We previously showed that MNMs can be used to open cellular membranes and synthetic lipid bilayers, destroy cancer cells, and disrupt bacterial cell wells (21,22) We sought to determine whether these MNMs can work effectively, retaining their disruptive nature in a spatially controlled manner when introduced to whole organisms. We used, and hairless nude mice to study the morphological effects of light-activated fast motor MNMs in small eukaryotes and mammalian tissue. We studied different destructive effects that MNMs cause in these eukaryotesusing several metrics including physiological and histopathological changes and mortality rates. Ourstudy expands the application of MNMs from their nanomechanical action oncell lines to whole organisms ranging from small microscopic invertebrates to larger mammalian vertebrates. Our results show that MNMs cause damage to cells and tissues in these eukaryotic species to varying degrees.

Hairless nude mice are used to study skin pathology, lesions, and ulcerations associated with skin cancers, including melanoma and adenocarcinoma. (16−19) These mouse skin models are used to observe skin histopathology together with fluorescent microscopy to identify localized cellular processes. (20)

species, commonly known as water fleas, are crustacean filter feeders in freshwater reservoirs.is a commonly used model organism in ecological studies examining different toxicities and environmental factors affecting freshwater systems. (13,14) More recently,andhave been used to study the effects of nanoparticles and their impact on the environment. (15) offers several advantages as a model system including ease of handling and visible characteristics for experimental measurements. Small invertebrate models such asandallow light to penetrate the organism and make it possible to study morphological changes.

is a hermaphrodite nematode worm, a free-living organism found on moist soil, and is a leading model organism used widely in neurobiology, biomedicine, genetics, and the study of host–pathogen interactions. (3−6) has a life span of 15–20 days at ambient temperatures from 20 to 22 °C. (7,8) It was first used as a model organism to study the mechanisms of aging. (9) Moreover,has a transparent body that permits the visualization of fluorescently tagged molecules, pathogens, and tissue pathologies. (6) Molecular work inover the past two decades has led to the discovery of important molecular mechanisms including programmed cell death, (10) RNAi techniques for gene silencing, (11) and expression of the green fluorescent protein (GPF). (12) These molecular characteristics makea desirable model to study physiological and pathological changes caused by external agents or treatments.

Molecular nanomachines (MNMs) are synthetically engineered molecules that have light-induced actuation (motorization). The design of these nanomachines was conceptually proposed in 1961, (1) but the recent award of the 2016 Nobel Prize for Chemistry for their discovery emphasizes the current high level of interest in their potential applications. (2) To better understand potential applications for MNM, it is important to evaluate how they can be applied in different cells and tissues. Of particular interest here is their functioningin multicellular organisms.

One potential limitation of the study is the use of 365 nm wavelength light to activate the MNM. This is in the UV-A spectrum and can sometimes have harmful effects on mammalian tissue over long exposure times. These MNM have been shown to be activated by two-photon excitation, which may be less likely to cause detrimental effects in mammalian cells. (33) More recently, we synthesized MNM that can be activated by visible light (395–425 nm) that will expand the utility of MNM with increased safety. Moreover, that study further demonstrated that the effects of the MNM were not due to increased generation of reactive oxygen species (ROS). In fact, the twisted alkene in the MNM are free radical traps. (34)

Furthermore, we recently showed the action of light-activated MNMin prokaryotic species, helping to increases the susceptibility of an extensively drug-resistantto otherwise ineffective antibiotics. (22) These studies, taken together, show the versatility of these light-activated MNMs and showcase their ability to disrupt the integrity of prokaryotic cell walls, eukaryotic cell membranes, and physiological functions of multicellular eukaryotes. While previous studies looked into applications of unidirectional molecular motors on surfaces of metal, in liquid crystalline environment and bilayers of lipids membranes, their applications in whole organisms have not been systematically studied. (2,31,32) Our findings suggest potential broader applications of MNMs in biological and physiological systems warranting further systemic studies into their applications.

This study extends the previous work showingactivity of MNMandin destroying prostate cancer cells and mouse embryonic fibroblast cell lines. (21) MNMs accelerated the death of these cells compared to the baseline necrosis observed by the effects of 365 nm light. In contrast, high mortality in eukaryotic species can be attributed to the fast drilling action of the three fast MNM, andwhile the control samples display much lower mortality during the same time. This was seen in bothand, wherefirst showed depigmentation andshowed physiological changes such as slowing heart rate and appendage movement and finally limb dismemberment and death. This shows that light-activated fast motor MNM can extend its destructive properties to cells present in multicellular eukaryotesas well.

Figure 12. Model summarizing results from the nanomechanical action of MNM in C. elegans , Daphnia pulex , and mouse skin. MNM causes damage to cells and tissues in eukaryotic species by nanomechanical drilling action of the fast motor. (a) C. elegans exposed to light-activated MNM 1 or 3 caused depigmentation, increased the fragility of the cuticle, increased autofluorescence, and increased mortality of the nematode by 2 days postexposure. (b) Daphnia exposed to light-activated MNM 1 or 5 caused a decrease in heart rate, decreased appendage movement, dismemberment, and an increase in mortality by day five postexposure. (c) Mouse skin exposed to light-activated MNM 1 or 3 caused epidermal damage, displayed accumulation of MNM in the epidermis, and increased autofluorescence in the dermis.

exposure to light-activated fast MNMorleads to depigmentation, shortening of their length, increasing of autofluorescence, and increasing mortality of the nematode by 2 days postexposure ( Figure 12 a).exposed to light-activated fast MNMorcauses a decrease in heart rate, decreased appendage movement, dismemberment of limbs, and an increase in mortality by 5 days postexposure ( Figure 12 b). Mouse skin exposed to light-activated fast MNMdisplays epidermal ulcerations and micro-lesions in the skin, accumulation of MNMin the epidermis, and increased autofluorescence in the dermal layer ( Figure 12 c). Our results show that the three fast MNM, andcause damage to cells and tissues in eukaryotic species most likely due to the nanomechanical drilling action of the fast motor.

These observations suggest that the nanomechanical action of light-activated MNM 3 can be used to penetrate and infiltrate mouse skin. The apparent micro-lesions and ulcerations in mouse skin suggest that light-activated MNM might have localized therapeutic use for skin growth (cosmetic) or early carcinomas or melanomas treatment, not only in the superficial layer but also in the epidermis. Its localized application and activation allow a controlled environment for the destruction of several layers of skin or epithelial cells.

Figure 11. Light-activated fluorescent MNM 3 can localize to mouse skin epidermal and dermal layers. (a–r) Unstained 4 μm thick mouse skin sections imaged using a Cytation 5 cell imaging multimode reader with a 4× 0.13 NA air objective using bright-field for skin tissue histology (a, d, g, j, m, and p), 469 nm excitation wavelength for MNM (b, e, h, k, n, and q), and overlay (c, f, i, l, o, and r). (a–f) Mouse skin exposed to acetonitrile without MNM: (a–c) no 365 nm light-activation, (d–f) activation with 365 nm light for 30 min. (g–l) Section of mouse skin exposed to MNM 4 dissolved in acetonitrile: (g–i) no 365 nm light-activation, (j–l) activation with 365 nm light for 30 min. (m–r) Mouse skin exposed to MNM 3 dissolved in acetonitrile. (m–o) no 365 nm light-activation, (p–r) activation with 365 nm light for 30 min. Black arrows indicate areas of MNM 3 localization to the epidermal layer. Black arrowheads show areas of MNM 3 localization in the dermal region. (s–t) Fluorescence of MNM localized to the epidermal layer. (s) Comparison of MNM fluorescence with and without light-activation in the no MNM, MNM 4 , and MNM 3 groups. (t) Relative change in MNM fluorescence with light activation compared among the three exposure groups; no MNM, MNM 4 , and MNM 3 . The data points represent the mean of each group using 16 epidermal sections, and error bars represent the standard error (SEM). P -values were obtained by using unpaired two-tail t tests, and the horizontal bars indicate the two groups compared. The scale bar at the lower right is the same for all images.

We examined mouse skin treated with MNMand MNMunder fluorescent microscopy ( Figure 11 ) to localize and quantify the MNM within the skin. Skin treated with nonactivated MNMand MNMdisplay only background fluorescence ( Figure 11 a–c,g–i,m–o). Light-activated MNMalso displayed baseline levels of fluorescence ( Figure 11 d–f,j–l). These observations suggest that there are only low levels of MNMor MNMpresent in the epidermal layer of skin in the absence of activation. Also, activation of MNMdoes not result in increased localization of the MNM. In contrast, we observed accumulation and localization of MNM(black arrows) in the epidermal layer of the light-activated MNMskin ( Figure 11 p–r). Compared to the light-activated MNMor no MNM, light-activated MNMskin displayed significantly higher BODIPY fluorescence ( Figure 11 s–t). These results suggest that light-activated MNMcan localize to the mouse epidermis and appear to penetrate deeper into the skin than in the absence of light activation or as compared to slow-rotating MNM

Figure 10. Mouse skin exposed to light-activated MNM 3 displays epidermal damage. Histopathology using 4 μm thick mice skin sections, stained with hematoxylin and eosin (H&E). Sections were examined by using an optical microscope with a 20× objective. Swiss nu/nu nude mice exposed to 100 μM MNM 3 or MNM 4 . The no MNM control was exposed to the solvent acetonitrile. (a–c) Sections of mouse skin that were not activated with 365 nm light. (a) Skin exposed to acetonitrile without MNM. (b) Skin exposed to MNM 4 dissolved in acetonitrile. (c) Skin exposed to MNM 3 dissolved in acetonitrile. (d–f) Sections of mouse skin that were activated with 365 nm light for 30 min. (d) Skin exposed to acetonitrile without MNM. (e) Skin exposed to light-activated MNM 4 dissolved in acetonitrile. (f) Skin exposed to light-activated MNM 3 dissolved in acetonitrile. Black arrows indicate epidermal ulceration and microlesions. The scale bar at the lower right is the same for all images.

Figure 9. Anesthetized mouse treatment experimental setup. Swiss nu/nu nude mouse skin on the dorsal surface incubated with MNM and subjected to light activation. Six areas on the skin were incubated with MNM in acetonitrile or acetonitrile alone while under anesthesia. (a) Mouse before 365 nm light exposure. (b) Mouse skin area not exposed to light was covered with aluminum foil. Mice were exposed to 365 nm light at 20 mW cm –2 for 30 min.

We examined the activity of light-activated fast motor MNM on larger vertebrate animals. We used the BODIPY-labeled MNMand MNMto treat hairless nude mouse skin ( Figure 9 ) so that we could both evaluate the effects of the rotation speed of MNM and track the localization of the MNM in tissues. Skin sections exposed to MNMor MNM, with or without light activation, were stained with hematoxylin and eosin for pathological examination ( Figure 10 ). Acetonitrile was used to apply the MNM, but it evaporated before the light exposure. Acetonitrile is a common solvent used for skin treatment purposes. (29,30) Histology sections in all three groups without light activation show intact epidermis (top purple layer) with preserved architecture and homogeneous thickness ( Figure 10 a–c). Similarly, mouse skin exposed light-activated slow MNMor no MNM groups also had intact epidermis ( Figure 10 d,e). In contrast, the epidermis of skin exposed to light-activated fast MNMdisplays widespread epidermal lesions and ulcerations (black arrows) ( Figure 10 f). The epidermis appeared thin, and the epidermal architecture was not fully intact in this exposure group. The skin segment exposed to light-activated MNMshows a slight thinning of the epidermal layer but does not show ulcerations as observed with light-activated MNM 10 Figure e). The control mouse skin exposed to 365 nm light with no MNM shows histological features similar to other controls, without any observable damage ( Figure 10 d). Overall, the histological examination suggests that light-activated MNMcan cause ulcerations and micro-lesions in the epidermis of mouse skin.

Light-activated MNMcauses physiological changes and damage inWe synthesized another MNM that is nearly identical to MNMbut carries a PEG arm to improve water solubility, and we examined its impact on. Adultwere incubated with MNMor MNMfor 10 min and activated with 365 nm light for 10 min.incubated with light-activated MNMshowed higher mortality (100%) than without MNM by using the same light exposure (20%) 5 days postexposure ( Figure 8 c). MNMdisplayed significantly higher mortality rates incompared to MNM. This is likely due to the higher solubility and bioavailability of MNMcompared to MNMin aqueous solution. While the killing oftakes time, the physiological changes that occur due to fast rotating MNMs most likely led to their death ( Figure 7 c). We also observed appendage dismemberment inexposed to light-activated MNM, suggesting the damage caused by MNMis very similar to that caused by MNM. Our results show thatexposed to light-activated MNMorled to limb dismemberment, reduced heart rate, reduced limb movement, and eventually the death of 8 Figure d). These observations demonstrate the ability of these light-activated fast motor MNMs to cause damage to cells and tissues in small eukaryotes.

Figure 8. Daphnia exposed to light-activated MNM 1 shows physiological changes, and MNM 5 causes increased mortality. Adult Daphnia incubated with MNM for 10 min and subjected to 365 nm light activation for 10 min. (a) Daphnia exposed either 1 or 10 μM MNM 1 with or without 365 nm light activation. Each bar represents one Daphnia to illustrate the changes in the rate of appendage movement before and after 365 nm light activation. (b) Five Daphnia exposed to no MNM (control) or 10 μM MNM 1 , with or without light activation. Changes in heart rate observed in Daphnia exposed to either no MNM or MNM 1 . A significant decrease in heart rate is observed in the group exposed MNM 1 after 365 nm light activation ( P = 0.0047) and 24 h postexposure ( P = 0.014) compared to no MNM control. (c) Ten adult Daphnia exposed to 365 nm light-activated with no MNM or fast MNM 5 were followed for 5 days. This contained 1% DMSO. Daphnia exposed to MNM 5 showed higher mortality as compared to no MNM ( P < 0.0001). (d) Schematic of a healthy adult Daphnia exposed to light-activated MNM 1 or 5 results in reduced heart rate, reduced limb movement, and limb dismemberment, eventually leading to its death. Black arrows indicate Daphnia appendages. Data points represent the mean of each group, and the error bars represent the standard error. P -values in (a, b) were generated by using unpaired two-tail t tests, and the horizontal bars indicate the two groups compared. The P -value in (c) was calculated with a log-rank Mantel–Cox test.

Figure 7. Representative Daphnia images from the experimental setup. (a–c) Daphnia in a water droplet incubated with 1 or 10 μM MNM 1 for 10 min as observed under a light microscope with a 4× 0.13 NA air objective. (a) A healthy adult Daphnia with all four of its appendages (arrows), as observed under a light microscope with a 4× 0.13 NA air objective. (b) An adult Daphnia as observed under a light microscope after light-activated MNM 1 treatment, showing only two of its four appendages intact (arrows). (c) A deceased adult Daphnia as observed under a light microscope 5 days post-light-activated MNM 1 treatment.

We evaluated thenanomechanical action of MNMin 1% DMSO in, housed in a freshwater reservoir, were grown to adulthood and exposed to MNM, in uniform water droplets, were incubated with 10 μM MNMfor 10 min and then exposed to 10 min of 365 nm light. We examined whether the nanomechanical action of MNMinis dose-dependent by assaying 1 and 10 μM concentrations. The appendage movement of eachwas measured before and after 365 nm light activation of MNM (28) Appendage movement inexposed to 1 μM MNMdid not change significantly ( Figures 7 a and 8 a). In contrast,exposed to 10 μM MNMshowed reduced appendage movement in those individuals that remained alive and no movement in those that were dead ( Figures 7 b,c and 8 a). The heart rate ofdecreased from 170 to 60 beats minafter light activation of MNM 8 Figure b). The heart rate remained close to 160 beats minin the control group without MNMs. Control groups without MNMs were exposed to either 0.1% or 1% DMSO, based on the concentration of MNM used in the experimental groups.were observed 24 h postexposure, and the heart rate of those exposed to light-activated MNMcontinued to remain significantly lower than those not exposed to MNM 8 Figure b). Representative individualare shown to illustrate the changes observed, as some had appendages, but no movement after exposure ( Figure 7 a–c). These data suggest that light-activated MNMcan cause physiological changes and severe appendage tissue damage in

Autofluorescence in animal tissue is mainly due to intracellular lysosome-derived granules, extracellular collagen, or mitochondrial products (NADPH and flavins). (25,26) Autofluorescent molecules lipofuscin and glycation end-products accumulate during the aging process in (25) Gut autofluorescence is also a marker for increased stress and stress response in (24,27) Our observations suggest that light-activated MNMcauses an increase in stress in, increasing their autofluorescence. This is to be expected as the light-activated fast-rotating MNM causes mechanical damage to the surrounding tissues.

Figure 6. High-resolution confocal images show an increase in autofluorescence in C. elegans exposed to light-activated MNM 3 . TP12 C. elegans (expressing eGFP in the cuticle) exposed to 100 μM MNM 4 or MNM 3 for 15 min in 10% DMSO were imaged with or without 365 nm light activation. A 40× 1.3 NA oil-immersion objective was used, with an excitation wavelength of 488 nm. At least four C. elegans were imaged for each group. Each nematode was imaged head (upper segment), trunk (midsegment), and tail (lower segment). Representative images are shown for each group. (a–f) C. elegans incubated with 10% DMSO (no MNM). (a–c) Without 365 nm light exposure. (d–f) With 15 min of light exposure. (g–l) C. elegans incubated with 100 μM MNM 4 with 10% DMSO. (g–i) Without 365 nm light activation. (j–l) With 15 min of light activation. (m–r) C. elegans incubated with 100 μM MNM 3 with 10% DMSO. (m–o) Without 365 nm light activation. (p–r) With 15 min of light activation. (s–u) Quantification of autofluorescence in C. elegans in each group. Four nematodes were used to evaluate three areas of fluorescence in each nematode, giving a mean fluorescence with 12 measurements for each group. The relative increase in fluorescence is shown after correcting for autofluorescence in C. elegans exposed to nonactivated MNM. (s) Relative increase in autofluorescence in no MNM, MNM 3 , and MNM 4 groups for each segment (upper, middle, and lower). C. elegans incubated with 365 nm light-activated MNM 3 showed a significant increase in autofluorescence. (t) Relative increase in autofluorescence for each of the segments (upper, middle, and lower) is similar in no MNM, MNM 3 , and MNM 4 . (u) Relative increase in autofluorescence for C. elegans as a whole organism. Light-activated MNM 3 showed a significant increase compared to both MNM 4 and no MNM groups. The bars represent the mean of each group, and the error bars represent the standard error. P -values are from unpaired two-tail t tests, and the horizontal bars indicate the two groups compared. The scale bar at the lower right is the same for all images.

We examined the physiological and pathological effects of MNMtreatment of. We used astrain expressing eGFP on its cuticle (TP12 strain) with MNMand MNMand examined them via confocal microscopy. While we did not observe widespread microlesions on the cuticular surface ofat this resolution, we observed a significant increase in gut autofluorescence inexposed to 365 nm light-activated fast MNMcompared toexposed to no MNM or slow MNM 6 Figure ). Gut autofluorescence, among other factors, is a marker of increased stress in (24) While there was an observable increase in gut autofluorescence in allsubjected to 365 nm light, the increase in autofluorescence was more widespread and intense in those exposed to light-activated MNM 6 Figure a–r).incubated with either no MNM or MNMdisplayed the same level of autofluorescence that was significantly lower than MNM 6 Figure d–f,j–l,s–u). These results suggest that while 365 nm light alone can cause some stress in, light-activated MNMcauses even greater stress ( Figure 6 p−r, s−u). The most likely source of stress inis the fast rotational movement of the light-activated MNMdamaging tissues.

We were interested in whether DMSO plays a role in the observedmortality when exposed to MNM(100 μM).exposed to light-activated MNM(100 μM) had higher mortality rates when incubated with 10% DMSO (70% mortality, Figures 4 c,d and 5 d–f) when compared to 1% DMSO (25% mortality, Figures 4 a,b and 5 a–c), 2 days postexposure.exposed to light-activated MNM(100 μM) in 1% DMSO continued to die, showing a mortality rate of 45% over the next 5 days. This showed that even though DMSO alone did not have a deleterious effect onwhen light-activated MNM(100 μM) were combined with 10% DMSO, they increased mortality from 25% to 70%. In the presence of 10% DMSO, the mortality rate oftreated with light-activated MNM(100 μM) also increased slightly to 20%. But this was not significantly different from the mortality caused by the no MNM, with a mortality rate of 15% ( Figure 4 d–f). In the absence of light activation, 10% DMSO with or without MNM did not cause an increase in mortality ( Figure 4 c,e). This suggests that DMSO, by itself, does not cause mortality. However, DMSO increases MNMmediated mortality. This is likely due to the increase in cell permeability caused by DMSO that allows MNMto gain access to cells and thereby cause more deleterious effects.

Figure 5. C. elegans exposed to 0, 1, and 10 μM MNM 1 in 1% (a–c) or 10% DMSO (d–f). (a–c) Three-day-old adult C. elegans incubated with no MNM, 1 μM, or 10 μM MNM 1 in 1% DMSO for 15 min and activated with 365 nm light. The life span and mortality rate were assayed by counting C. elegans daily. Each group had 20 nematodes. (a) Without light activation ( P = 0.987). (b) 5 min of light activation ( P = 0.244). (c) 15 min of light activation ( P = 0.016). (d–f) Three-day-old adult C. elegans incubated with no MNM or 100 μM MNM 1 in 10% DMSO for 15 min and activated with 365 nm light. The life span and mortality rate were assayed by counting C. elegans daily. Each group had 20 nematodes. (d) Without light activation ( P = 0.372). (e) 5 min of light activation ( P = 0.887). (f) 15 min of light activation ( P = 0.019). P -values in (a–f) were calculated with log-rank Mantel–Cox tests.

We examined the dose–response of light activation on mortality caused by MNM(100 μM) through exposingto 5 min of light activation, 15 min of light activation, or no light activation. Activation of MNM(100 μM) with 365 nm light showed a time-dependent increase in mortality ( Figure 5 ). The mortality ofwas also dependent on the concentration of activated MNM 5 Figure a–c).exposed to MNMat 100 μM with 15 min of light activation showed a 65% mortality compared to 25% with 5 min of light activation and 10% mortality without light activation, 2 days postexposure ( Figure 5 e,f). These results further confirm that mortality caused by MNM(100 μM) is due to their fast rotational movement triggered by 365 nm light activation, and the concentration and time dependence suggests a dose–response relationship.

Figure 4. Light-activated MNM 1 increases mortality in C. elegans . Three-day-old adult C. elegans incubated with 100 μM MNM 1 , MNM 2 , or no MNM control for 15 min and exposed to 365 nm light for 15 min. The life span and mortality rate were assayed by counting C. elegans daily. Each group had three replicates of 20 nematodes each (total 60 per group). (a, b) C. elegans incubated with no MNM, MNM 2 , and MNM 1 , in 1% DMSO. (a) Without light activation ( P = 0.432). (b) With 15 min of 365 nm light activation ( P = 0.309). (c, d) C. elegans incubated with no MNM, MNM 2 , and MNM 1 , in 10% DMSO. (c) Without light activation ( P = 0.981). (d) With 15 min of light activation ( P = 0.005). (e, f) Survival fraction of C. elegans incubated with MNM 1 , MNM 2 , or no MNM, in 10% DMSO, with or without 365 nm light activation, 2 days postexposure. C. elegans exposed to 15 min of light-activated MNM 1 had a 70% mortality rate. (e) Comparing differences in survival fraction between no MNM, MNM 2 , and MNM 1 . (f) Comparing differences in survival fraction between light-activated and non-light-activated in each of the MNMs. The bars represent the mean of each group, and the error bars represent the standard error. P -values in (a–d) calculated with log-rank Mantel–Cox tests and (e, f) with unpaired two-tail t tests and horizontal bars indicate the two groups compared.

To study the effects of MNMon mortality, we incubatedwithout MNM, with 100 μM MNM, or with 100 μM MNMfor 15 min in 1% or 10% DMSO and activated with 365 nm light for 15 min. Without light activationexposed to no MNM, MNM(100 μM), or MNM(100 μM) in 1% DMSO displayed similar life spans ( Figure 4 a). With light activation,exposed to MNM(100 μM) in 1% DMSO showed a mortality rate of 25% 2 days postexposure ( Figure 4 b). In groups exposed to light-activated MNM(100 μM) with 10% DMSO, we observed a mortality rate of 70% 2 days postexposure compared to groups not activated by light ( Figure 4 c–f). This was in contrast to mortality rates that were seen with MNM(100 μM) (22.5%) and no MNM control (10%) groups ( Figure 4 c–f).exposed to 100 μM MNMorwithout light activation showed no increase in mortality, similar to that of the no MNM control group ( Figure 4 a,c). These results suggest that MNM induced mortality is specifically driven by light activation of fast MNM, and the effect is concentration-dependent.exposed to 365 nm light for 15 min ( Figure 4 b,d) showed a slight increase in mean life span compared to those not exposed to 365 nm light ( Figure 4 a,c). Such an increase in life span has been observed before and is attributed to the induction of stress proteins whenis exposed to a stressor such as heat or light energy. (8,23)

in control groups that were exposed to either no MNM ( Figure 3 b,d) or slow MNM(100 μM) control ( Figure 3 f, h), 2 days postexposure, retained their dark pigmentation and body length and survived the initial exposure. In contrast, those exposed to light-activated MNM(100 μM) ( Figure 3 l) showed pathological changes 2 days postexposure. This included loss of dark pigmentation (arrow), a decrease in length (arrow), and eventually leading to death (arrowhead). This was a gradual process over the first 2 days postexposure, with loss of dark pigmentation observed even 1 day after exposure ( Figure 3 k). Our results indicate that MNM(100 μM) can cause pathological changes inwith as little as 15 min of light activation.exposed to light-activated MNMunderwent depigmentation, displayed a shortened length, had a >70% reduction in laying of eggs, and resulted in the death of 3 Figure m).

Figure 3. Light-activated MNM 1 can cause pathological changes in C. elegans . C. elegans exposed to either nonactivated or 365 nm light-activated MNM 1 , MNM 2 , or no MNM observed under a light microscope (40× 1.3 NA) to identify morphological and pathological changes. C. elegans in all groups were incubated with MNM in 10% DMSO for 15 min prior to light activation. Three representative C. elegans are shown for each group; 1 day and 2 days postexposure to MNM. (a, b) Incubated with only 10% DMSO (no MNM), without light activation. (c, d) Incubated with no MNM, with light activation. (e, f) Incubated with MNM 2 , without 365 nm light activation. (g, h) Incubated with MNM 2 , with light activation. (i, j) Incubated with MNM 1 , without light activation. (k, l) Incubated with MNM 1 , with light activation. (l) White arrow shows C. elegans with loss of pigmentation, and the white arrowhead shows two dead C. elegans , 2 days postactivation. The scale bar in the lower right is the same for all images. (m) Schematic of a healthy 3 day old C. elegans exposed to light-activated MNM 1 showing depigmentation, shortened length, and reduced progeny, eventually leading to its death.

Figure 2. C. elegans experimental setup. Nematodes incubated with MNM for 15 min were transferred onto NGM agar plates and exposed to 365 nm light. (a) Light source with a wavelength of 365 nm. (b) The light source was placed directly above the NGM agar plate to cover the entire plate and ensure high and constant energy delivery. (c) C. elegans in NGM agar plate with 365 nm light source at a constant distance of 1.27 cm above the agar plate to deliver a constant energy of 10–15 mW/cm 2 . (d) NMG agar plates with E. coli (OP50) as a food source used to maintain C. elegans after MNM and light exposure.

We used a 365 nm light source to activate MNMs with constant light flux (10–15 mW/cm) delivered at a constant distance from our model organisms ( Figure 2 ). To study morphological and pathological effects caused by the fast rotational action of MNM, we exposed N2(wild type) in 10% DMSO with no MNMs, MNM(100 μM), or MNM(100 μM) with and without light activation for 15 min. DMSO was needed at 1% or 10% to dissolve MNMs according to their concentrations.were then observed under a light microscope (40× magnification) to identify changes caused by MNM ( Figure 3 ). Control groups ofwere exposed to no MNM or MNM(100 μM) with and without light activation, resulting in healthy nematodes with appropriate dark pigmentation and no apparent pathological changes ( Figure 3 a–h). In addition, it was observed that their ability to lay eggs was intact, 1 day and 2 days postexposure. In contrast, we observedexposed to light-activated MNM(100 μM) showed a notable decrease in the number of eggs laid (>70% reduction in eggs laid compared to nonexposed) and a decrease in pigmentation when compared to non-light-activated MNMexposed 3 Figure i–l) at 1 day and 2 days postexposure. Our observations suggest that light-activated MNMcan cause deleterious effects inas early as 1 day postexposure.

Figure 1. MNMs used in this study. MNM 1 : a fast motor with a rotor that can rotate at 2–3 MHz when activated with 365 nm light. MNM 2 : a slow motor with a rotor that can rotate 1.8 revolutions h –1 when activated with 365 nm light at 60 °C but only undergoes cis/trans isomerization at lower temperatures, not a full rotation. MNM 3 : a fast motor with BODIPY fluorophores attached to two arms in its stator. MNM 4 : a slow motor with BODIPY fluorophore attached to two arms in its stator. MNM 5 : a fast motor with one PEG to increase water solubility.

When MNMs ( Figure 1 ) were added in micromolar concentrations (1–10 μM) into mammalian cells cultured in media, and activated by 365 nm light, they embeded into the cell membrane and made pores by using their fast-rotational movement. The rotational speed of MNMis 2–3 million rotations s. MNMs that bear peptides for specific cell targeting can be activated at 355–365 nm to destroy prostate adenocarcinoma cells (PC-3) and mouse embryonic fibroblast cells (NIH 3T3) (21) The variations of MNMs used in this study are shown in Figure 1 . The slow motor (MNM) rotates at ∼1.8 revolutions hand allows us to control for the effects of MNMs other than fast rotational movement. We attached BODIPY fluorophores to the stator of MNMto form MNM, and we useH NMR spectra to characterize the half-rotation of slow MNM Figures S1 and S2 ). Hence, MNMand MNMserve as controls to confirm that the deleterious effects observed with MNMand MNMare due to their drilling action via fast rotational movements. MNMprovides a complementary water-soluble pendant to the fast motor core of MNM

In summary, our study shows that the nanomechanical action of light-activated fast-rotating MNM ( 1 , 3 , and 5 ) causes varying degrees of damage to cells and tissues of eukaryotic species: C. elegans , Daphnia , and mice. These MNM also increase mortality in C. elegans (70%) and Daphnia (100%) over a few days postexposure. The depigmentation of C. elegans and the dismemberment of Daphnia limbs suggest that the action of MNM first causes nanomechanical disruption of cells and tissues and then causes death in organisms that cannot recover from MNM damage. Decreased heart rate and appendage movement in Daphnia also highlight the impact of light-activated fast motor MNM on physiology in small eukaryotes. Lesions and ulcerations caused by topical application of light-activated MNM onto mouse skin demonstrate the ability of MNM to function in larger eukaryotes. Light-activated MNMs have potent in vivo activity against microscopic eukaryotes and skin tissue, suggesting the potential to be applied to industrial or environmental parasite control, local treatment of diseases including skin cancer, and cosmetic application on the skin.

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Light Source to Activate MNMs 2.(22) A 365 nm light source (Sunlite Science & Technology, Inc., www.powerledlighting.com ) was used to activate MNMs. MNMs were activated with a constant light flux by placing the light source directly above at a constant distance to the eukaryotic organism ( Figure 2 a–d). The 365 nm light source had a narrow wavelength spectrum of 360–376 nm, with a peak intensity at 368 nm and a constant power output of 10–15 mW/cm

Synthesis of the BODIPY Slow Motor 4 1, 2, and 3 were described previously.2 (13.0 mg, 0.022 mmol), BODIPY dye 4 ·5H 2 O(s) (0.55 mg, 0.022 mmol), and sodium ascorbate (0.93 mg, 0.007 mmol) was sealed with a rubber septum cap. A well-degassed mixture of CH 2 Cl 2 (0.1 mL) and water (0.1 mL) was added to the vial, and the vial was shaken by a wrist-action shaking machine for 36 h. The mixture was partitioned between CH 2 Cl 2 (5 mL) and water (5 mL). The organic phase was dried over anhydrous MgSO 4 and filtered, and the filtrate was concentrated under vacuum. The crude product was purified by preparative TLC (silica gel, 4% MeOH in CH 2 Cl 2 ) to afford the desired compound 4 as an orange solid (27 mg, 87%). Complete spectra are provided in the Syntheses of MNM, andwere described previously. (21,35) A 2 mL vial charged with motor(13.0 mg, 0.022 mmol), BODIPY dye (36) (20.0 mg, 0.049 mmol), CuSO·5HO(s) (0.55 mg, 0.022 mmol), and sodium ascorbate (0.93 mg, 0.007 mmol) was sealed with a rubber septum cap. A well-degassed mixture of CHCl(0.1 mL) and water (0.1 mL) was added to the vial, and the vial was shaken by a wrist-action shaking machine for 36 h. The mixture was partitioned between CHCl(5 mL) and water (5 mL). The organic phase was dried over anhydrous MgSOand filtered, and the filtrate was concentrated under vacuum. The crude product was purified by preparative TLC (silica gel, 4% MeOH in CHCl) to afford the desired compoundas an orange solid (27 mg, 87%). Complete spectra are provided in the Supporting Information (Figures S2–S4).

C. elegans, Daphnia, and Mice Strains N2 (Bristol, wild type) and TP12 [kaIs12(col-19::GFP)] C. elegans strains were used in this study. The N2 strain was used to observe pathological changes and for mortality assays. The TP12 strain was used for confocal microscopy imaging with fluorescent MNM-BODIPY in C. elegans. The Daphnia strain was obtained from Carolina Biological Supply Company (Burlington, NC). Female Swiss nu/nu nude mice were maintained and handled using protocols approved by the Institutional Care and Use Committee (IACUC) at the MD Anderson Cancer Center (MDACC).

C. elegans Exposure to MNM C. elegans (N2) from a nonstarved NGM plate was subjected to a bleaching procedure to obtain nematode eggs.C. elegans nematodes recovered with M9 buffer were used, and about 200 worms each were placed Eppendorf tubes in 1 mL of M9 buffer. C. elegans were exposed to MNM 1, MNM 2, or a DMSO control without MNM at concentrations of 1, 10, or 100 μM. Nematodes were incubated at room temperature for 15 min with gentle shaking on a test tube rocker. Worms were pipetted onto small NGM plates seeded with OP50 and exposed to 365 nm light for 15 min or not exposed to light. The worms were then placed at room temperature (20–22 °C) overnight and followed for the duration of the worms’ life spans. Worms were counted daily and transferred onto new small NGM agar plates for the first 3 days after light exposure and then transferred onto new plates every other day. Adult(N2) from a nonstarved NGM plate was subjected to a bleaching procedure to obtain nematode eggs. (37) Age synchronized 3-day-old adult N2nematodes recovered with M9 buffer were used, and about 200 worms each were placed Eppendorf tubes in 1 mL of M9 buffer.were exposed to MNM, MNM, or a DMSO control without MNM at concentrations of 1, 10, or 100 μM. Nematodes were incubated at room temperature for 15 min with gentle shaking on a test tube rocker. Worms were pipetted onto small NGM plates seeded with OP50 and exposed to 365 nm light for 15 min or not exposed to light. The worms were then placed at room temperature (20–22 °C) overnight and followed for the duration of the worms’ life spans. Worms were counted daily and transferred onto new small NGM agar plates for the first 3 days after light exposure and then transferred onto new plates every other day.

Imaging of C. elegans TP12 [kaIs12(col-19::GFP)] C. elegans expressing cuticular eGFP under the control of gene col-19 were exposed to MNM 3 or 4 as detailed above. After exposure, C. elegans were fixed with 4% paraformaldehyde and washed once with M9 buffer. C. elegans were then mounted onto slides, and 4–6 worms were imaged in each exposure group. Each nematode was imaged at the head (upper), trunk (middle), and tail (lower) region, at a magnification of 400× and a resolution of 1024. Representative images are presented to show differences observed after exposure to nonactivated and activated MNM. A confocal microscope (Nikon A1R+/A1+) with a FITC fluorescence filter (excitation 488 nm, emission 525/50 nm) and 40× oil immersion objective were used. Confocal microscopy used the Galvanometric scanning method controlled by a 1/4 frame/s (fps: 0.25; frame time 4 s), immersion oil type F (η = 1.518), a numerical aperture of 1.3, and a pinhole of 0.6 AU (19.2 μm). The size of the images was 1024 × 1024 pixels, mono 12 bit (0.31 μm/px), and an average of two images were taken.

Confocal Microscopy Quantification NIH ImageJ (version 1.4.3.67) software was used to quantify fluorescent intensities of images obtained by confocal imaging. Confocal images were obtained with a FITC fluorescence filter (excitation 488 nm, emission 525 nm) and 40× 1.3 NA oil immersion objective. For quantification of fluorescence, each group containing four nematodes with three different segments (head, trunk, and tail) were used. The fluorescence signal in three regions within each segment was quantified by using the same area, and averages were calculated. For each group, the ratios of non-light-activated to light-activated MNM were obtained, and their averages were calculated. Results presented are an average of four images taken for each nematode segment in each group. The total sample size was at least four C. elegans per exposure group, with images of the upper, middle, and lower sections of each worm. When more than four C. elegans were imaged, it is stated in the results. For quantification, the worm images were further divided into three areas to give a total of 12 representative areas for each C. elegans segment.

Daphnia Exposure to MNM Adult Daphnia were incubated with MNM 1 for 10 min and exposed to 365 nm light for 10 min, similar to the setup described for C. elegans. Heart rate and appendage movement were measured on the same Daphnia, before and after light activation of MNM 1. Observations were made under 20× magnification with a light microscope for 15 or 30 s, and the rates were calculated per minute. Ten adult Daphnia were incubated with MNM 5 for survival assay. The Daphnia exposed either to no MNM or MNM 5 were followed over the course of 5 days for mortality and survival.

Mouse Skin Exposure to MNM 3, MNM 4 or no MNM in acetonitrile, over six delimited areas on their skin while anesthetized with isoflurane administered. After 5 min of incubation for the acetonitrile solvent to allow it to evaporate, half the mouse skin that had been topically treated with MNM was exposed to 365 nm light at 20 mW/cm2 for 30 min and the other half was covered with aluminum foil to protect it from light exposure (3 (fast motor-BODIPY) without light activation; (2) MNM 4 (slow motor-BODIPY) without light activation; (3) no MNM control (acetonitrile) without light exposure; (4) MNM 3 (fast motor-BODIPY) with light activation; (5) MNM 4 (slow motor-BODIPY) with light activation; (6) no MNM control (acetonitrile) with light exposure. Immediately after the light activation, the mice were euthanized and skin segments were harvested. MD Anderson institutional animal care and use committee (IACUC) protocols were followed when handling the mice. Female Swiss nu/nu nude mice were obtained from the Experimental Radiation Oncology Mouse Facility at the MD Anderson Cancer Center. They were treated with 10 μL of 100 μM MNM, MNMor no MNM in acetonitrile, over six delimited areas on their skin while anesthetized with isoflurane administered. After 5 min of incubation for the acetonitrile solvent to allow it to evaporate, half the mouse skin that had been topically treated with MNM was exposed to 365 nm light at 20 mW/cmfor 30 min and the other half was covered with aluminum foil to protect it from light exposure ( Figure 9 ). The six exposure areas were as follows: (1) MNM(fast motor-BODIPY) without light activation; (2) MNM(slow motor-BODIPY) without light activation; (3) no MNM control (acetonitrile) without light exposure; (4) MNM(fast motor-BODIPY) with light activation; (5) MNM(slow motor-BODIPY) with light activation; (6) no MNM control (acetonitrile) with light exposure. Immediately after the light activation, the mice were euthanized and skin segments were harvested. MD Anderson institutional animal care and use committee (IACUC) protocols were followed when handling the mice.

Histopathology of Mouse Skin Exposed to MNM Mouse skin segments were fixed in 10% buffered formalin for 24 h and embedded in paraffin blocks. The skin tissues were processed as 4 μm thick sections and subsequently stained with hematoxylin and eosin (H&E). H&E stained slides were imaged using an optical microscope (AMEX-1100, Advance Microscopy Group) with a 20× objective.

Fluorescent Microscopy of Mouse Skin Exposed to MNM Mouse skin segments were fixed in 10% buffered formalin for 24 h and embedded in paraffin blocks prior to cutting 4 μm thick sections. These unstained slides were imaged for NM-BODIPY emission by using an inverted fluorescence microscope (DMI6000B, Leica, Germany) with a 20× objective, 0.4, L5 blue light filter cube with excitation filter 480/40 nm and suppression filter 527/30 nm. The tissue autofluorescence was imaged with N3 green light filter cube with excitation filter 546/12 nm and suppression filter 600/40 nm. The fluorescence intensities were quantified by using ImageJ with a similar method as used in C. elegans. Fluorescence images for localization were prepared on a Cytation 5 cell imaging multimode reader with a 4× objective using bright-field for skin tissue histology and 469 nm excitation wavelength for BODIPY-MNM.