m/s for SWV (p = 0.013;Fig. 1C), but largest at 1.75 m/s for postural sway variability (p = 0.029;

Figure 5 .
5Correlations between changes in step width variability (SWV) and those in gluteus medius (GM) activity during (A) terminal swing, (B) midstance, and (C) early stance, pooled across walking speeds and perturbation amplitudes.

Figure 6 .
6Correlations between changes in postural sway variability and those in (A) external oblique (EO) and (B) gluteus medius (GM) activity, both during terminal swing. (C) Correlations between concurrent changes in gluteus medius (GM) activity and those in external oblique (EO) activity during terminal swing. All correlations pooled across walking speeds and perturbation amplitudes.
Scientific RepoRts | 7: 808 | DOI:10.1038/s41598-017-00942-x
© The Author(s) 2017
AcknowledgementsThis study was supported by a grant from the National Center for Advancing Translational Sciences (UL1TR001111). We also gratefully acknowledge Dr. Debby Givens for her helpful instruction on electrode placement for postural control muscles.Author ContributionsH.E.S., J.D.T., and J.R.F. contributed equally to the: (1) conception and design of the work, (2) acquisition, analysis, or interpretation of data for the work, and (3) drafting the work or revising it critically for important intellectual content.Additional InformationCompeting Interests: The authors declare that they have no competing interests.Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Active control of lateral balance in human walking. C E Bauby, A D Kuo, 10.1016/S0021-9290(00)00101-9doi:10.1016/ S0021-9290(00Journal of biomechanics. 33Bauby, C. E. & Kuo, A. D. Active control of lateral balance in human walking. Journal of biomechanics 33, 1433-1440, doi:10.1016/ S0021-9290(00)00101-9 (2000).

Mechanical and metabolic requirements for active lateral stabilization in human walking. J M Donelan, D W Shipman, R Kram, A D Kuo, 10.1016/j.jbiomech.2003.06.002Journal of biomechanics. 37Donelan, J. M., Shipman, D. W., Kram, R. & Kuo, A. D. Mechanical and metabolic requirements for active lateral stabilization in human walking. Journal of biomechanics 37, 827-835, doi:10.1016/j.jbiomech.2003.06.002 (2004).

Coupling of posture and gait: mode locking and parametric excitation. B A Kay, W H WarrenJr, 10.1007/PL00008002Biological cybernetics. 85Kay, B. A. & Warren, W. H. Jr. Coupling of posture and gait: mode locking and parametric excitation. Biological cybernetics 85, 89-106, doi:10.1007/PL00008002 (2001).

A neuromechanical strategy for mediolateral foot placement in walking humans. B L Rankin, S K Buffo, J C Dean, 10.1152/jn.00138.2014Journal of neurophysiology. 112Rankin, B. L., Buffo, S. K. & Dean, J. C. A neuromechanical strategy for mediolateral foot placement in walking humans. Journal of neurophysiology 112, 374-383, doi:10.1152/jn.00138.2014 (2014).

Two independent contributions to step variability during over-ground human walking. S H Collins, A D Kuo, 10.1371/journal.pone.0073597PloS one. 873597Collins, S. H. & Kuo, A. D. Two independent contributions to step variability during over-ground human walking. PloS one 8, e73597, doi:10.1371/journal.pone.0073597 (2013).

Direction-dependent control of balance during walking and standing. S M O'connor, A D Kuo, 10.1152/jn.00131.2009Journal of neurophysiology. 102O'Connor, S. M. & Kuo, A. D. Direction-dependent control of balance during walking and standing. Journal of neurophysiology 102, 1411-1419, doi:10.1152/jn.00131.2009 (2009).

Effects of visual focus and gait speed on walking balance in the frontal plane. A Goodworth, K Perrone, M Pillsbury, M Yargeau, 10.1016/j.humov.2015.04.004Human movement science. 42Goodworth, A., Perrone, K., Pillsbury, M. & Yargeau, M. Effects of visual focus and gait speed on walking balance in the frontal plane. Human movement science 42, 15-26, doi:10.1016/j.humov.2015.04.004 (2015).

Energetic cost of walking with increased step variability. S M O'connor, H Z Xu, A D Kuo, 10.1016/j.gaitpost.2012.01.014Gait & posture. 36O'Connor, S. M., Xu, H. Z. & Kuo, A. D. Energetic cost of walking with increased step variability. Gait & posture 36, 102-107, doi:10.1016/j.gaitpost.2012.01.014 (2012).

Gait variability in healthy old adults is more affected by a visual perturbation than by a cognitive or narrow step placement demand. C A Francis, J R Franz, S M O'connor, D G Thelen, 10.1016/j.gaitpost.2015.07.006Gait & posture. 42Francis, C. A., Franz, J. R., O'Connor, S. M. & Thelen, D. G. Gait variability in healthy old adults is more affected by a visual perturbation than by a cognitive or narrow step placement demand. Gait & posture 42, 380-385, doi:10.1016/j.gaitpost.2015.07.006 (2015).

. 10.1038/s41598-017-00942-xScientific RepoRts |. 7808Scientific RepoRts | 7: 808 | DOI:10.1038/s41598-017-00942-x

Visuomotor Entrainment and the Frequency-Dependent Response of Walking Balance to Perturbations. J R Franz, C Francis, M Allen, D G Thelen, 10.1109/TNSRE.2016.2603340IEEE Trans Neural Syst Rehabil Eng. Franz, J. R., Francis, C., Allen, M. & Thelen, D. G. Visuomotor Entrainment and the Frequency-Dependent Response of Walking Balance to Perturbations. IEEE Trans Neural Syst Rehabil Eng. doi:10.1109/TNSRE.2016.2603340, (2016).

Advanced age brings a greater reliance on visual feedback to maintain balance during walking. J R Franz, C A Francis, M S Allen, S M O'connor, D G Thelen, 10.1016/j.humov.2015.01.012Human movement science. 40Franz, J. R., Francis, C. A., Allen, M. S., O'Connor, S. M. & Thelen, D. G. Advanced age brings a greater reliance on visual feedback to maintain balance during walking. Human movement science 40, 381-392, doi:10.1016/j.humov.2015.01.012 (2015).

Intersegmental coordination elicited by unexpected multidirectional slippinglike perturbations resembles that adopted during steady locomotion. F Aprigliano, D Martelli, S Micera, V Monaco, 10.1152/jn.00327.2015doi:10.1152/ jn.00327.2015Journal of neurophysiology. 115Aprigliano, F., Martelli, D., Micera, S. & Monaco, V. Intersegmental coordination elicited by unexpected multidirectional slipping- like perturbations resembles that adopted during steady locomotion. Journal of neurophysiology 115, 728-740, doi:10.1152/ jn.00327.2015 (2016).

Interaction between age and gait velocity in the amplitude and timing of antagonist muscle coactivation. T Hortobagyi, 10.1016/j.gaitpost.2008.12.007Gait & posture. 29Hortobagyi, T. et al. Interaction between age and gait velocity in the amplitude and timing of antagonist muscle coactivation. Gait & posture 29, 558-564, doi:10.1016/j.gaitpost.2008.12.007 (2009).

The influence of visual perturbations on the neural control of limb stiffness. J Wong, E T Wilson, N Malfait, P L Gribble, 10.1152/jn.90371.2008Journal of neurophysiology. 101Wong, J., Wilson, E. T., Malfait, N. & Gribble, P. L. The influence of visual perturbations on the neural control of limb stiffness. Journal of neurophysiology 101, 246-257, doi:10.1152/jn.90371.2008 (2009).

How does age affect leg muscle activity/coactivity during uphill and downhill walking?. J R Franz, R Kram, 10.1016/j.gaitpost.2012.08.004Gait & posture. 37Franz, J. R. & Kram, R. How does age affect leg muscle activity/coactivity during uphill and downhill walking? Gait & posture 37, 378-384, doi:10.1016/j.gaitpost.2012.08.004 (2013).

Effects of age and walking speed on coactivation and cost of walking in healthy adults. D S Peterson, P E Martin, 10.1016/j.gaitpost.2009.12.005Gait & posture. 31Peterson, D. S. & Martin, P. E. Effects of age and walking speed on coactivation and cost of walking in healthy adults. Gait & posture 31, 355-359, doi:10.1016/j.gaitpost.2009.12.005 (2010).

Kinematic variability and local dynamic stability of upper body motions when walking at different speeds. J B Dingwell, L C Marin, 10.1016/j.jbiomech.2004.12.014Journal of biomechanics. 39Dingwell, J. B. & Marin, L. C. Kinematic variability and local dynamic stability of upper body motions when walking at different speeds. Journal of biomechanics 39, 444-452, doi:10.1016/j.jbiomech.2004.12.014 (2006).

Two simple methods for determining gait events during treadmill and overground walking using kinematic data. J A ZeniJr, J G Richards, J S Higginson, 10.1016/j.gaitpost.2007.07.007Gait & posture. 27Zeni, J. A. Jr., Richards, J. G. & Higginson, J. S. Two simple methods for determining gait events during treadmill and overground walking using kinematic data. Gait & posture 27, 710-714, doi:10.1016/j.gaitpost.2007.07.007 (2008).

Gait Analysis: Normal and Pathological Function. Second Edition. Gait Analysis: Normal and Pathological Function. J Perry, J Burnfield, Second Edition 1-551Perry, J. & Burnfield, J. M. Gait Analysis: Normal and Pathological Function. Second Edition. Gait Analysis: Normal and Pathological Function, Second Edition 1-551 (2010).

Introduction to surface electromyography. J R Cram, G S Kasman, J Holtz, Aspen PublishersCram, J. R., Kasman, G. S. & Holtz, J. Introduction to surface electromyography. (Aspen Publishers, 1998).

Gait changes in older adults: predictors of falls or indicators of fear. B E Maki, 10.1111/jgs.1997.45.issue-3Journal of the American Geriatrics Society. 45Maki, B. E. Gait changes in older adults: predictors of falls or indicators of fear. Journal of the American Geriatrics Society 45, 313-320, doi:10.1111/jgs.1997.45.issue-3 (1997).

Gait variability and fall risk in community-living older adults: a 1-year prospective study. Archives of physical medicine and rehabilitation. J M Hausdorff, D A Rios, H K Edelberg, 10.1053/apmr.2001.2489382Hausdorff, J. M., Rios, D. A. & Edelberg, H. K. Gait variability and fall risk in community-living older adults: a 1-year prospective study. Archives of physical medicine and rehabilitation 82, 1050-1056, doi:10.1053/apmr.2001.24893 (2001).

Responses of human hip abductor muscles to lateral balance perturbations during walking. A L Hof, J Duysens, 10.1007/s00221-013-3655-5Experimental brain research. 230Hof, A. L. & Duysens, J. Responses of human hip abductor muscles to lateral balance perturbations during walking. Experimental brain research 230, 301-310, doi:10.1007/s00221-013-3655-5 (2013).

Contributions of feed-forward and feedback strategies at the human ankle during control of unstable loads. J M Finley, Y Y Dhaher, E J Perreault, 10.1007/s00221-011-2972-9Experimental brain research. 217Finley, J. M., Dhaher, Y. Y. & Perreault, E. J. Contributions of feed-forward and feedback strategies at the human ankle during control of unstable loads. Experimental brain research 217, 53-66, doi:10.1007/s00221-011-2972-9 (2012).

Association between muscle activation and metabolic cost of walking in young and old adults. T Hortobagyi, A Finch, S Solnik, P Rider, P Devita, 10.1093/gerona/glr008J Gerontol A Biol Sci Med Sci. 66Hortobagyi, T., Finch, A., Solnik, S., Rider, P. & DeVita, P. Association between muscle activation and metabolic cost of walking in young and old adults. J Gerontol A Biol Sci Med Sci 66, 541-547, doi:10.1093/gerona/glr008 (2011).

Foot orientation affects muscle activation levels of ankle stabilizers in a single-legged balance board protocol. R De Ridder, T Willems, S De Mits, J Vanrenterghem, P Roosen, 10.1016/j.humov.2013.12.008Human movement science. 33De Ridder, R., Willems, T., De Mits, S., Vanrenterghem, J. & Roosen, P. Foot orientation affects muscle activation levels of ankle stabilizers in a single-legged balance board protocol. Human movement science 33, 419-431, doi:10.1016/j.humov.2013.12.008 (2014).

Biomechanical motor patterns in normal walking. D A Winter, 10.1080/00222895.1983.10735302J Mot Behav. 15Winter, D. A. Biomechanical motor patterns in normal walking. J Mot Behav 15, 302-330, doi:10.1080/00222895.1983.10735302 (1983).

Basic gait parameters: reference data for normal subjects, 10-79 years of age. T Oberg, A Karsznia, K Oberg, J Rehabil Res Dev. 30Oberg, T., Karsznia, A. & Oberg, K. Basic gait parameters: reference data for normal subjects, 10-79 years of age. J Rehabil Res Dev 30, 210-223 (1993).

Joint angle parameters in gait: reference data for normal subjects, 10-79 years of age. T Oberg, A Karsznia, K Oberg, J Rehabil Res Dev. 31Oberg, T., Karsznia, A. & Oberg, K. Joint angle parameters in gait: reference data for normal subjects, 10-79 years of age. J Rehabil Res Dev 31, 199-213 (1994).

General and Specific Strategies Used to Facilitate Locomotor Maneuvers. M Wu, J H Matsubara, K E Gordon, 10.1371/journal.pone.0132707PloS one. 10132707Wu, M., Matsubara, J. H. & Gordon, K. E. General and Specific Strategies Used to Facilitate Locomotor Maneuvers. PloS one 10, e0132707, doi:10.1371/journal.pone.0132707 (2015).


A/C magnetic hyperthermia of melanoma mediated by iron(0)/iron oxide core/shell magnetic nanoparticles: a mouse study

Sivasai Balivada
Raja Shekar Rachakatla
Hongwang Wang
Thilani N Samarakoon
Raj Kumar Dani
Marla Pyle
Franklin O Kroh
Brandon Walker
Xiaoxuan Leaym
Olga B Koper
Masaaki Tamura
Viktor Chikan
Stefan H Bossmann
Deryl L Troyer
A/C magnetic hyperthermia of melanoma mediated by iron(0)/iron oxide core/shell magnetic nanoparticles: a mouse study
R E S E A R C H A R T I C L E Open Access
Background: There is renewed interest in magnetic hyperthermia as a treatment modality for cancer, especially when it is combined with other more traditional therapeutic approaches, such as the co-delivery of anticancer drugs or photodynamic therapy.Methods: The influence of bimagnetic nanoparticles (MNPs) combined with short external alternating magnetic field (AMF) exposure on the growth of subcutaneous mouse melanomas (B16-F10) was evaluated. Bimagnetic Fe/ Fe 3 O 4 core/shell nanoparticles were designed for cancer targeting after intratumoral or intravenous administration. Their inorganic center was protected against rapid biocorrosion by organic dopamine-oligoethylene glycol ligands. TCPP (4-tetracarboxyphenyl porphyrin) units were attached to the dopamine-oligoethylene glycol ligands.Results: The magnetic hyperthermia results obtained after intratumoral injection indicated that micromolar concentrations of iron given within the modified core-shell Fe/Fe 3 O 4 nanoparticles caused a significant anti-tumor effect on murine B16-F10 melanoma with three short 10-minute AMF exposures. We also observed a decrease in tumor size after intravenous administration of the MNPs followed by three consecutive days of AMF exposure 24 hrs after the MNPs injection.Conclusions:These results indicate that intratumoral administration of surface modified MNPs can attenuate mouse melanoma after AMF exposure. Moreover, we have found that after intravenous administration of micromolar concentrations, these MNPs are capable of causing an anti-tumor effect in a mouse melanoma model after only a short AMF exposure time. This is a clear improvement to state of the art.

Background

Recently, questions have emerged regarding whether anticancer drug development is headed in the right direction and whether opportunities that are off the accepted path are being overlooked [1]. Largely due to the increasing insight into the series of mutations associated with the development of cancer, drug development has moved into the "molecular target" area. There have been initial successes (e.g. imatinib mesylate for the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors); however, the genetic complexity and diversity of tumor cells, including the occurrence of cancer stem cells, have prevented molecular targeting from becoming universally successful. Because the progression of a normal cell to a cancer cell involves numerous genetic mutations, targeting one or even several gene products may be ineffective. Furthermore, many biological processes feature alternate pathways which can be upregulated, if needed, thus thwarting molecularly targeted therapies [1]. To overcome these obstacles, a successful cancer therapy has to combine several approaches. Molecular targeting can be a viable component of this methodology. However, other approaches, such as stem cell delivery, hyperthermia, photodynamic therapy, and the design of multifunctional platforms that combine cancer diagnostics and treatment (theranostics) have not received full attention during the last decade.

We and others are working toward cost-effective treatment methods based on non-conventional combinations of known and proven techniques. We asked whether it is possible to achieve cancer localization by using porphyrin labels for the delivery of iron-containing superparamagnetic nanoparticles to tumor tissue. Tumor cells selectively uptake porphyrins, which they need as prosthetic groups in their elevated sugar metabolism, via over-expression of porphyrin receptors in their cell membranes [2]. There is a strong positive correlation between the cell uptake of a variety of chemically defined, synthetic and natural porphyrins and their octanol/water distribution coefficients [3,4]. These findings support the paradigm that there indeed exists a porphyrin uptake mechanism other than endocytosis in cancer cells. The LDL (lowdensity lipoprotein) receptor, which is over-expressed in cancer cells, has the ability to take up porphyrins as well, either alone or simultaneously with other porphyrin receptors. Localized hyperthermia is a powerful therapeutic modality. When administered selectively, hyperthermia treatment can be very potent against many types of cancer because it is not based on the intake of drugs by cancer cells, but on the application of heat. When heated to 45°C, vital proteins of the cancer cell become damaged (e.g. misfolded) and/or the cell membrane partially dissolves in the surrounding aqueous medium [5]. A multitude of heatinduced deviations from the "normal" metabolism of a cancer cell can eventually lead to apoptosis (programmed cell death). Although many cancer types are slightly more susceptible to hyperthermia than healthy cells, the latter essentially share the same fate when heated [6]. Therefore, the development of methods to localize hyperthermia to cancer cells remains one of the challenges in this field. This is important when attempting to treat solid tumors within the human body as well as for treatment of metastasizing cancers. The use of tethered porphyrins as "bait" may provide an effective (and low cost) alternative to using antibodies for getting magnetic nanoparticles effectively into tumors, which is currently a common tumor targeting method.

Magnetic nanoparticles dump thermal energy into the system, therefore providing heating (magnetic hyperthermia) [7]. Heating takes place by power absorption of magnetic particles due to an A/C magnetic field [8]. The important factor for magnetic heating experiments is the specific absorption rate (SAR), which is determined by SAR = C × ΔT/Δt, where C is the specific heat capacity of the sample and T and t are the temperature and time, respectively. SAR is very sensitive to the material properties. While in multi-domain particles the dominant heating is hysteresis loss due to the movement of domain walls, this is not the case with small, single domain particles. The two main contributing mechanisms of SAR in single domain magnetic NPs are the Brownian (rotation of the entire nanoparticle) and Néel (random flipping of the spin without rotation of the particle) relaxations [9,10]. The transition between the two mechanisms occurs between 5-12 nm for various materials, but it also varies with frequency [11] Melanoma incidence has reached almost epidemic proportions worldwide. When it is present as disseminated metastatic disease it is not curable using current clinical tools; traditional chemotherapy is often ineffective due to inherent drug resistance [12]. We have selected the B16-F10 melanoma model in syngeneic C57BL/6 female mice because this type of melanoma has proven to be quite resistant to treatment. In order to evaluate the efficacy of hyperthermia treatment against cancer, we did not want to select a less resistant cancer cell line/host system. In addition, since hyperthermia often enhances the immune response, an immunocompetent host was desirable.

In the work reported here, we have used (bi)magnetic iron/iron oxide core/shell nanoparticles, synthesized by NanoScale Corporation (Manhattan, KS), for A/C (alternating current)-magnetic cancer therapy, because they exhibit superior properties in several areas: The nanoscopic size (d<15 nm) of the stealth-protected Fe/Fe 3 O 4 core/shell nanoparticles will permit passive tumor targeting from the bloodstream by using the EPR (enhanced permeation and retention) effect [13,14]. In addition, we surmise that our porphyrin coating may enhance MNP uptake in tumors, because cancer cells are in constant need of porphyrins as prosthetic groups in their elevated sugar metabolism [2] and overexpress the LDL(low-density lipoprotein) receptor. This remains an area for future clarification. Here, we compared the effects of intratumoral (IT) and intravenous (IV) core/ shell porphyrin-tethered nanoparticle treatment followed by A/C exposure on B16-F10 melanoma growth in a mouse model.

Methods

Cell lines and animals

B16-F10 melanoma cells were purchased from ATCC (Manassas, VA) and maintained in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified 37°C incubator at 5% CO 2 .

6-8 week old female C57/BL6 mice were purchased from Charles River Laboratories (Wilmington, MA). Mice were maintained according to approved institutional IACUC guidelines in the Comparative Medicine Group Facility of Kansas State University. All animal experiments were conducted according to these IACUC guidelines.

Transmission Electron Microscopy

The sizes of the different nanoparticles were determined by using TEM. This was achieved employing a Philips CM-200 TEM instrument operating at 100 kV. 1-2 micrograms of the MNPs were dissolved in anhydrous tetrahydrofuran THF (5 mL) and one drop of the resulting nanoparticle solution was spread over a copper grid (300 mesh size) supporting a thin film of amorphous carbon. To reduce the damage from the electron beam, the sample was cooled to liquid nitrogen temperature during data collection.

Porphyrin-tethered Stealth-Coated (Bi) Magnetic Fe/Fe 3 O 4 Nanoparticles

The synthesis of the stealth-coated dopamine-labeled Fe/Fe 3 O 4 nanoparticles featuring tethered 4-tetracarboxyphenyl porphyrins (TCPP) is reported in a separate publication (Wang et al., ACS Nano, 2010, in preparation). Briefly, Fe/Fe 3 O 4 -core/shell nanoparticles were synthesized by NanoScale Corporation and then coated with dopamine-anchored ligands. The structure of the nanoparticles is shown in Figure 1, and results from electron microscopy are shown in Figure 2. The diameter of the Fe(0)-core was 5.4 ± 1.1 nm; the diameter of the inorganic Fe/Fe 3 O 4 -nanoparticles was determined to be 7.2 ± 2.8 nm. Using the program IMAGE (NIH), we have determined the polydispersity index of the Fe/ Fe 3 O 4 -nanoparticles to be 1.31. Note that the stealth ligand has a length of 2.5 nm (AM1-Chemdraw Ultra 3D package, Cambridge Soft Corporation, Cambridge, MA 02140), so that the resulting bimagnetic nanoparticles are 12 ± 3 nm in size. The porphyrin-labels have a diameter of 1.95 nm (AM1). Note that the dopamineanchored tetraethylene glycol ligand (I) and the TCPPlinked dopamineanchored tetraethylene glycol ligand (II) have been synthesized separately. The binding of the ligands to the Fe 3 O 4 layer was achieved in anhydrous THF under argon; the molar ratio of ligands I/II was 100/4. The reaction procedure is described in detail in a separate paper (Wang et al., ACS Nano, 2010, in preparation). We assume a statistical distribution of the ligands at the Fe 3 O 4 surface. Assuming a Poisson distribution, 96.4 percent of the Fe/Fe 3 O 4 NPs at the chosen ratio feature at least one chemically linked TCPP unit, which will act as "bait" for the B16-F10 cancer cells. The solubility of the organically coated Fe/Fe 3 O 4 NPs was determined to be 0.35 mg ml -1 and the Specific Adsorption Rate (SAR) at the field conditions described here was 64 ± 2 Wg -1 (Fe).

Determination of iron concentration in MNPs

Iron concentration in MNPs was measured using the Ferrozine-based spectrophotometric iron estimation method [15]. For this method, 50 μl of MNPs were diluted to 1 ml with distilled water. MNPs were then lysed by incubating for 2 hours at 65-70°C after the addition of 0.5 ml of 1.2 M HCl and 0.2 ml of 2 M ascorbic acid. After incubation, 0.2 ml of reagent containing 6.5 mM Ferrozine, 13.1 mM neocuproine, 2 M ascorbic acid, and 5 M ammonium acetate was added and incubated for 30 minutes at room temperature. After 30 minutes, the optical density of the samples was measured using a UV-VIS spectrophotometer at 562 nm. A standard curve was prepared using 0, 0.1, 0.2, 0.5, 1, 2, 5 μg/ml ferrous ammonium sulfate samples. Water with all other reagents is used as blank.

Magnetic Heating

The nanoparticles used in these experiments are dominated by Néel relaxation due to the superparamagnetic nature of the iron(0) cores. The hyperthermia apparatus (Superior Induction Company, Pasadena, CA) used here has a "heavy duty" induction heater converted to allow measurement of the temperature change of a sample. In the setup, a remote fiber optic probe (Neoptix, Quebec, Canada) is used to monitor the temperature change. The frequency is fixed (366 kHz, sine wave pattern); field amplitude is 5 kA/m. The coil diameter is 1 inch, 4 turns continuously water cooled. For all in vivo experiments, the mice were placed into the induction coil using a specially designed Teflon supporter so that tumors were located exactly in the region of the AMF possessing the highest field density.

Cytotoxicity of Magnetic Nanoparticles on B16-F10 cells

Potential cytotoxic effects of MNPs were studied by incubating cells in differing concentrations of MNPs. B16-F10 cells were incubated overnight with MNPs amounts corresponding to 5, 10, 15, 20, and 25 μg/mL iron. After incubation, the medium was removed and the cells were washed twice with DMEM and cells were counted via hemocytometer with Trypan blue staining. This method also allows counting non-viable cells since only they allow the blue stain into the cell. All experiments were run in triplicate and repeated at least twice.

Temperature measurements on mice

MNPs containing 100 μg of iron in 100 μl of distilled water were injected into the rear limb muscle of one mouse and the leg was then exposed to AMF for 10 min. An optical temperature probe was inserted intramuscularly at the injection site and the temperature increase was measured during AMF exposure. At the same time, the body temperature was monitored with a separate temperature probe.

Intratumoral Hyperthermia

Ten mice were transplanted subcutaneously into each rear limb above the stifle with 1 × 10 6 B16-F10 melanoma cells suspended in PBS. 120 μL of saline were injected into melanomas on the left leg of all mice and 120 μl MNPs containing 1 mg Fe/mL were injected into right leg tumors of all mice in three injections on day 4, 5, 6 (total of 360 μg iron). Because the MNPs were dilute and tumor volumes were quite small, 3 injections instead of 1 were needed in order to reach this level of MNPs. Both left (saline) and right (MNPs) leg tumors of five of the mice were exposed to AMF for 10 minutes soon after injections. Tumors on the remaining five mice were not exposed to AMF. Based on this, there were 4 groups which tested the effects of MNPs with and without AMF and of AMF alone: Group 1: Intratumoral saline injection, not exposed to AMF (left legs of first five mice); Group 2: Intratumoral injection of saline, exposed to AMF (left legs of remaining five mice); Group 3: Intratumoral injection of MNPs, not exposed to AMF (right legs of first five mice); Group 4: Intratumoral injection of MNP, exposed to AMF (right legs of remaining five mice). After three AMF exposures, tumor sizes were measured with a caliper on days 8 to 14, and tumor volume was calculated using the formula 0.5aXb 2 (a = longest diameter; b = smaller diameter). After 14 days mice were euthanized, tumors were excised, and tumor weights were measured.

Intravenous administration of MNPs with AMF exposure

On day 0, 0.35 × 10 6 B16-F10 melanoma cells were injected subcutaneously into the right legs of 27 mice. Mice were randomly divided into three groups: Group 1, IV MNPs, no AMF; Group II, IV MNPs, AMF; Group III, DMEM control, no AMF. On days 6, 9, and 11 after tumor cell transplant, MNPs corresponding to 226 mcg of iron were injected intravenously into each mouse in groups I and II. On the same day, DMEM was injected intravenously into group III. For group II, tumors were exposed to AMF for 10 minutes one day after each I.V. MNPs injection (total of three AMF treatments). MNPs not coated with porphyrins were not sufficiently soluble in water, so we were not able to use them as control. Tumor sizes were measured using a caliper on days 14 and 18, and tumor volume was calculated as described above. On day 18 all mice were euthanized, tumors were excised, and tumor weights were measured.

Histological Analysis

After euthanizing mice, lung, liver, and tumors were collected and snap frozen. 8-10 μm sections were made in a cryostat (Leitz Kryostat 1720). Staining for iron content on these sections was carried out by using Perl's Prussian blue staining kit (Polysciences, Inc., Warrington, PA). Apoptosis was evaluated using a DeadEnd Fluorometric TUNEL kit. (Promega Corp., Madison, WI) following the manufacturer's instructions.

Statistical analysis

Statistical analyses were performed by Macanova 4.12 (School of Statistics, University of Minnesota, Minneapolis, MN). The means of the experimental groups were evaluated to confirm that they met the normality assumption. To evaluate the significance of overall differences in tumor volumes and tumor weights between all in vivo groups, statistical analysis was performed by analysis of variance (ANOVA). A p-value less than 0.1 was considered as significant. Following significant ANOVA, post hoc analysis using least significance difference (LSD) was used for multiple comparisons. Significance for post hoc testing was set at p < 0.1. All the tumor volumes and weight data were represented as mean +/-standard error (SE) on graphs.

Results

Cytotoxicity of Magnetic Nanoparticles on B16-F10 cells

MNP toxicity was tested after overnight incubation of B16-F10 cell cultures in 24-well plates with various MNPs concentrations, as measured by iron concentration. There was a dose-dependent cytotoxicity of the MNPs. B16-F10 cancer cell viability assessment in the presence of varying concentrations of MNPs is shown in Figure 3. The MNPs showed a pronounced cytotoxic effect on B16-F10 cells at >10 μg iron levels. (p-value < 0.05)

Temperature measurements on mice after intramuscular MNPs injection

We observed 11°C temperature increase subcutaneously at the MNPs injection site within 10 minutes of AMF exposure. There was no increase in core body temperature ( Figure 4). These data demonstrate specific magnetic hyperthermia. However, we did not directly measure temperature change in tumors during experiments because the necessary skin opening for the probe caused leakage of the gelatinous melanoma tumor parenchyma, introducing increased potential variability in tumor volumes.

Intratumoral Magnetic Hyperthermia

After three AMF exposures, tumor sizes were measured from days 8 to 14; the comparison is shown in Figure 5. We identified decreased tumor size both in tumor-bearing mice treated with MNPs and with MNPs+AMF. The tumors with MNPs+AMF showed a significant reduction in tumor volume at 8,9,11, & 14 days (p < 0.1) compared to the saline treated group. A decrease in size  with only MNP treatment (no AMF) relative to the saline controls was also noted; however, this decrease was not significant. Since earlier intramuscular injections and optical probe measurements revealed hyperthermia after AMF, the probable cause for the tumor attenuation shown here is local hyperthermia.

Intravenously administered MNPs and AMF exposure

Tumor-bearing mice with intravenously injected MNPs were exposed to AMF treatments three times and after 18 days were euthanized. Tumor weights were obtained and compared to controls ( Figure 6). A significant decrease in tumor weight (p < 0.1) was observed in the intravenous MNPs+AMF group and was most likely due to heat generated from MNPs in tumors, based on earlier optical probe experiments in anesthetized mice. Some tumor weight decrease was also observed in intravenous MNPs without AMF treatment. On days 14 and 18, tumor volumes were recorded and were markedly attenuated in the mice with MNPs with AMF; however this was not significant (Additional file 1). After tumors were harvested and sectioned, MNPs in tumor sections and other tissues were identified as Prussian blue positive material in tumor bearing mice intravenously injected with MNPs ( Figure 7A-D).

Apoptosis assay

Histological analysis after apoptosis assay with the modified TUNEL assay showed the most apoptotic positive cells in the intratumoral MNP+AMF treatments ( Figure  7G), intermediate apoptosis levels in mice that received intravenous MNP+AMF ( Figure 7F), and the fewest apoptotic cells in the saline+AMF group ( Figure 7E).

Discussion

The major finding of this study was that there is a significant decrease in tumor size after systemic (intravenous) administration of low (microgram iron content) amounts of the porphyrin-tethered MNPs and AMF exposure compared to tumors in animals given intravenous DMEM. We have found the MNPs in the melanomas, leading us to surmise that the porphyrins attached to them may facilitate MNP uptake. However, further studies are needed to confirm the porphyrin-facilitated MNP uptake in tumors. As already discussed, cancer cells over-express porphyrin receptors, because they require more porphyrins as prosthetic groups in their elevated sugar metabolism than normal cells [2]. Tumor localization of MNPs by urokinase plasminogen activator receptor (uPAR) is reported by Yang et al. [16].

Our intratumoral magnetic hyperthermia results showed that microgram amounts of iron delivered by the core-shell Fe/Fe 3 O 4 nanoparticles caused an antitumor effect on melanoma with short-time AMF exposures (10 min.). This is a clear improvement with respect to current protocols, which are defined by milligram amounts of MNPs and much longer exposure times, usually 30 minutes [17]. We have also observed a trend toward decreased tumor size after MNP administration without AMF exposure. This is not surprising, since our in vitro work indicated that the MNPs have a pronounced cytotoxic effect on B16-F10 cells. In in vitro studies, we used a total of 10 μg of ironcontaining MNP (in 1 ml of medium) for 500,000 cells. So theoretically, each cell is exposed to 0.00002 μg of iron. However, in vivo, a detectable, three-dimensional tumor contains  many more cells, and the tumor interstitium offers a vastly different microenvironment than the monolayer of cells. A tumor volume of 1 cm 3 contains approximately 10 9 cells [18]. In the in vivo studies, we injected a total of 360 μg of ironcontaining MNPs intratumorally when mean tumor size was about 50 mm 3 (an estimated 50 million cells). Extrapolating our in vitro results, 1000 μg of iron-containing MNPs would be required for similar toxicity to 50 million cells. Therefore, we feel that the effective amount of core/shell MNPs in the tumor on a per cell basis was actually much less than in the in vitro studies. Minamimura et al. gave intratumoral iron oxide MNP injections and noted that their MNPs alone exerted a noticeable anti-melanoma effect [19]. In the study reported here, the effect of MNPs alone is most probably due to the biocorrosion of the MNPs and the subsequent release of iron(II) and iron(III) cations, which is known to cause cell damage via iron(II/III)-enhanced chemistry of reactive oxygen species (ROS) [20]. Thus, we propose a two-pronged effect: magnetic hyperthermia and the additional generation of free radicals by manifold Fenton-type reactions [20].

It must be noted that we have found significant amounts of iron in lungs and liver, as indicated by Prussian blue staining ( Figure. 7B and 7C). Despite this widespread distribution of MNPs in vivo, we emphasize that there were no fatalities due to the blocking of arteries or exposure to AMF. This may be because the AMF was only applied to the melanoma region, preventing unwanted hyperthermia in other tissues. Our findings of minimal side effects after systemic administration of MNPs are corroborated by other work testing superparamagnetic NP for MRI capability and possible toxic effects. Wiegand et al. showed that 250 or 500 nm ferrofluid given intravenously to normal rabbits resulted in normal serum iron and enzymes for liver and kidney function at 1-72 hours after administration [21]. Kim et al. showed that silica over coated magnetic nanoparticles of 50 nm size did not cause apparent toxicity or alter the blood-brain barrier in mice after four weeks [22]. Pharmacokinetic data of the dependence of iron or iron oxide nanoparticles on their respective sizes are not yet available. Therefore, we have to rely on the available data on the distribution of stealthcoated Au-NPs (sizes: 15 nm, 50 nm and 160 nm) in rats and rabbits [23]. The larger the diameter of the Au-NPs, the higher their concentration in liver and spleen 24 h after IV-injection. The smallest Au-NPs did more evenly distribute within the mammalian bodies. Furthermore, they remained in the bloodstream distinctly longer. A longer residence time of our stealth Fe/Fe 3 O 4 nanoparticles (diameter including the stealth ligands: 12 ± 3 nm) in the bloodstream may enhance the EPR effect (enhanced permeation and retention).

Conclusion

The findings reported here indicate that ligand modified nanoparticles given systemically or intratumorally at low concentrations can significantly attenuate subcutaneous B16-F10 tumors in mice after repetitive short AMF exposure. Hence, it may be possible to exploit upregulated porphyrin uptake by cancer cells to facilitate getting core/shell bimagnetic nanoparticles to them, but further work is required to confirm this. After exposure to an AMF, which itself causes no harm, localized hyperthermia of cancer tissue attenuates the tumor without the undesirable side effects associated with traditional chemotherapy. This approach also circumvents failure of molecularly-targeted approaches due to redundant systems and failure of chemotherapeutic approaches due to cancer cell multidrug resistance. Furthermore, we surmise that AMF treatment is augmented by the release of iron within the tumor regions due to biocorrosion, increasing the intratumoral concentration of reactive oxygen species. Thus, localized hyperthermia after systemic administration of porphyrin labeled stealth MNPs may hold promise for future clinical therapy of melanomas. Our future goals are to further modify the MNPs to improve accumulation in tumors relative to other tissues and organs, and to test these improved MNPs in treatment of a preclinical metastatic model.

Figure 1
1Composition of the 4-tetracarboxyphenyl porphyrin (TCPP)-labeled, dopamine-anchored tetraethylene glycol ligands. Nanoparticles of 7.2 nm (outer diameter) require approximately 120 dopamine-anchored ligands (assuming a monolayer).

Figure 2
2TEM image of Fe/Fe 3 O 4 core/shell nanoparticles featuring an organic protective dopamine-anchored stealth layer.

Figure 3
3In vitro cell viability of B16-F10's cultured in medium containing increasing concentrations of MNPs, as measured by iron concentration. *Statistically significant (pvalue less than 0.05).

Figure 4
4Graph depicting temperature change at MNP injection site and in body core during AMF exposure, measured with a fiber optic temperature probe.

Figure 5
5Effect on tumor burden of intratumoral injection of MNPs followed by alternating magnetic field (AMF) treatments. Graph depicting average tumor volumes over time of B16-F10 tumor bearing mice which were later injected with either saline or MNP intratumorally and with or without AMF treatments. *Statistically significant (pvalue less than 0.1).

Figure 6
6Effect of intravenous injection of MNP and AMF on tumor weight. *Statistically significant difference (p-value less than 0.1) between control and IV MNP+AMF groups.

Figure 7
7Prussian blue staining and evaluation of apoptosis of tissues after the in vivo experiment. Green fluorescence indicates apoptosis positive and blue is DAPI counterstaining (E-G). A-C: IV MNP + AMF in tumor, lung, and liver, respectively. D: Intratumoral MNP, tumor section. E-G: Apoptosis assay pictures. E: Control tumor section. F: Tumor section with intravenous MNP administration followed by AMF. G: Tumor section with intratumoral MNP administration followed by AMF. (Scale bar = 100 μm) Additional file 1 Title: Tumor volume measurements on day14 and 18 of intravenously administered MNPs and AMF exposure experiment. Description: Day 14 and 18 tumor volumes of individual groups are compared. (not significant)

Additional file 1 :
1Tumor volume measurements on day14 and 18 of intravenously administered MNPs and AMF exposure experiment. Day 14 and 18 tumor volumes of individual groups are compared. (not significant). Abbreviations MNPs: Magnetic Nanoparticles; TCPP: 4-tetracarboxyphenyl porphyrin; AMF: Alternating magnetic field; SAR: Specific absorption rate; IT: Intratumoral; IV: Intravenous.

Is anticancer drug development heading in the right direction?. T W Hambley, W N Hait, Cancer Res. 694Hambley TW, Hait WN: Is anticancer drug development heading in the right direction? Cancer Res 2009, 69(4):1259-1262.

Molecular mechanism for cancer-associated induction of sialyl Lewis X and sialyl Lewis A expression-The Warburg effect revisited. R Kannagi, Glycoconj J. 205Kannagi R: Molecular mechanism for cancer-associated induction of sialyl Lewis X and sialyl Lewis A expression-The Warburg effect revisited. Glycoconj J 2004, 20(5):353-364.

Accumulation of porphyrins in cells: influence of hydrophobicity aggregation and protein binding. G Oenbrink, P Jurgenlimke, D Gabel, Photochem Photobiol. 484Oenbrink G, Jurgenlimke P, Gabel D: Accumulation of porphyrins in cells: influence of hydrophobicity aggregation and protein binding. Photochem Photobiol 1988, 48(4):451-456.

On the Correlation Between Hydrophobicity, Liposome Binding and Cellular Uptake of Porphyrin Sensitizers. S Ben-Dror, I Bronshtein, A Wiehe, B Röder, M O Senge, B Ehrenberg, Photochem Photobiol. 82Ben-Dror S, Bronshtein I, Wiehe A, Röder B, Senge MO, Ehrenberg B: On the Correlation Between Hydrophobicity, Liposome Binding and Cellular Uptake of Porphyrin Sensitizers. Photochem Photobiol 2006, 82:695-701.

Heat injury to cells in perfused systems. D P Orgill, S A Porter, Taylor Ho, Ann N Y Acad Sci. 1066Orgill DP, Porter SA, Taylor HO: Heat injury to cells in perfused systems. Ann N Y Acad Sci 2005, 1066:106-118.

Functional magnetic particles for medical application. M Shinkai, J Biosci Bioeng. 946Shinkai M: Functional magnetic particles for medical application. J Biosci Bioeng 2002, 94(6):606-613.

Presentation of a new magnetic field therapy system for the treatment of human solid tumors with magnetic fluid hyperthermia. A Jordan, R Scholz, K Maier-Hauff, M Johannsen, P Wust, J Nadobny, H Schirra, H Schmidt, S Deger, S Loening, W Lanksch, R Felix, Journal of Magnetism and Magnetic Materials. 225Jordan A, Scholz R, Maier-Hauff K, Johannsen M, Wust P, Nadobny J, Schirra H, Schmidt H, Deger S, Loening S, Lanksch W, Felix R: Presentation of a new magnetic field therapy system for the treatment of human solid tumors with magnetic fluid hyperthermia. Journal of Magnetism and Magnetic Materials 2001, 225:118-126.

Inductive heating of ferrimagnetic particles and magnetic fluids: physical evaluation of their potential for hyperthermia. A Jordan, P Wust, H Fahling, W John, A Hinz, R Felix, Int J Hyperthermia. 91Jordan A, Wust P, Fahling H, John W, Hinz A, Felix R: Inductive heating of ferrimagnetic particles and magnetic fluids: physical evaluation of their potential for hyperthermia. Int J Hyperthermia 1993, 9(1):51-68.

Time-Domain Study of Brownian and Neel Relaxation in Ferrofluids. R Kotitz, P C Fannin, L Trahms, Journal of Magnetism and Magnetic Materials. 149Kotitz R, Fannin PC, Trahms L: Time-Domain Study of Brownian and Neel Relaxation in Ferrofluids. Journal of Magnetism and Magnetic Materials 1995, 149:42-46.

Effects of surfactant friction on Brownian magnetic relaxation in nanoparticle ferrofluids. A B Pakhomov, Y P Bao, K M Krishnan, 97:310Q305/301-310Q305/303J Appl Phys. Pakhomov AB, Bao YP, Krishnan KM: Effects of surfactant friction on Brownian magnetic relaxation in nanoparticle ferrofluids. J Appl Phys 2005, 97:310Q305/301-310Q305/303.

Magnetic nanoparticle design for medical diagnosis and therapy. S Mornet, S Vasseur, F Grasset, E Duguet, J Mat Chem. 14Mornet S, Vasseur S, Grasset F, Duguet E: Magnetic nanoparticle design for medical diagnosis and therapy. J Mat Chem 2004, 14:2161-2175.

NPropionyl-Cysteaminylphenol-Magnetite Conjugate (NPrCAP/M) Is a Nanoparticle for the Targeted Growth Suppression of Melanoma Cells. Myt Sato, M Ohkura, Y Osai, A Sato, T Takada, H Matsusaka, I Ono, Y Tamura, N Sato, Y Sasaki, A Ito, H Honda, K Wakamatsu, S Ito, K Jimbow, J Invest Dermatol. 129Sato MYT, Ohkura M, Osai Y, Sato A, Takada T, Matsusaka H, Ono I, Tamura Y, Sato N, Sasaki Y, Ito A, Honda H, Wakamatsu K, Ito S, Jimbow K: NPropionyl-Cysteaminylphenol-Magnetite Conjugate (NPrCAP/M) Is a Nanoparticle for the Targeted Growth Suppression of Melanoma Cells. J Invest Dermatol 2009, 129:2233-41.

Exploiting the enhanced permeability and retention effect for tumor targeting. A K Iyer, G Khaled, J Fang, H Maeda, Drug Discov Today. 11Iyer AK, Khaled G, Fang J, Maeda H: Exploiting the enhanced permeability and retention effect for tumor targeting. Drug Discov Today 2006, 11(17- 18):812-818.

Nanoparticles for hyperthermia treatment of cancer. Fabrication and Bio-Application of Functionalized Nanomaterials(ahead to print). S H Bossmann, Bossmann SH: Nanoparticles for hyperthermia treatment of cancer. Fabrication and Bio-Application of Functionalized Nanomaterials(ahead to print) 2009.

Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells. J Riemer, H H Hoepken, H Czerwinska, S R Robinson, R Dringen, Anal Biochem. 3312Riemer J, Hoepken HH, Czerwinska H, Robinson SR, Dringen R: Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells. Anal Biochem 2004, 331(2):370-375.

Molecular imaging of pancreatic cancer in an animal model using targeted multifunctional nanoparticles. L Yang, H Mao, Z Cao, Y A Wang, X Peng, X Wang, H K Sajja, L Wang, H Duan, C Ni, Gastroenterology. 1365Yang L, Mao H, Cao Z, Wang YA, Peng X, Wang X, Sajja HK, Wang L, Duan H, Ni C, et al: Molecular imaging of pancreatic cancer in an animal model using targeted multifunctional nanoparticles. Gastroenterology 2009, 136(5):1514-1525, e1512.

Effects of magnetic fluid hyperthermia (MFH) on C3H mammary carcinoma in vivo. A Jordan, R Scholz, P Wust, H Fahling, J Krause, W Wlodarczyk, B Sander, T Vogl, R Felix, Int J Hyperthermia. 136Jordan A, Scholz R, Wust P, Fahling H, Krause J, Wlodarczyk W, Sander B, Vogl T, Felix R: Effects of magnetic fluid hyperthermia (MFH) on C3H mammary carcinoma in vivo. Int J Hyperthermia 1997, 13(6):587-605.

On the growth rates of human malignant tumors: implications for medical decision making. S Friberg, S Mattson, J Surg Oncol. 654Friberg S, Mattson S: On the growth rates of human malignant tumors: implications for medical decision making. J Surg Oncol 1997, 65(4):284-297.

Tumor regression by inductive hyperthermia combined with hepatic embolization using dextran magnetite-incorporated microspheres in rats. T Minamimura, H Sato, S Kasaoka, T Saito, S Ishizawa, S Takemori, K Tazawa, K Tsukada, Int J Oncol. 166Minamimura T, Sato H, Kasaoka S, Saito T, Ishizawa S, Takemori S, Tazawa K, Tsukada K: Tumor regression by inductive hyperthermia combined with hepatic embolization using dextran magnetite-incorporated microspheres in rats. Int J Oncol 2000, 16(6):1153-1158.

. Balivada, BMC Cancer. 10Balivada et al. BMC Cancer 2010, 10:119 http://www.biomedcentral.com/1471-2407/10/119

Toxicity of iron and hydrogen peroxide: the Fenton reaction. C C Winterbourn, Toxicol Lett. Winterbourn CC: Toxicity of iron and hydrogen peroxide: the Fenton reaction. Toxicol Lett 1995, 82-83:969-974.

Evaluation of the tolerance and distribution of intravenously applied ferrofluid particles of 250 and 500 nm size in an animal model. S Wiegand, T Heinen, A Ramaswamy, A M Sesterhenn, C Bergemann, J A Werner, A S Lubbe, J Drug Target. 173Wiegand S, Heinen T, Ramaswamy A, Sesterhenn AM, Bergemann C, Werner JA, Lubbe AS: Evaluation of the tolerance and distribution of intravenously applied ferrofluid particles of 250 and 500 nm size in an animal model. J Drug Target 2009, 17(3):194-199.

Toxicity and tissue distribution of magnetic nanoparticles in mice. J S Kim, T J Yoon, K N Yu, B G Kim, S J Park, H W Kim, K H Lee, S B Park, J K Lee, M H Cho, Toxicol Sci. 891Kim JS, Yoon TJ, Yu KN, Kim BG, Park SJ, Kim HW, Lee KH, Park SB, Lee JK, Cho MH: Toxicity and tissue distribution of magnetic nanoparticles in mice. Toxicol Sci 2006, 89(1):338-347.

Circulation and distribution of gold nanoparticles and induced alterations of tissue morphology at intravenous particle delivery. G S Terentyuk, G N Maslyakova, L V Suleymanova, B N Khlebtsov, B Y Kogan, G G Akchurin, A V Shantrocha, I L Maksimova, N G Khlebtsov, V V Tuchin, J Biophotonics. 25Terentyuk GS, Maslyakova GN, Suleymanova LV, Khlebtsov BN, Kogan BY, Akchurin GG, Shantrocha AV, Maksimova IL, Khlebtsov NG, Tuchin VV: Circulation and distribution of gold nanoparticles and induced alterations of tissue morphology at intravenous particle delivery. J Biophotonics 2009, 2(5):292-302.

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