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Category : Nanomedicine

Nanoparticle interactions with immune cells dominate tumor retention and induce T cellmediated tumor suppression in models of breast cancer – Science…


Nanoparticles provide unique opportunities and challenges for cancer therapy and diagnosis. They have the potential to interact with the immune system and solid tumor microenvironment (TME) in unexpected ways to ultimately and critically affect performance and tumor response (13). The premise that nanoscale materials can be engineered to selectively detect and destroy cancer cells in solid tumors is undergoing a critical reevaluation (411). Yet, relatively little analysis of nanoparticle fate and intratumor accumulation across biological models and immune cell or tumor compartments has been completed, particularly with histology or flow cytometry (6).

As with many cancer drug development scenarios, nanotechnology-based formulations are often tested and optimized using a specific mouse model of human cancer. These xenograft tumor studies rely on immunodeficient animal models, which provide a permissive environment for cross-species tissue grafting. Therefore, how well these models predict the potential and mechanisms for nano-targeting becomes a relevant question when the construct itself demonstrates strong interactions with the recipients immune system (13, 6).

Polysaccharide (starch or dextran)coated iron oxide nanoparticles have been used for decades in biomedicine as agents for parenteral anemia therapy, magnetic resonance contrast, cancer hyperthermia, drug delivery, cell sorting, and most recently for inducing ferroptosis in cancer cells (4, 5, 1219). Thus, they present an interesting and important class of nanoparticles for applications in medicine.

Here, we show that host immune status and the immune components of the TME are key factors influencing retention of 100-nm hydroxyethyl starchcoated iron oxide nanoparticles in orthotopic mammary tumors. When labeled with an antibody, the nanoparticles were retained by tumors regardless of the presence of the target antigen, whereas retention of the unlabeled counterpart was not substantial. Additional experiments demonstrated that systemic exposure of tumor-bearing immune competent mice to the nanoparticles induced immune-mediated tumor growth inhibition with evidence of later infiltration by CD8+ T cells. Both plain and antibody-labeled nanoparticles initiated similar immune responses with similar tumor growth inhibition and T cell infiltration into tumors, despite different tumor retention. This suggests that complex interdependencies exist between host and tumor immune responses to nanoparticle exposure. Together, these results offer intriguing possibilities to explore nanoparticle targeting of the tumor immune microenvironment, and they demonstrate an exciting potential to develop nanoparticles as cancer immune therapy platforms.

We used amine-functionalized starch-coated bionized nanoferrite (BNF) nanoparticles with trastuzumab (BH), a humanized antihuman epidermal growth factor receptor 2 (HER2/neu) monoclonal antibody approved for clinical use in the management of HER2+ breast cancer (Fig. 1A). The ability of trastuzumab to target HER2+ cancer cells in tumors has been validated and well documented, as has its use for nanoparticle-targeting studies (20, 21). The precursor BNF-Plain (BP) nanoparticles comprise a magnetic iron oxide core that is coated with hydroxyethyl starch (core shell) to provide biocompatibility and colloid stability in biological media (1519).

(A) Schematic of particle chemistry showing amine functionalization of BP nanoparticles using maleimide precursors for conjugation with thiol moieties of the antibody (trastuzumab). (B) Western blot analysis showing HER2 protein expression by human breast cancer cell lines used in the study. (C) Immunofluorescence results showing HER2 protein surface expression in six human breast cancer cell lines. MDA-MB-231 is a triple-negative ER/PR/HER2- cell line. MCF7/neo and MCF7/HER2 are an isogenic pair with HER2-expressing (MCF7/HER2) variant having a single copy of HER2 gene and HER2- (MCF7/neo), which received a scrambled gene. Other cell lines are wild type and express varying amounts of HER2 protein. (D) In vitro iron content analysis (ferene-s assay) after exposure of cells to BP and BH nanoparticles shows a positive correlation with HER2 protein level and iron uptake in the breast cancer cells. For the assay, cells were incubated at 37C for 3 hours with BP or BH nanoparticles (0.5 mg/ml) and evaluated for total iron content after washing unbound particles. Untreated cells, Herceptin alone, and BNF-IgG were used as controls. The average of three independent experiments is shown. Statistical differences among BP, BH, and BNF-IgG were obtained by two-tailed Students t test (*P < 0.05 and **P < 0.01). (E) Schematic of the overall study design using mouse models of human breast cancers. See text for details.

The choice of 100-nm BNF nanoparticles was motivated from our previous study, which demonstrated higher accumulation by the 100-nm nanoparticles to tumors than with 30-nm nanoparticles, despite the longer blood circulation time of the latter construct (1519). When measured by dynamic light scattering, BP nanoparticles had a mean measured (z average) hydrodynamic diameter of 99 nm (3) with a mean polydispersity index of 0.07 (0.02) (table S1). Zeta potential, a measure of surface charge density, was slightly negative (2.2 0.2 mV) at pH 7.4. Overall, addition of trastuzumab to the BP nanoparticles had only a modest effect on the measured physical properties of the nanoparticles. Several of antibody-labeled nanoparticles were prepared and assayed using a modified in vitro test to confirm selective binding of the BH construct. In all cases, successful binding of antibody was confirmed by a modified bicinchoninic acid assay (BCA) and immunofluorescence (tables S2 to S5 and fig. S1, A and B). A BNFimmunoglobulin G (IgG) construct was synthesized with a nonspecific human polyclonal antibody, as an additional control. The measured physical properties of the BNF-IgG nanoparticles were similar to those of BH nanoparticles (tables S2 and S4).

We limited our selection of cancer models to those for which a stable transmembrane protein/marker is well documented and for which multiple cell lines and mouse models are readily available. In general, HER2+ breast cancer biology has been extensively studied, providing numerous human and mouse cell lines to yield xenograft, syngeneic, and spontaneous models (22, 23). For in vitro and xenograft studies, we selected six human breast cancer cell lines (Fig. 1, B and C, and table S3). HER2 protein expression was verified by Western blotting (Fig. 1B). We used an isogenic pair derived from a HER2- MCF7 parental line, MCF7/HER2 (+), and MCF7/neo () (Fig. 1, B and C). The variable total protein and surface expression of HER2 were evident in three HER2+ lines: HCC1954, BT474, and SKBR3 from both Western blotting and immunofluorescence, whereas MDA-MB-231 cells showed no HER2 expression.

Residual iron concentration was measured in cells using a modified ferene-s assay (24) and correlated with HER2/neu expression following exposure to BH nanoparticles. Both iron concentration and HER2/neu expression followed the same progression: MCF7/HER2 < HCC1954 < BT474 < SKBR3 (Spearman correlation coefficient, = 0.89, P = 0.03; Fig. 1D, inset, and fig. S1C), confirming that in vitro targeting occurred via the expected antibody-antigen binding.

We used two immunodeficient strains of mice [athymic nude and nonobese diabetic/severe combined immunodeficiency (NOD/scid) gamma (NSG)] engrafted with five human breast cancer cell lines: two HER2- cell lines (MDA-MB-231 and MCF7/neo) and three HER2+ lines (MCF7/HER, HCC1954, and BT474). The xenograft study design is illustrated in Fig. 1E, and details are provided in Materials and Methods and table S6. Visibly evident 24 hours after injection by discoloration of tumors, BH nanoparticles were retained by tumors to a greater extent than were BP nanoparticles (Fig. 2A).

(A) Gross morphology of tumors following intravenous injection with BP or BH nanoparticles shows different tissue color. Darker (black) color indicates greater particle uptake. Tumors from NOD/scid (NSG) mice show more BH than BP. Photo credit: Preethi Korangath, Johns Hopkins University. (B) Representative images of HER2 immunohistochemistry (IHC) from breast xenografts showing that expression correlates with in vitro expression. (C and D) Inductively coupled plasma mass spectrometry (ICP-MS) of Fe recovered from tumors excised from mice injected with BH nanoparticles demonstrates consistently higher Fe content than tumors from mice injected with BP nanoparticles regardless of HER2 status of the tumor. Recovered iron was higher in tumors excised from NSG mice (D) than that from athymic nude mice (C) (*P < 0.05, **P < 0.01, and ***P < 0.001). (E and F) Prussian bluestained tissue slides recovered from the same tumors as in (C) and (D) and digitally analyzed for percent positive area that showed a similar trend as observed with ICP-MS. (G and H) ICP-MS analysis of Fe from the livers showed higher iron content in mice injected with BP nanoparticles than mice injected with BH nanoparticles, mirroring the results of Fe recovered from tumors (**P 0.01 and ***P < 0.0001).

Volumetric analysis of iron by inductively coupled plasma mass spectrometry (ICP-MS) recovered from tumors grown in nude mice corroborated our observations of gross tumor presentation. HER2 status/expression of tumors was confirmed by immunohistochemistry (IHC) (Fig. 2B). Higher iron concentrations were present in tumors of mice injected with BH relative to phosphate-buffered saline (PBS) or BP-injected mice (P < 0.001) irrespective of HER2 status (Fig. 2C). In contrast, intratumor iron concentrations measured from mice receiving BP was only slightly higher than PBS-injected controls (MCF7/HER, HCC1954, and BT474; P > 0.05; see Fig. 2C). Iron recovered from nude mice bearing MCF7/neo tumors injected with BH was comparable to those recovered from MCF7/HER tumors. Comparable iron recovery in these two isogenic (HER2+/) tumor models following injection with BH, which was higher than either PBS- or BP-injected controls, suggests that biological factors other than antibody-antigen binding were responsible for nanoparticle retention. In other words, the BH nanoparticle targeting observed in vitro was not evident in vivo.

This pattern of retention was also measured in tumors recovered from NSG mice (Fig. 2D and fig. S2); however, HER2 expression by the tumor slightly correlated more with BH retention in NSG mice than in nude mice (Fig. 3, A and B). In contrast to results obtained from nude mice, iron recovered from HCC1954 and BT474 tumors in NSG mice was slightly higher than in MCF7/HER, consistent with higher HER2/neu protein expression in these cell lines (Fig. 1, B and C).

(A) Analysis of Prussian bluepositive (nanoparticle-rich) areas of tumors from nude mice injected with BH nanoparticles reveals only weak correlation with HER2 expression. (B) Conversely, this correlation is stronger in tumors from NSG mice. (C and D) Weak or no correlation was observed between BH nanoparticle presence and CD31+ (vascular endothelium) regions. (E) Representative histology images of sequential sections showing IBA-1+ cells associated with Prussian bluepositive areas in HCC1954 (HER2+) tumors grown in NSG mice and treated with BH (a) hematoxylin and eosin (H&E), (b) Prussian blue, (c) HER2 IHC, (d) IBA-1 IHC, (e) CD-31 IHC, (f) H&E of another area from same tumor, (g) sequential section stained for Prussian blue shows positive staining for iron nanoparticles, and (h) immunofluorescence (IF) staining for IBA-1 shows positivity in the nanoparticle accumulated region. (F and G) Iron recovery from HER2+ (HCC1954) or HER2 (MDA-MB-231) tumors is similar whether BNF nanoparticles have trastuzumab (anti-HER2) or human IgG (polyclonal), suggesting that antibody-antigen binding does not drive intratumor nanoparticle accumulation. ns, not statistically significant.

We analyzed tumor tissue sections stained with Perls reagent (also known as Prussian blue) to visualize the nanoparticle-rich regions across all models (Fig. 2, E and F, and fig. S3). The trends observed with gross presentation and ICP-MS were consistent with tumor histopathology (Fig. 2, C to F, and fig. S2) and also revealed notable spatial heterogeneity of iron localization. Nevertheless, all tumor models studied showed significantly more nanoparticle retention when mice were injected with BH, but localization to cancer cells was not evident.

As previously observed, a substantial amount of systemically injected nanoparticles will accumulate in the liver (611, 2527). It is widely held that resident macrophages (liver) and circulating macrophages along with other phagocytic immune cells will sequester nanoparticles of about 100 nm in diameter, clearing them from blood circulation and depositing them into the liver and other organs. Our ICP-MS analysis of iron recovered from the livers showed that all mice injected with nanoparticles exhibited higher iron concentration in the liver than PBS-injected controls. However, the livers of mice injected with BP had higher iron content than the livers of mice injected with BH (Fig. 2, G and H). We conclude that BH retention in tumors (and perhaps other tissues not assayed) contributed to the reduced liver content when compared with BP-injected mice.

Higher tumor retention of Herceptin (Her/trastuzumab)labeled nanoparticles having varied composition and sizes (15 to 500 nm) following systemic delivery into nude female mice bearing MCF7 tumors has been noted (2831). It is worth emphasizing, however, that MCF7 cancer cells express no HER2 antigen on their membranes, begging the question of the mechanisms of targeting observed in these previous studies. Together, results reported here and elsewhere indicate that retention of nanoparticles in (xenograft human-mouse) tumors may depend on complex biological responses that are intertwined with the host immune system. We note in our results that immune status of the mouse seemed to play a role in nanoparticle retention in tumors, whereas antigen expression by cancer/tumor cells seemed to have very little influence, especially in nude mice (Fig. 2 and fig. S2). Further study was needed to determine nanoparticle association with cell type.

We analyzed comparable regions of stained serial tissue sections in detail by scoring to determine whether intratumor nanoparticle localization correlated with tumor-specific factors. Digitally scored Prussian bluestained sections were compared with manual scoring of the corresponding HER2-stained tumor sections using Spearmans rank correlation coefficient from mice injected with BH nanoparticles (Materials and Methods). A positive but weak correlation was found between BH localization and HER2/neu protein expression in nude mice ( = 0.3827; Fig. 3A). We measured a stronger, positive correlation between BH localization with HER2+ sections in tumors from NSG mice ( = 0.8462; Fig. 3B). These results were consistent with both ICP-MS and digital scoring of Prussian bluestained slides among all tumor models (Fig. 2, C to F) further supporting our observations that immune status of the host animal was an important factor determining BH retention in tumors but not for BP (fig. S4A).

To test whether BH nanoparticle retention in tumors correlated with the tumor microvascular network, we compared Prussian bluestained areas with corresponding sections stained with CD31 for visualizing the vascular endothelium (32). No correlation was found between BH score and CD31+ score in sections obtained from nude mice ( = 0.018; Fig. 3C), but a weak positive correlation was measured in sections obtained from NSG mice ( = 0.3241; Fig. 3D). By contrast, slight positive correlations were found with CD31+ regions in both nude and NSG mice injected with BP (fig. S4A).

Both athymic nude and NSG mice lack mature T cells, but NSG mice, in addition, also lack functional components of their innate immune system (table S6) (33). We speculated that subpopulations of innate immune cells in the TME contributed to BH retention. We compared Prussian bluestained sections with corresponding sections stained for ionized calcium-binding adapter molecule 1 (IBA-1), a pan-(murine) macrophage marker that also labels other myeloid cells including subpopulations of dendritic cells, monocytes, activated neutrophils, and some types of endothelial cells (Fig. 3E) (34). Comparing IBA-1+ tissue sections with Prussian bluepositive regions revealed that antibody-labeled nanoparticles were found in similar locations as IBA-1+ regions within the TME in both nude and NSG mice (HCC1954 tumor grown in NSG mice, Fig. 3E; BT474 tumor grown in NSG mice, fig. S4B). However, we found no significant differences in the content (number) of IBA-1+ cells among any of the tumor models or treatment (fig. S4C).

Next, we tested the notion that antibody-antigen binding to cancer cells does not determine tumor localization of BH to tumors by using BNF nanoparticles labeled with a nonspecific human polyclonal IgG. BNF-IgG nanoparticles were intravenously injected into cohorts of both nude and NSG mice bearing HER2+ (HCC1954) and HER2 (MDA-MB-231) tumors. ICP-MS analysis of tissue iron content of tumors extracted from mice injected with BNF-IgG was similar to that measured from mice injected with BH in both tumor models and immune backgrounds of mice (Fig. 3, F and G, and fig. S5, A and B). These results support that retention of antibody-labeled nanoparticles (i.e., BH or BNF-IgG) was independent of antibody-antigen binding.

From the results obtained across the five human tumor xenograft models in two immunodeficient mouse strains and with two antibody nanoparticle types (trastuzumab and nonspecific IgG), we hypothesized that BNF nanoparticle retention by tumors was determined by active biological processes influenced (or directed) by cells of the innate immune system, residing within the TME and reacting to the presence of an antibody on the nanoparticle surface. Our analysis of xenograft tumors of the IBA-1stained tissue sections provided no evidence of measurable (aggregate innate) immune cell infiltration into or depletion from the tumors following nanoparticle exposure. To the contrary, the area of IBA-1+ regions among PBS- and nanoparticle-injected cohorts was comparable (fig. S4C), indicating that tumor-associated immune cell subpopulations internalized antibody-labeled nanoparticles (trastuzumab or IgG; see Fig 3, F and G). To test whether macrophages were responsible for these observations, we depleted macrophages by treatment with clodronate liposomes in athymic nude mice growing HCC1954 tumors and injected with BH (35). Unexpectedly, macrophage depletion alone failed to decrease the amount of BH nanoparticles retained in tumors (fig. S5C), suggesting involvement by other immune cells.

BNF nanoparticle localization in tumors across multiple xenograft mouse models suggested that immune status contributed to, and perhaps dominated, nanoparticle retention. To test this concept further, we used a syngeneic tumor model derived from the transgenic huHER2 mouse (Fig. 4A) and transplanted to NSG, nude, and immune competent FVB/N mice. HER2 protein expression in the tumors was confirmed by IHC (Fig. 4B).

(A) Schema of transgenic huHER2 tumor allograft development and IHC confirmation of HER2 protein expression on cancer cells in tumors. (B) IHC analysis demonstrates that HER2 protein expression in syngeneic huHER2 allografts is comparable among the range of immune strains of mice tested: FVB/N, athymic nude, and NSG mice. (C) Gross appearance of huHER2 allograft tumors grown to 150 to 200 mm3 in FVB/N, athymic nude, or NSG mice 24 hours after they were injected via tail vein with BP or BH nanoparticles shows that BH accumulation is greatest in tumors growing in immune competent host(s). Photo credit: Preethi Korangath, Johns Hopkins University. (D) ICP-MS results showing absolute iron recovery from tumors grown in all mice reveals highest accumulation of BH nanoparticles in FVB/N mice (*P < 0.05, **P < 0.005, and ***P 0.0001). (E) Histology analysis revealed that Prussian bluepositive area was seen in stromal area and colocalized more with IBA-1+ cells than HER2+ tumor cells.

The intensity of coloration, 24 hours after injection by BH nanoparticles into FVB/N mice, was visibly greater than that displayed by tumors in either NSG or nude mice (Fig. 4C). Iron content analysis by ICP-MS and analysis of Prussian bluestained slides demonstrated a notable uptake of BH by huHER2 allograft tumors grown in FVB/N mice (Fig. 4D and fig. S6, A and B). Similar to results obtained from xenograft models (Fig 2), FVB/N mice showed retention of less BH in the liver than BP, and higher iron content was detected in the lymph nodes and spleens of both BP- and BH-injected mice (fig. S6, C to E). Prussian bluepositive areas appeared more prominently in stromal regions associated with IBA-1+ areas than in the HER2+ regions (Fig. 4E and fig. S7). These results provided strong evidence that immune status of mouse models is a critical biological variable for nanoparticle targeting studies; however, the nature of this interaction was unclear.

Across all models studied, the presence of immune cells within tumors was detected. Colocalization of nanoparticles and IBA-1+ cells occurred at the tumor periphery (Fig. 3E and fig. S3) in xenograft tumors and in tumor-stromal interfaces in the immune competent huHER2 allograft model (Fig. 4E and fig. S7). It has been well documented that the cancer tissue boundary of tumors often exhibits proinflammatory features (36). We hypothesized that tumor-associated immune cells exhibiting an inflammatory phenotype preferentially sequestered and retained antibody-labeled nanoparticles.

To test this hypothesis and to further elucidate the mechanism of nanoparticle retention in the TME, we performed tests in vitro with murine macrophages and neutrophils. Macrophages were activated with lipopolysaccharide (LPS) and interferon- (IFN-) to mimic a T helper 1 (TH1)type induction (M1) or with interleukin-4 (IL-4) to mimic a TH2-type induction (M2). When exposed to either BP or BH, macrophages always sequestered more BH than BP; however, M1 macrophages sequestered significantly more nanoparticles, especially BH (Fig. 5A). Uninduced neutrophils showed no preference for either construct; however, when activated with LPS (TH1-type induction), neutrophils demonstrated significantly greater preference for BH (Fig. 5B).

(A) Undifferentiated RAW 264.7 (M0) or differentiated M1 or M2 (LPS + IFN- or IL-4, respectively) macrophages were incubated for 24 hours with BP or BH, and ferene-s assay was conducted to measure the total amount of iron uptake per cell. As a control, BP and Her, added together, were also used. As shown in the figure, BH nanoparticles were taken up more significantly than BP by macrophages irrespective of their phenotype. The uptake was significantly higher in M1 macrophages than either M0 or M2, which indicates that proinflammatory macrophages take up more BP and BH nanoparticles with preference toward BH. (B) Likewise, LPS-activated neutrophils (induced) preferentially sequestered BH over BP, whereas no difference in uptake was observed with nave bone marrow neutrophils (uninduced). (C) Total cell count obtained from magnetically separated BP- or BH-injected tumors shows significant difference. Immune competent FVB/N mice (n = 3 per group, two tumors each) bearing huHER2 tumors were intravenously injected with BP or BH. After 24 hours, tumors were harvested and digested to isolate single cells and were magnetically separated to collect nanoparticle-associated cells to determine the total cell count. (D) Analysis of magnetically sorted cells obtained from in vivo tumors showed that nanoparticles were associated with immune cells, not tumor cells. Immune competent FVB/N mice (n = 5 to 8 per group) bearing huHER2 tumors were intravenously injected with PBS, BP, or BH. After 24 hours, tumors were harvested and digested to isolate single cells and were magnetically separated to collect nanoparticle-associated cells for analysis by flow cytometry. Gating strategy is provided in fig. S8. Cell numbers measured from BP- and BH-injected mice are shown as change in ratio relative to PBS-injected mice (PBS ratio = 1). (a) Populations of cancer cells were not changed in nanoparticle-associated cancer cells. Ratios of NK cells (b), monocytes (c), TAMs (d), neutrophils (e), and dendritic cells (f) are increased in nanoparticle fractions, suggesting uptake of nanoparticles by immune cells rather than tumor cells. (*P 0.05, **P 0.01, and ***P < 0.001).

Magnetic nanoparticles provide a unique tool to query biological responses because they enable magnetic sorting to isolate specific cell populations containing the nanoparticles. To further elucidate the in vivo tumor immune response to BNF nanoparticle exposure, tumor digests were placed on a permanent magnet. Cells containing nanoparticles were sedimented, whereas cells devoid of nanoparticles remained suspended. Sedimented (nanoparticle-associated) cells were collected and analyzed for total number (Fig. 5C). Consistent with in vitro results, the total number of cells containing iron was higher in tumors of mice injected with BH than in those injected with BP. To distinguish among tumor-associated cell populations that sequestered nanoparticles, both sedimented (nanoparticle associated) and suspended (supernatant, no nanoparticle) cells were collected and analyzed by polychromatic flow cytometry. Figures S8 and S9 provide graphical gating strategy and complete results of analysis, respectively. Results of magnetic sorting of equal (initial) numbers of tumor-derived cell populations are displayed in Fig. 5D (a to f) as ratios of cell number by type and fraction relative to cell numbers obtained from PBS-injected mice. PBS ratios are expressed as unity and all others as <1 or >1 depending on the number of cells detected in each fraction. Among cancer cells, it is notable that for either BP or BH, numbers were lower than from PBS-injected controls, indicating little nanoparticle association with the HER2+ cancer cells (Fig. 5D, a). This is consistent with histopathology (Fig. 3E). Following intravenous delivery, evidence indicates that nanoparticle association with cancer cells was minimal regardless of HER2+ expression, further confirming the different performance of antibody-labeled nanoparticles in vivo versus in vitro.

On the basis of the evidence, nanoparticle retention in the studied models was likely determined by tumor-associated leukocytes, but what effect did systemic exposure to nanoparticles have on the tumor immune microenvironment? We used polychromatic flow cytometry to identify changes of individual tumor immune cell populations in huHER2 allograft tumors growing in FVB/N mice following injection with nanoparticle or free antibody (Fig. 6A and fig. S8, A and B). Twenty-four hours after intravenous injection, we measured a slight decrease of live cell populations in tumors derived from mice receiving either BP or BH relative to PBS-injected controls. No measurable differences were detected in cancer cell populations among the four cohorts, but a significant decrease in CD45+ population was noted (fig. S9B, a to c).

Immune competent FVB/N mice (n = 5 to 8 per group) bearing huHER2 tumors were intravenously injected with PBS, BP, BH, or Herceptin (Her). After 24 hours, tumors were harvested and digested to isolate single cells and evaluated by polychromatic fluorescence-activated cell sorter (FACS). Gating strategy is provided in fig. S8. (A) Relative decreases in T cell (a) and B cell (b) populations were observed following injection of nanoparticles. By contrast, relative increases were measured in many innate immune cell populations within the TME: NK cells (c), neutrophils (d), TAMs (e), and monocytes (f) 24 hours after nanoparticle exposure. Except for TAMs, no significant increase was seen in any other immune cell population after Her injection. (*P 0.05 and **P 0.01). (B) Graphic representation of distributions of nanoparticle-associated CD45+ immune cells among the cohorts.

Nanoparticle exposure induced many changes across a number of tumor immune cell lineages, with a notable decrease in T cells and an increase in the relative fraction (i.e., ratio) of innate immune cells initiating a restructuring of the immune compartment of the TME (Fig. 6A, a). B cell populations also decreased in BH- and Her-treated groups (Fig. 6A, b). Relative to PBS controls, natural killer (NK) cell and monocyte fractions increased following BH injection but not in mice receiving BP or Her (Fig. 6A, c and f). Populations of other phagocytic innate immune cells, specifically neutrophils, and tumor-associated macrophages (TAMs) increased with either BP or with BH injection relative to controls (Fig. 6A, d and e, and fig. S9B), but dendritic cell populations remained relatively unchanged 24 hours after injection (fig. S9B, d) as did the fraction of T cells (GD T cells) (fig. S9B, e). However, we found no evidence in histology data indicating that depletion or infiltration of innate immune cells carrying nanoparticles to or from tumors occurred after nanoparticle injection, suggesting capture of nanoparticles by the resident population(s) of innate immune cells in the TME (Fig. 3 and fig. S4C) (37). Nevertheless, for conclusive quantification of this process, further study is needed. Exposure to free trastuzumab (Her) elevated TAMs, reflecting a specific interaction (Fig. 6A, e).

Trastuzumab is a humanized monoclonal antibody with a human IgG1 (hIgG1) that can elicit a response in murine macrophages (38). Furthermore, it is recognized that Fc receptors on murine macrophages can recognize hIgG1 (38), and the response observed in our flow cytometry with free trastuzumab (Her) is consistent with this observation (Fig. 6A, e). Note that, however, macrophages were the only tumor immune population that elevated within 24 hours following injection with free trastuzumab, whereas multiple immune cell subpopulations responded to BP and BH exposure (Fig. 6, A and B, and fig. S9B). The tumor immune response to BH was more complex than that to free trastuzumab (Her)including T cells, NK cells, monocytes, neutrophils, dendritic cells, and macrophagesand it was similar to that of BP. Thus, while the potential exists for specific interactions between murine macrophages and hIgG1-containing nanoparticles, our evidence demonstrates that labeling the surface of a nanoparticle with a hIgG1 monoclonal antibody alters the immune response to recognize the nanoparticle-antibody construct as an entity distinguishable from free antibody.

The data indicate that, in addition to macrophages (TAMs), many other lineages of phagocytic innate immune cellsNK cells, monocytes, neutrophils, and dendritic cellsreside in the TME sequestered nanoparticles (Fig. 5D, b to f, and fig. S9A, b to i). It seemed that an intact immune system is a critical component in determining the retention of nanoparticles in solid tumors. To challenge this notion, we pretreated tumor-bearing mice with a pan-leukocyte inhibitor, azathioprine (39, 40), before injecting with BH. Iron recovered from tumors in azathioprine-treated mice was significantly reduced and similar to BP-injected mice (fig. S10, A and B), confirming the role of a wider immune involvement in nanoparticle retention.

These results support a model that tumor-associated phagocytic immune cells significantly influence the degree of retention of systemically delivered nanoparticles within the TME. Furthermore, our results demonstrate that an intact host immune system can manifest decidedly different tumor retention when compared with comparable immunodeficient models, raising an important question about clinical relevance of studies performed in the latter. Depending on environmental chemical cues, tumor-associated leukocytes may display a greater sensitivity to the chemical signatures of nanoparticles than their counterparts residing in other tissues. This offers potential for tumor targeting with nanomedicines.

In a complex manner, while the restructuring of the immune compartment of the TME, likely mirroring a systemic immune response to nanoparticle exposure, was similar for both BH and BP nanoparticles, it is only the BH nanoparticles that were significantly retained within the TME. These complex and seemingly contradictory immune responses may indicate potential for anticancer effects.

To explore the potential clinical relevance of our findings, we used the huHER2 allograft tumor model to ascertain effects of nanoparticle exposure on tumor growth in FVB/N and athymic nude mice. Five days after implantation of huHER2 tumors, FVB/N or athymic nude mice received a single intravenous injection of PBS, BP, BH, or Her as previously described. Exposure to either BP or BH significantly delayed tumor growth in FVB/N mice but not in athymic nude mice (Fig. 7, A to C, and fig. S11, A to C). As expected, trastuzumab alone was effective to significantly inhibit tumor growth in both FVB/N and athymic nude mice, however, its mode of action involves direct binding via HER2 antigen to cancer cell membranes. Our evidence shows that neither BP nor BH nanoparticles associated appreciably with cancer cells in vivo; thus, the therapeutic effect seen only in FVB/N mice due to nanoparticle exposure must involve an alternate mechanism that we hypothesized was mediated by the adaptive immune system. To gain further insight, we repeated the experiment in FVB/N mice and conducted flow cytometry analysis of immune populations in tumors 3, 7, and 14 days after injection. Beginning at 7 days after injection, significant increases in activated T cells (CD3+/CD4+/CD8+) were measured in tumors, reversing the depletion observed at 24 hours and 3 days and supporting a model of immune-mediated tumor suppression induced by systemic exposure to nanoparticles (Fig. 7, D and E, and figs. S11D to S14). Immune cells known to be involved in adaptive immune signaling, i.e., dendritic and T cells, displayed a complex time-dependent patternincreasing to day 3 and decreasing thereafterconsistent with adaptive immune signaling response (Fig. 6 and figs. S9 and S13) (41, 42). On the other hand, phagocytic effector immune cells, i.e., macrophages and monocytes, initially displayed relatively elevated numbers at day 1 but displayed no such increases afterward relative to PBS controls (Fig. 6 and fig. S9 and S14). These complex and time-dependent immune cell responses observed in the TME resemble systemic responses observed in mice following acute and nonlethal infection by some pathogens, i.e., Listeria monocytogenes, which can also lead to anticancer immune stimulation (41, 42). Note that both BH and BP nanoparticles induced similar effects on tumor immune cell populations and on tumor growth, despite the fact that BP nanoparticles were not significantly retained within the tumor. This suggests that exposure to nanoparticles has the potential to induce both systemic and local (tumor) effects, which bear further study and offer potential for developing another paradigm in cancer nanomedicine (fig. S15).

(A) Female FVB/N mice bearing huHER2 allograft tumors (n = 7 to 18 per group) were intravenously injected with either PBS, BP, BH (5 mg per mouse), or Herceptin (175 g per mouse) 5 days after tumor implantation (day 0). Growth of the tumors was monitored by caliper measurements twice per week for 4 weeks (means SEM). Final tumor weight is given in inset (**P < 0.005 and &P 0.0001). (B) On day 28, all mice were euthanized, and representative images of tumors are shown. Photo credit: Preethi Korangath, Johns Hopkins University. [C (a and b)] Female athymic nude mice bearing huHER2 allograft tumors (n = 6 to 7 per group) were similarly treated as above, and 3 weeks of tumor growth and tumor weight is reported (means SEM, *P < 0.05). [D (a and b) and E (a and b)] Flow analysis of tumors: As in (A), mice (n = 5 per group) were intravenously injected with either PBS, BP, BH (5 mg per mouse), or Herceptin (175 g per mouse) on the 10th day after tumor implantation. Seven days after injection, mice were euthanized; tumors were harvested, and single cells were isolated and evaluated by FACS. Infiltration of CD3+ T cells with increases in CD8+ T cells was measured following nanoparticle exposure, likely leading to growth inhibition observed in (A) (*P < 0.05). FITC, fluorescein isothiocyanate.

In summary, targeting nanoparticles has been a topic of considerable debate, even controversy, in the cancer nanomedicine community (17, 16, 2531). In most previous studies, the biology of tumor and/or host was not studied in detail with analysis of tissue histology and flow cytometry, thus motivating our efforts to understand the role of host biology in nanoparticle-tumor interactions (610). Across all models studied, we found strong evidence pointing to immune status of the host as a key factor determining the retention of antibody-labeled nanoparticles in tumors. Using an immune intact model, we discovered that the retention of nanoparticles in tumors was dominated by multiple lineages of tumor-associated immune cells when the nanoparticles included an antibody and found no in vivo evidence supporting a mechanism of antibody-antigen binding (i.e., the mechanism operating in vitro) to cancer cells in the tumor. Yet, the amount of nanoparticle retained by the tumor within 24 hours was most pronounced in an immune intact model, further emphasizing the significance of an intact immune system in studies of nanoparticle delivery to solid tumors. Our results demonstrate that the host immune system can be a substantial factor in studies of cancer nanomedicine and that macrophages are only one among many immune cell lineages that determine nanoparticle fate. It was only when we pharmacologically inhibited the entire host immune system that we measured a reduced retention of the BH nanoparticles. While these findings reveal new insights, they also raise many questions regarding complexities of nanoparticleimmune cell interactions in vivo across the many biological models used in cancer research and how immune cell receptors distinguish among nanoparticle coatings.

Related to this, but in a different manner, we observed that the immune response to nanoparticle exposure measured in tumors was equally profound and seemed insensitive to nanoparticle composition (BP or BH). As measured by population changes of immune cells in the TME, the immune response included an initial T cell depletion and later T cell infiltration into the tumor with significant tumor growth inhibition.

The presence of immune cells within an established solid tumor implies that immune cells are performing surveillance and homeostasis functions to support the growth and maintenance of the tumor. Our results show that exposure to nanoparticles can disrupt this delicate balance, potentially enabling a transient immune recognition of the tumor. In an immune-intact model of cancer, the systemic delivery of a nanoparticle construct can initiate a complex immune response, which can affect tumor growth regardless of retention. These results highlight the notion that the biology of the host and cancer tumor forms an interconnected and inextricably linked biological network that interacts in complex ways to determine the biological fate and retention of nanoparticles. Host immune status and, consequently, composition of the immune compartment(s) within the TME are critical variables in developing and testing the performance of cancer nanomedicines. Results presented here motivate more questions of mechanism of host and tumor immune cell interactions with nanoparticles. They also point to new possibilities for nanoparticle anticancer immunotherapy technologies.

MDA-MB-231 [ER/PR/HER2 () negative], MCF7 [ER/PR (+) positive/HER2 () negative], and BT474 [ER/PR/HER2 (+) positive] were purchased from the American Type Culture Collection (ATCC; Manassas, VA) and maintained according to the suppliers recommendations. They were grown in Dulbeccos modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS). HCC1954 [ER/PR () negative/HER2 (+) positive] was grown in RPMI containing 10% FBS. MCF/neo and MCF7/HER were provided by K. Osborne (University of Texas Health Science Center). All cell lines were authenticated using short tandem repeat analysis (data provided upon request) and matched against ATCC and Deutsche Sammlung von Mikroorganismen und Zellkulturen databases to ensure the genetic origins.

The nanoparticles used for this study are commercially available aqueous suspensions of hydroxyethyl starchcoated magnetite (Fe3O4) core-shell particles (BNF; Micromod Partikeltechnologie GmbH, Rostock, Germany). The synthesis and physical characterization of the BNF particles have been extensively documented (1519). Briefly, BNF particles were produced by precipitating ferric and ferrous sulfate salts from solution at high pH in a high-pressure homogenization reaction vessel, which controls both crystal formation and aggregation. According to the manufacturer, they have a mean hydrodynamic diameter of ~100 nm and an iron content of >50% (w/w) [or iron oxide of >70% (w/w)].

The mean hydrodynamic diameter of the magnetic iron oxide nanoparticles (BNF) and their zeta potential were measured in 1 mM PBS buffer (pH 7.4) with a Zetasizer Nano ZS90 (Malvern Instruments Limited, UK) at an iron concentration of 0.1 mg/ml. The mean particle diameter Z(Ave) is given as a result of the cumulative analysis of the autocorrelation function. The polydispersity index is a measure of the quality of the size distribution. Monodisperse suspensions have a polydispersity index of <0.25.

The monoclonal anti-HER2/neu antibody (Her), or trastuzumab (trade name) (Genentech, South San Francisco, CA), was purchased from Johns Hopkins Pharmacy and was shipped to micromod for conjugation with BNF particles to form BH. The Her was formulated according to the prescribing information. The lyophilized powder that contained 440 mg of Her was dissolved in 20 ml of bacteriostatic water for injection (provided). The Her solution was purified by washing with PBS buffer (pH 4) using a desalting column (PD-10, GE Healthcare, UK) to remove the stabilizing agents. The obtained Her solution was thiolated with Trauts reagent (2-iminothiolane) as follows: The antibody solution (390 l, 1.7 mg/ml in PBS buffer) was mixed with 160 l of 1.4 mM 2-iminothiolane in 450 l of PBS-EDTA buffer. After shaking for 1 hour at room temperature, the excess of 2-iminothiolane was removed by washing with PBS-EDTA buffer (PBS buffer, 1 mM EDTA) in a desalting column (G-25, GE Healthcare, UK). In parallel, an aqueous suspension of 80-nm BNF-starch nanoparticles with amino groups on the surface (2.25 ml, [Fe] = 8.0 mg/ml; product code: 10-01-801, micromod Partikeltechnologie GmbH) was mixed with 250 l of 10 PBS-EDTA buffer. Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) (3.6 mg) was dissolved in 100 l of dimethyl sulfoxide and added to the BNF-starch suspension. After 1 hour of shaking at room temperature, the excess of sulfo-SMCC was removed by washing with PBS-EDTA buffer in a PD-10 desalting column. The maleimide-functionalized nanoparticles were mixed with the thiolated antibody solution and shaken for 3 hours at room temperature. Then, 200 l of 20 mM cysteine solution was added to quench the remaining maleimide groups on the nanoparticle surface. Last, the nanoparticles were washed by magnetic separation in a high-gradient magnetic field column (QuadroMACS with LD columns, Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany) with 5 ml of PBS-Tween buffer (pH 7.4, 0.05% Tween 20) and 5 ml of PBS buffer (pH 7.4) per column filling. The magnetic column was removed from the magnet, and the nanoparticles were eluted with 2 ml of water per column filling. The high gradient magnetic field (HGMF) wash was repeated until the suspension was completely washed. The suspension was filtered using 0.22-m polyethersulfone filter (Carl Roth GmbH, Karlsruhe, Germany).

After conjugation, BH nanoparticles were rigorously characterized for their physical and biological properties in vitro to ensure nanoparticle stability, and BNF-Her binding was successful and retained sufficient protein. Antibody immunoreactivity of the BH construct was separately tested using a cell culturebased assay (see below).

The iron content of the antibody-conjugated nanoparticles (BH) was determined after the digestion of a 20 l of sample with 80 l of concentrated HCl. After addition of 4.9 ml of a citrate phosphate buffer (pH 3.6), the iron concentration was calorimetrically determined with the Spectroquant Kit (Merck, Germany) against a Titrisol Iron Standard (Merck, Germany).

The amount of the conjugated antibody in the sample was measured by a modified BCA method. The BCA reagents were obtained from Thermo Fisher Scientific (Germany). The calibration curve was obtained by adding increasing amounts of an albumin standard solution to aminated BNF-starch particles (without antibody on the surface) at a constant iron concentration of 0.25 mg/ml. The antibody-conjugated nanoparticles were adjusted to the same iron concentration of 0.25 mg/ml and developed with the BCA reagent together with the calibration curve for 2 hours at 37C.

Polyclonal normal hIgG was purchased from R&D Systems (Minneapolis, MN) for conjugation with BNF nanoparticles for BNF-IgG nanoparticles. Methods to conjugate the IgG antibody to BNF nanoparticles were same as for trastuzumab, except that proportions of reagents were altered to accommodate differences between the antibodies. The lyophilized hIgG (2 mg) was dissolved in 1 ml of PBS buffer (pH 7.4) and purified by washing with PBS buffer (pH 4) using a desalting column (G-25, GE Healthcare, UK). The antibody solution used was 510 l (1.3 mg/ml) in PBS buffer and was mixed with 160 l of 1.4 mM 2-iminothiolane in 330 l of PBS-EDTA buffer. After shaking for 1 hour at room temperature, the excess 2-iminothiolane was removed by washing with PBS-EDTA buffer in a desalting column. In parallel, BP nanoparticles with amino groups on the surface were prepared as described above. The maleimide-functionalized nanoparticles were mixed with the thiolated antibody solution, reacted, washed, and purified as above.

The detailed protocol for conducting the modified ferene-s measurement of iron associated with cells after exposure to BNF nanoparticles has been previously described (24). Briefly, cells were trypsinized and washed with PBS thoroughly and were incubated at 37C with BP (0.5 mg/ml), BH, or trastuzumab (Her 2 g/ml) alone for 3 hours in growth media (DMEM + 10% FBS) with occasional shaking/tapping of tubes to maximize distribution and prevent settling of cells. After incubation, cells were pelleted by centrifugation and washed with PBS to remove unbounded particles and again pelleted by centrifugation. This washing with PBS was repeated three more times. The final cell pellet was resuspended in PBS and counted using a Cellometer (Nexcelom Bioscience, Lawrence, MA) to obtain the total number of cells. The cells in the tubes were then centrifuged, and the supernatant was removed to add working solution (acetate buffer with ascorbic acid). Cell pellets were digested in working solution by incubating at room temperature for at least 20 hours before reading in a colorimeter. A known quantity of ferene-s was used along with other external standard reference materials to quantify the iron concentration of the test samples according to previously described procedures (24). For the entire study, we used only those batches of BH showing more than fourfold retention by SKBR3 cells, as measured by iron concentration with the ferene-s assay when compared to BP (table S5). In addition, we used MDA-MB-231 (HER2-, control) to confirm that nonspecific binding of BH particles by those cells was minimal (<1 pg of Fe per cell).

Cells were trypsinized and washed in PBS and incubated in DMEM + 10% FBS at 37C with trastuzumab (2 g/ml) for 3 hours with occasional shaking/tapping of tubes to maximize distribution and prevent settling of cells. After incubation, cells were washed four times with PBS and plated on poly-lysinecoated coverslips in six-well plates. After overnight incubation, they were washed with PBS, fixed with methanol for 10 min, and blocked with 1% bovine serum albumin for 30 min at 37C. Dye-labeled secondary antibody (anti-human Alexa Fluor 488, Life Technologies, Eugene, OR) was added and incubated for 1 hour in the dark at room temperature, followed by washing three times in PBS and mounting with mounting media containing DAPI (4,6-diamidino-2-phenylindole). They were then visualized and photographed using a fluorescent microscope (Zeiss Axioimager Z1, Carl Zeiss Microscopy GmbH, Jena, Germany). To visualize BNF-HER nanoparticles alone, 30 l of BNF-HER or BP nanoparticles was separated on a magnet for 2 hours at 4C. The particles suspended in 1 ml of PBS volume and the concentration of BH nanoparticle suspensions were incubated with anti-human Alexa Fluor 488 secondary antibody (1:1000) for 1 hour at room temperature. The particles were then separated on a magnet for 1 hour, washed with PBS, and dropped on a clean slide to mount and visualize with a fluorescent microscope.

Cells were lysed with radioimmunoprecipitation assay buffer (Sigma-Aldrich, St. Louis, MO) containing protease and phosphatase inhibitors on ice for 30 min. The lysates were centrifuged at 13,000 rpm for 15 min. The supernatant was collected and quantified by BCA (Thermo Fisher Scientific, Waltham, MA) assay. Thirty to 50 g of total protein were used for SDSpolyacrylamide gel electrophoresis gel after being heated with sample buffer. The proteins were then transferred to nitrocellulose membranes. After blocking with 5% milk solution in PBS-T (1% Tween 20) for 30 min, the membranes were blotted with primary antibody (anti-human HER2 antibody, 1:1000; Cell Signaling Technology, 29D8) overnight and with secondary horseradish peroxidase (HRP)conjugated antibody (GE Healthcare, UK) for 1 hour. The membranes were developed using chemiluminescence reagent (Amersham Biosciences, Marlborough, MA).

RAW264.7 cells were purchased from the ATCC (Manassas, VA) and maintained in DMEM with 10% heat-inactivated FBS. Low-passage cells were used for the study (P3 to P5). For M1 macrophage activation, cells were treated with LPS (100 ng/ml; Sigma-Aldrich, St. Louis, MO) and IFN- (50 ng/ml; Miltenyi Biotech, Germany) for 24 hours. To differentiate cells into M2, phenotype cells were treated with IL-4 (10 ng/ml; Miltenyi Biotech, Germany) for 24 hours (43). Induced and uninduced cells (1 million) were collected and treated with either BP or BH nanoparticles (0.5 mg/ml) or cotreated with BP and Her (16.3 g/ml; equivalent to protein content of BH) for 24 hours. After incubation, cells were washed thoroughly with PBS four times and processed for iron content analysis with the ferene-s assay as described above. Experiments were repeated three times.

Neutrophils were activated in vivo with LPS by the method described by Rnnefarth et al. (44). Briefly, 50 g of LPS was intraperitoneally injected into FVB/N mice (n = 3). After 18 hours, activated peritoneal neutrophils were collected by injecting 5 ml of PBS to peritoneum, cells were harvested, and red blood cells (RBCs) were lysed with ammonium-chloride-potassium (ACK) lysis buffer and thoroughly washed. Nave neutrophils were prepared using methods described by Mcsai et al. (45). For this, bone marrow cells were collected to Hanks balanced salt solution (HBSS) from femur and tibia of FVB/N mice (n = 3). RBCs were lysed from bone marrow cells with ACK lysis buffer, and cells were passed through a 70-m strainer. These cells were then centrifuged after layering on 62.5% freshly prepared Percoll in HBSS for 30 min at 1000g without brake. The cloudy pellet of neutrophils was collected. Uninduced bone marrowderived neutrophils and activated peritoneal-derived neutrophils were incubated with BP or BH nanoparticles (0.5 mg/ml) for 24 hours, and ferene-s assay was conducted to measure the amount of iron uptake per cell as described above.

All animal studies were approved by the Institutional Animal Care and Use Committee at Johns Hopkins University and were conducted using female mice. All mice were fed normal diet and water ad libitum. They were maintained in the normal 12-hour light/12-hour dark cycle. All animals were closely monitored for any distress or pain throughout the study period. The weight range of animals during the study was 20 to 30 g. Strains of mice used in all studies were athymic nude (Charles River Laboratories, Frederick, MD), NSG (Sydney Kimmel Comprehensive Cancer Center colony, Johns Hopkins University School of Medicine, Baltimore, MD), and FVB/N (Jackson laboratory, Bar Harbor, ME); all mice were aged 6 to 8 weeks. The characteristics of cell lines and mice used are provided above and in tables S3 and S6. A schematic of the xenograft tumor study design is provided in Fig. 1E. An overview of the numbers of mice divided by strain and group used for the studies is provided in table S10. Depending on cohort, 3 106 MDA-MB-231 or HCC1954 or 5 106 MCF-7(HER/neo) or BT474 cells were suspended in 50 l of PBS and Matrigel (1:1) and injected into the fourth mammary gland on either side of female mice under anesthesia. For MCF-7(HER/neo) and for BT474 xenograft studies, mice received estrogen by implanting a 60-day release estrogen pellet (0.72 mg per pellet; Innovative Research of America, Sarasota, FL) 5 days before cell line injection on the dorsal neck region through a small subcutaneous insertion made using sterile scissors while mice were under ketamine/xylazine anesthesia[ketamine (10 mg/ml) Vedco Inc., St. Joseph, MO] and xylazine (2 mg/ml; Lloyd Inc., Shenandoah, IA) mixed in sterile PBS and intraperitoneally injected at 0.01 ml/g body weight. Tumor volume was monitored by caliper measurements twice weekly when tumors became palpable. When the measured tumor volume was 125 to 200 mm3, mice were randomly assigned into cohorts containing five animals in each group. Group I received intravenous (tail vein) injections of PBS and served as (negative) control. Group II received intravenous injections to tail vein of BP (5 mg of Fe per animal), and group III received intravenous tail vein injections of BH (5 mg of Fe per animal). Group IV received injections of BNF-IgG (intravenous tail vein injections; 5 mg of Fe per animal) only for mice bearing either MDA-MB-231 or HCC1954 xenografts. The total volume of injection was 150 l in all cases. Twenty-four hours after injection, all mice were euthanized to collect tumors and liver for analysis.

Athymic nude mice growing HCC1954 tumors (n = 3 with two tumors each) were treated with two consecutive doses of clodronate liposome (CL) (Liposoma, Netherlands) via intraperitoneal (300 l per animal) injection. After the second dose of CL, BH nanoparticles were injected (5 mg of Fe per mouse intravenously) and euthanized 24 hours later to harvest tumors for ICP-MS.

The second half of each tumor and whole livers were weighed, lyophilized, and stored at 20C until analysis by ICP-MS using methods previously described (46). Briefly, each tissue sample was transferred to a 7-ml Teflon microwave digestion vessel (Savillex Corporation, Eden Prairie, MN) to which 1 ml of optima-grade HNO3 (Fisher Scientific, Columbia, MD) was added. The vessel was sealed and placed into a 55-ml Teflon microwave digestion vessel (CEM Corporation, Matthews, NC) to which 10 ml of ultrapure H2O (Millipore Corporation, Billerica, MA), and samples were digested in a MARS5 Xpress microwave (CEM Corporation, Matthews, NC) using a single-stage ramp-to-temperature of 15-min ramp to 130C with a hold of 30 min. After cooling, each sample was diluted: 35 l of sample digest and 300 l of HNO3 were added to 14.665 ml of ultrapure H2O to achieve a final HNO3 concentration of 2% (w/v). External reference standards scandium (CPI Incorporated, Santa Rosa, CA) and Seronorm Trace Elements Whole Blood (SERO AS, Billingstad, Norway) were added to normalize instrument counts and sample iron content, respectively. In addition, four reagent blanks were digested and analyzed in each run to correct for background iron content.

An Agilent 7500ce ICP-MS (Agilent Technologies, Santa Clara, CA) was used to measure iron content of each sample. Measurements were blank-corrected using the average iron value of the reagent blanks and corrected using external standard reference materials. An eight-point calibration curve (0, 1, 5, 10, 50, 100, 500, and 1000 g/liter) was obtained from Standard Reference Material (SRM) measurements. The analytical limit of detection (LOD) was calculated by multiplying the SD of the lowest detectable calibration standard (1 g/liter) by three. For samples with values below the analytical LOD, one-half of the LOD was substituted (46).

Fresh tumors were fixed in 10% formalin and sectioned on positively charged slides. For HER2 staining, a VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA) was used to perform IHC. After hydration with serial dilutions of ethanol, antigen retrieval was performed using 10 mM citrate buffer. The sections were then treated with 3% hydrogen peroxide for 10 min and incubated with normal serum to block nonspecific binding. The sections were later incubated overnight with anti-human HER2 antibody (1:400; Cell Signaling Technology, 29D8). Secondary antibody (provided in the kit) was added the next day after washing, followed by incubation with ABC reagent and developed with 3,3-Diaminobenzidine (DAB) (DAB peroxidase substrate kit, Vector Laboratories, Burlingame, CA) reagent and counterstained with hematoxylin (Dako North America Inc., Carpinteria, CA.) as specified by the manufacturer. For CD31 (Dianova, DIA 310), and IBA-1 (Wako, 019-19741), after deparaffinization and hydration, the slides were steamed in HTTR or EDTA buffer for 45 min in a steamer followed by washing in PBS containing Tween. They were then blocked in peroxidase solution and incubated with CD31 (1:40) or IBA-1 (1:2500) antibody for 45 min at room temperature. After washing, sections were incubated with secondary antibody (PowerVision Poly-HRP anti-Rabbit IHC Detection Systems Novocastra, Leica Biosystems, Buffalo Grove, IL) for 30 min at room temperature followed by washing. The slides were then washed and developed with DAB fast (Sigma-Aldrich, St. Louis, MO) for 20 min at room temperature and counter stained with hematoxylin.

One-half of each tumor was fixed with 10% formalin and submitted for paraffin embedding and sectioning for hematoxylin and eosin (H&E) staining, Prussian blue (also known as Perls reagent) staining to visualize nanoparticle (iron oxide) distributions, and IHC (HER2, CD31, and IBA-1). All stained slides were evaluated by a pathologist (B.W.S.) and quantitated in a blinded study. For manual analysis, HER2 immunostains were semiquantiatively scored to determine the percentage of tumor cells with positive, membranous staining. For automated image analysis, whole slides were digitized using the Aperio ScanScope At or CS system (Aperio, Vista CA) at 40 magnification. Analysis was performed using Aperio ImageScope software (version with the included Positive Pixel Count algorithm. Images were manually annotated to select a region of interest representing a full cross section of each graft and a 50-m border of surrounding subcutaneous tissue. Artifacts and necrotic regions of the tumor were excluded from analysis. Default hue values (brown, positive; blue, negative) were used for immunostains (DAB Chromogen) and were adjusted for Prussian blue (blue, positive; pink, negative). Digital analysis settings that were used are provided in tables S7 and S8. One slide per condition per tumor was analyzed, and results represent as percent positive pixels over negative pixels in region of interest.

Transgenic (huHER2) mice (FVB/N background) that develop mouse mammary tumor virusdriven mammary-specific human HER2overexpressing tumors were provided under a material transfer agreement (Genentech, South San Francisco, CA). These mice are well characterized for their tumor development and response to trastuzumab as described elsewhere (22, 23). The primary tumor from a donor mouse was collected in normal media and finely minced. Approximately 3 to 4 mm3 of the mash were implanted into the fourth mammary gland on either side of FVB/N females (Jackson laboratory, Bar Harbor, ME) at 6 to 8 weeks of age under anesthesia. Tumor growth was monitored twice weekly by caliper measurements. When the measured tumor volume was ~1000 mm3, tumors were collected and minced to repeat the transplantation into other FVB/N recipient mice for expansion by serial transplantation for up to six generations. At each generation, a section of tumor was fixed in formalin and was analyzed for tumor morphology by H&E and (human) HER2/neu expression by IHC. Nanoparticle uptake studies commenced when a sufficient number of tumors was established to ensure completion of the huHER2 study design. To establish tumors for the nanoparticle studies, huHER2 tumors were collected from five to eight FVB/N donor mice and minced. Portions of the mashes (3 to 4 mm3) were implanted into the fourth mammary gland on either side of female recipient mice comprising immune strains FVB/N, athymic nude, or NSG (18 to 24 animals in each group) under anesthesia. When the measured tumor volume reached 150 to 200 mm3, animals were randomly assigned into cohorts comprising five to nine animals in each group and treated according to their cohort as described for the xenograft studies (see above). For tumor growth delay, huHER2 allografts were implanted in either FVB/N or athymic nude mice and intravenously treated with PBS, BP, BH (5 mg per animal, or Her 175 g/ml, equivalent dose of Her on BH particles) 5 days after implantation (day 0). Tumors were measured and recorded twice weekly up to 28 days. On day 28, all animals were euthanized, tumors were collected, and weight was recorded.

FVB/N female mice (five to eight animals per group) growing single huHER2 allograft tumors of 150 to 200 mm3 received intravenous (tail vein) injections of PBS, BP, or BH (5 mg of Fe per mouse) or trastuzumab (Her; 175 g per mouse). Mice were euthanized 24 hours after injection. For the later time point (14 day after injection) flow analysis, mice bearing huHER2 allograft tumors were injected with PBS, BP, BH, or Her (same concentrations as above) 10 days after tumor implantation. Tumors were minced with a sterile blade in a petri dish and transferred to a 50-ml conical tube containing digestion media [DMEM + 10% FBS (heat inactivated) and 0.1% collagenase + 0.005% hyaluronidase]. The tubes were rotated at 37C for 30 min, and the dissociated tissue was filtered through a 100-m filter. After centrifugation at 1400 rpm for 10 min, the pellets were washed with 30 ml of DMEM with 10% serum and centrifuged again for 10 min. Supernatant was discarded, and RBCs were lysed with ACK lysis buffer at room temperature for 3 to 5 min. An additional 30 ml of media was added, and the mixture was centrifuged at 1400 rpm for 10 min. The supernatant was discarded, and 500 l of media was added to resuspend the pellet. An aliquot of this whole tumor was removed and labeled with the panel of flow cytometry antibodies (see table S9 for list). The remaining single cells were incubated on a rare-earth permanent magnet at 37C for 30 min. After incubation, the supernatant was carefully separated to a fresh tube for flow cytometry. Three milliliters of media was added to the adhered (remaining) fraction cells, which contained magnetic (nanoparticle) material. Media was added to all sample tubes to make up a final volume of 3 ml, and they were then centrifuged at 1400 rpm for 10 min. The supernatant was discarded, and pellets were suspended in PBS and counted to measure the total number of cells. Cells (1 106 to 2 106) were collected and treated with Fc blocker (2 l of 100 l of PBS; anti-CD16/32, BioLegend, San Diego, CA) and incubated on ice for 10 min. Samples were then centrifuged at 1400 rpm for 3 min, after which the cells were incubated with LIVE/DEAD solution (1 l of 100 l of PBS; Zombie Aqua, BioLegend, San Diego, CA) at room temperature in the dark for 30 min and then centrifuged. After centrifugation, cells were washed with 100 l of PBS with 5% heat-inactivated FBS and again centrifuged. Cells were then stained with 50 l of the solution of panel of antibodies (table S9) in appropriate dilution and incubated at room temperature in the dark for 30 min, centrifuged at 1400 rpm for 3 min, washed with 100 l of PBS and 5% heat-inactivated FBS, and again centrifuged. Cells were then fixed with 50 l of fix/perm solution and incubated at room temperature in the dark for 30 min, centrifuged, and washed. Two hundred microliters of PBS with 5% heat-inactivated FBS was added, and the suspensions were stored at 4C until they were measured by polychromatic flow cytometry (LSR-II, BD Biosciences, San Jose, CA). The gating and selection of quadrants were based on fluorescence minus one controls. Analysis of data was done using FlowJo (version 10) software. Gating strategy is provided in figs. S8 and S11.

FVB/N female mice (three animals per group with two tumors each) growing huHER2 allograft tumors at ~100 mm3 received intravenous (tail vein) injections of BP or BH (5 mg of Fe per mouse). Mice were euthanized 24 hours after injection. Tumors were processed as above for flow cytometry and placed on permanent magnet for 30 min. After discarding supernatant, total numbers of magnetically attached cells were counted in a cell counter (Nexcelom, MA).

huHER2 allograft tumors were grown in FVB/N female mice (n = 4). When the tumor volumes reached ~150 mm3, mice were treated with subcutatneous injections of azathioprine (Sigma-Aldrich, St. Louis, MO), a pan leukocyte inhibitor at a dose of 200 mg/kg body weight for three consecutive days (39, 40). On the third day and 4 hours after azathioprine injection, mice were intravenously injected with BH (5 mg of Fe per animal). Mice were euthanized 24 hours after BH injection to collect tumor for ICP-MS analysis.

Results of all statistical analyses are provided in tables S11 to S33. ICP-MS and Prussian blue Aperio scored data were modeled as following log-normal distributions on the basis of proper exploratory analyses. Generalized mixed-effect models were used, with treatment, strain, and cell line as the fixed effects and mice as the random effect (intercept), such that the ratios between any two levels of fixed effects may be reported directly from the mixed-effect models. Models with fixed effect only and with two-way and three-way interactions were considered. Multiple comparison adjustments were made using the Bonferroni method to strongly control the overall family-wise type I error at 0.05.

Flow cytometry and tumor growth analysis. For flow cytometry and tumor growth data, it seemed unreasonable to assume commonly used parametric distributions. Therefore, pure nonparametric comparisons were made across all comparisons with Dwass-Steel-Critchlow-Fligner procedure for the pairwise comparisons to properly adjust for the potential inflation of family-wise type I errors.

In vitro cell count and ferene-s assay. All in vitro cell count and ferene-s assay data were analyzed by Students t test.

Correlation analysis. Rank-based, nonparametric Spearman correlation was performed using SAS 9.4, R, and Graphpad software.

Supplementary material for this article is available at

Supplementary Materials and Methods

Table S1. Summary of analytical data of (80 nm) BP nanoparticles.

Table S2. Summary of analytical data of all BNF-HER nanoparticles prepared.

Table S3. Characteristics of breast cancer cell lines used in the study.

Table S4. Summary of analytical data of BNF-IgG nanoparticles.

Table S5. Summary of analytical data of BNF-HER nanoparticles that passed in vitro qualification testing.

Table S6. Summary of immune modifications in mouse strains used for study.

Table S7. Summary of Aperio imaging settings used for digital analysis of tissue sections.

Table S8. Definitions of parameters used for Aperio imaging settings.

Table S9. Antibodies used for flow cytometry and their dilutions.

Table S10. Summary of numbers and strains of mice used in the study.

Table S11. Summary of one-factor model statistical analysis of iron measurements in xenograft models.

Table S12. Summary of two-factor model statistical analysis of iron measurements in xenograft models.

Table S13. Summary of three-factor model statistical analysis of iron measurements in xenograft models.

Table S14. Summary of one-factor model statistical analysis of Prussian blue histopathology analyses in xenograft models.

Table S15. Summary of two-factor model statistical analysis of Prussian blue histopathology analyses in xenograft models.

Table S16. Summary of three-factor model statistical analysis of Prussian blue histopathology analyses in xenograft models.

Table S17. Summary of statistical analysis of whole tumor digests flow cytometry in huHER2 allograft model.

Table S18. Summary of statistical analysis of nanoparticle-associated fractions (magnetic-sorted sediment) from flow cytometry in huHER2 allograft model.

Table S19. Summary of statistical analysis of nanoparticle-depleted fractions (magnetic-sorted supernatant) from flow cytometry in huHER2 allograft model.

Table S20. Summary of statistical analysis of iron measurements (ICP-MS) obtained from the livers of xenograft models.

Table S21. Ratio of Fe level between groups (treatment).

Table S22. Ratio of Fe level between groups (strains).

Table S23. Statistical analysis of ICP-MS huHER2-FVB/N lymph node data.

Table S24. Statistical analysis of ICP-MS huHER2-FVB/N spleen data.

Table S25. Statistical analysis of ICP-MS huHER2-FVB/N liver data.

Table S26. Ratio of percent positive between groups.

Table S27. Statistical analysis of tumor weight in huHER2-FVB/N.

Table S28. Statistical analysis of tumor growth in huHER2-FVB/N.

Table S29. Statistical analysis of whole tumor flow data third day.

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Nanoparticle interactions with immune cells dominate tumor retention and induce T cellmediated tumor suppression in models of breast cancer - Science...

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Global Nanomedicine Market Outlook, Strategies, Manufacturers, Countries, Type and Application, Global Forecast To 2026 – Stop Smoking Lounge

The latest report on Global Nanomedicine Market now available at Report Ocean, explains the contemporary and upcoming trends besides details associated with the regional landscape of the nanomedicine market that includes several regions. The report further emphasizes intricate details regarding the demand and supply analysis, contributions by leading industry players and market share growth of the nanomedicine market industry. Comprehensive secondary research was done to collect information on the market and its parent and ancillary markets. Further, primary research was performed to validate the assumptions and findings obtained from secondary research with key opinion leaders (KOL) and industry experts.

The report is a universal account of the major insights related to the geographical landscape of this business as well as the companies that have a reputable status in the nanomedicine market.

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In this report, we analyze the nanomedicine market industry from two aspects.

1. Production In terms of its production, we analyze the production, revenue, gross margin of its main manufacturers and the unit price that they offer in different regions from 2014 to 2019.

2. Consumption In terms of consumption, we analyze the consumption volume, consumption value, sale price, import and export in different regions from 2014 to 2019.

We also make a prediction of its production and consumption in coming 2020-2026.

At the same time, we classify different nanomedicine market based on their definitions. Upstream raw materials, equipment and downstream consumers analysis is also carried out. It also focuses on market influencing factors, competitive landscape, data, trends, information, and exclusive vital statistics of the market.

The market study focuses on various key parameters that include:

Market Segmentation

Regional Segmentation

In-Depth study of Market Determinants

360-Degree Economic Analysis

Regulatory Analysis

Company Profiling and others

Competitive Landscape:

The competitive analysis of major market players is another notable feature of the nanomedicine market industry report; it identifies direct or indirect competitors in the market. The report offers company profile of market players alongside product picture and its specifications, nanomedicine market industry market plans, and technology adopted by them, future development plans. In addition, strength and weaknesses analysis of nanomedicine market industry competitive firms gives competitive advantages so that the efficiency and the productivity of companies are improved.

Market Segmentation:

The segmentation is used to decide the target market into smaller sections or segments like product type, application, and geographical regions to optimize marketing strategies, advertising technique and global as well as regional sales efforts of nanomedicine market. The common characters are also being considered for segmentation such as global market share, common interests, worldwide demand and supply of Access Control devices. Moreover, the report compares the production value and growth rate of Global Nanomedicine Market across different geographies.

This report studies the top producers and consumers, focuses on product capacity, production, value, consumption, market share and growth opportunity in these key regions, covering

North America (United States, Canada and Mexico)

Europe (Germany, France, UK, Russia and Italy)

Asia-Pacific (China, Japan, Korea, India and Southeast Asia)

South America (Brazil, Argentina, Colombia)

Middle East and Africa (Saudi Arabia, UAE, Egypt, Nigeria and South Africa)

The research methodology adopted by analysts to study the market include inputs derived from industry professionals across the value chain and various other secondary research methods, along with primary research as a major tool for market study.

Some of the Major Highlights of TOC covers:

Executive Summary

Global Nanomedicine Market Insights

Global Nanomedicine Market forecast by different Segments and Regions

Manufacturing Cost Structure Analysis

Development and Manufacturing Plants Analysis of Global Nanomedicine Market

Key Figures of Major Manufacturers

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The report would provide an in-depth analysis on the current and future market trends

Analysis on global, regional and country level markets

Key strategic initiatives taken by major players operating in the market along with ranking analysis for the key players

Analysis based on historical information along with the current trends to estimate the future of the market

Analysis of the impact of constantly changing global market scenarios

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Global Nanomedicine Market Executive Summary and Analysis by Top Players 2020 – 2025 : GE Healthcare, Johnson & Johnson – Stock Market Herald

A market study dependent on the Nanomedicine Market over the globe, as of late added to the storehouse of Market Research, is titled Worldwide Nanomedicine Market 2019. The exploration report examinations the chronicled just as present execution of the overall Nanomedicine industry and makes expectations on the future status of Nanomedicine advertise based on this investigation.

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Top Companies Include (from a broad pool of working players over the globe):GE Healthcare, Johnson & Johnson, Mallinckrodt plc, Merck & Co. Inc., Nanosphere Inc., Pfizer Inc., Sigma-Tau Pharmaceuticals Inc., Smith & Nephew PLC, Stryker Corp, Teva Pharmaceutical Industries Ltd., UCB (Union chimique belge) S.A

The report reads the business for Nanomedicine over the globe taking the current business chain, the import and fare measurements in Nanomedicine advertise and elements of interest and supply of Nanomedicine into thought. The Nanomedicine examine study covers every single part of the Nanomedicine showcase comprehensively, which begins from the meaning of the Nanomedicine business and creates towards Nanomedicine advertise divisions. Further, every fragment of the Nanomedicine advertise is grouped and broke down based on item types, applications, and the end-use businesses of the Nanomedicine showcase. The land division of the Nanomedicine business has likewise been canvassed finally in this report.

Market Size Segmentation by Type (Customizable):Regenerative Medicine, In-vitro & In-vivo Diagnostics, Vaccines, Drug Delivery

Market Size Segmentation by Application (Customizable):Clinical Cardiology, Urology, Genetics, Orthopedics, Ophthalmology

The focused scene of the overall market for Nanomedicine is controlled by assessing the different business members, creation limit, Nanomedicine markets creation chain, and the income produced by every producer in the Nanomedicine advertise around the world.

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The worldwide Nanomedicine showcase 2020is additionally examined based on item evaluating, Nanomedicine creation volume, information with respect to request and Nanomedicine supply, and the income accumulated by the item. Different precise instruments, for example, speculation returns, plausibility, and market engaging quality investigation has been utilized in the exploration to introduce a far-reaching investigation of the business for Nanomedicine over the globe.

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Global Nanomedicine Market Executive Summary and Analysis by Top Players 2020 - 2025 : GE Healthcare, Johnson & Johnson - Stock Market Herald

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Nanoparticle Therapy Might Help Reduce Brain Swelling in… : Neurology Today – LWW Journals

Article In Brief

Mice with an open- and closed-traumatic brain injury were injected with immunomodulatory nanoparticles that reduced brain swelling and damage on MRI.

Investigators used a novel approach to prevent the swelling that can occur after traumatic brain injury (TBI) in a mouse model: they injected nanoparticles that trick white blood cells into going after them instead of rushing to the injured brain and causing an inflammatory and immune response.

Mice with TBI that were given three injections of the immunomodulatory nanoparticles beginning two to three hours after injury showed less brain swelling and damage on MRI as compared with mice with TBI that did not get the nanoparticles; the treated mice also performed better on functional tests.

The immunomodulatory nanoparticle treatment, if further proven in preclinical trials and human trials, would not undo damage from the initial injury to the brain. But it could help prevent the body from setting off a cascade of immune and inflammatory cells in reaction to the injury, which in turn can cause brain swelling and even more damage to brain tissue.

We certainly haven't gone and magically prevented that initial damage, said Jack Kessler, MD, professor of neurology at Northwestern University Feinberg School of Medicine and the senior author of the paper. What we can do is prevent the secondary damage, which is substantial.

Predicting which TBI patients will develop edema of the brain isn't easy, so having a preventive treatment like the nanoparticles that could be administered upfront could be life-altering, Dr. Kessler said.

He said some patients with head injuries come into the hospital walking and talking, but then their brain swells, and they die.

According to background in the study, published January 10 online in Annals of Neurology, each year more than 2.5 million people in the US have a traumatic brain TBI and more than five million Americans live with at least one sequela of TBI.

After the primary injury, there is substantial secondary injury attributable to infiltrating immune cells, cytokine release, reactive oxygen species, excitotoxicity, and other mechanisms, the study authors wrote. Despite many preclinical and clinical trials to limit such secondary damage, no successful therapies have emerged.

The nanoparticles tested in the mouse experiments are made of material used in biodegradable sutures. The paper specifically described the particles as highly negatively charged, 500 nm-diameter particles composed of the Food and Drug Administration (FDA)-approved biodegradable biopolymer carboxylated poly (lactic-co glycolic) acid.

The nanoparticles (IMPs), which seem like foreign invaders to the body's immune system, attract the attention of large white blood cells known as monocytes, which have been implicated in the secondary damage that occurs with TBI.

IMPs bind to the macrophage receptor with collagenous structure (MARCO) on monocytes and monocytes bound to IMPs no longer home to sites of inflammation but rather are sequestered in the spleen, where the cells die, the study authors wrote.

The mouse study involved two types of head injury. In some of the mice, the researchers performed a craniotomy to create a controlled cortical impact. Other mice received a closed head injury involving a direct blow to the head. Both types of injuries were meant to mimic what occurs in humans with TBI.

Injections of the nanoparticles were given two to three hours after the brain injury, and again at 24 hours and 48 hours post-injury. Control animals with similar brain injuries were given saline solution at the same time points.

Outcomes for the mice who received the nanoparticles were better by multiple measures, including MRI and a motor function test called the ladder rung walking test that is used in mouse experiments.

IMP administration resulted in remarkable preservation of both tissue and neurological function, in both models of head injury, the paper said. After acute treatment, there was a reduction in the number of immune cells infiltrating into the brain, mitigation of the inflammatory status of the infiltrating cells, improved electrophysiological visual function, improved long-term motor behavior, reduced edema formation as assessed by magnetic resonance imaging, and reduced lesion volumes on anatomic examination.

Dr. Kessler said that in the case of mice with an open head injury, the size of their brain lesion was 50 percent smaller in the treated animals compared with those that did not get the nanoparticles.

He said MRI showed significantly less brain swelling and less compression of the ventricles, both signs that secondary damage was minimized.

Dr. Kessler said that right now the only recourse for severe brain swelling is to do a craniotomy to relieve pressure in the skull.

He said one of the appeals of the nanoparticle treatment is that an emergency medical technician could do it in the field or the emergency room personnel could inject it.

But Dr. Kessler is also cautious about too many predications based on a pre-clinical study, saying he is fond of telling medical students that if I had a nickel for every mouse we cured, I'd be a rich man.

Sripadh Sharma, PhD, an MD-PhD student at Northwestern and the study's first author, said the nanoparticle therapy needs to be tested further in animal models before it could go into human testing. The researchers also want to learn more about how the nanoparticles bring about a reduced immune response in the body.

Dr. Sharma noted that while immune responses are a good thing in the face of injury or infection, sometimes nature doesn't always get it right, so too much of a good thing is a bad thing. And that can be the case with TBI.

He said it has been shown by another collaborator on the study, Stephen Miller, PhD, that when the scavenger receptors on the monocytes detect the light negative charge of the nanoparticles, the monocytes engulf and bind to the particles and apoptose in the spleen instead of going to the site of injury.

More studies need to be done to optimize what dose and what time these particles need to be given following a head injury, said Dr. Sharma.

Similar nanoparticle therapy is being tested for other medical conditions, including celiac disease and myocardial infarction, Dr. Kessler said.

Michael J. Schneck, MD, FAAN, professor of neurology (and neurosurgery) at Loyola University Chicago, said the study was well-designed and thorough, using two different head injury models and multiple outcome measures, including brain imaging, functional testing, and brain tissue analysis. Dr. Schneck said the paper made him wonder whether a similar approach using immune-modulating nanoparticles could reduce inflammatory-related damage following stroke and spinal cord injury.

Dr. Schneck said the concept of trying to dampen the immune response after TBI to prevent edema is not new, but the Northwestern researchers took the idea in a new direction. The nanoparticle therapy is particularly intriguing, he said, because it is fairly simple and involves the use of a material that is already approved by the US FDA, which could mean that it would take less time to move the therapy from the laboratory into clinical trials.

This is a very elegant study with interesting translational potential, he said. But it is a mouse model and its application to (human) TBI and other forms of central nervous system injury remains to be validated.

Jiangbing Zhou, PhD, associate professor of neurosurgery and biomedical engineering at Yale University, said that as someone who does research in the field of nanomedicine, he was surprised by the study's findings and wants to understand how this simple formulation particle could achieve this marked efficacy.

The study looks very exciting, but I want to know more about the mechanism, said Dr. Zhou, whose research focuses on developing translational nanomedicine, gene therapy, and stem cell therapy for neurological disorders including TBI.

He had these and other questions about the study: Why do the particles interact specifically with the inflammatory monocytes but not the others? How do the particles, which are made of safe biomaterials, efficiently kill the inflammatory monocytes in the spleen? What is happening and why?

Javier Crdenas, MD, director of the Barrow Concussion and Brain Injury Center at the Barrow Neurological Institute, said the study on the immune-modulating nanoparticle therapy for TBI was very promising, though he stressed that he is always cautiously optimistic when he sees a mouse study.

It is definitely a novel approach to addressing the secondary sequelae of brain injury and they might have something that minimizes that and hopefully improves outcomes, Dr. Crdenas said.

He said the study also raises some questions, including how the immune-modulating approach would fare in patients who have multiple injuries, not just to the head.

Dr. Crdenas said brain injuries often do not happen in isolation, with patients also having broken bones, lacerations, and other organ damage.

We don't know how this (nanoparticle treatment) would affect other organs, other immune responses elsewhere in the body, he said.

Dr. Crdenas said the field of TBI research has been disappointed before by studies of new therapies that looked promising in animal models and clinical testing but ultimately failed. He noted, for instance, that progesterone and hypothermia did not turn out to be good at preventing brain swelling.

We will wait and see, he said of the nanoparticles.

Drs. Sharma, Schneck, Zhou, and Crdenas had no disclosures.

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Nanoparticle Therapy Might Help Reduce Brain Swelling in... : Neurology Today - LWW Journals

Recommendation and review posted by Alexandra Lee Anderson

What swarm robotics has taught me about leading a team working on swarm robotics –

As a roboticist and engineer, one of the growing areas Im most excited about is the advancement of swarm robotics. I love the idea of taking inspiration from biological systems to enhance what autonomous robots can achieve collectively, and re-contextualizing social animal behaviors like flocking, foraging, and transport to create new possibilities for humanity. From nanomedicine and environmental monitoring to search and rescue and space exploration, theres no shortage of beneficial applications for swarms.

While robot swarm intelligence might sound intimidatingly high tech, its predicated on a very relatable concept. Just as robots within a swarm must operate collaboratively to solve shared problems, human teams need to work together to accomplish common objectives. In my experience working with a multidisciplinary team to commercialize autonomous aerial robots, Ive learned that you can transpose a number of key principles from robot swarm intelligence to enhance the approach to team leadership.

Organization >> IndividualIn the swarm model, the primary intention is to produce meaningful behavior at the grouprather than the individuallevel. While the individual robot is capable of operating on its own, its also intelligent enough to understand that when the group works together, the whole result becomes greater than the sum of its parts. Take one biological inspiration as an example: starlings. Scientists believe they form flocks primarily for safety because individually, they are quite agile and quick to avoid predators. But by forming a murmuration, they actually increase every individuals chance of survival by confusing predators, and increase the safety of the group as a whole.

This type of thinking is also true for an organization in which people work together in orchestrated harmony toward a common goal, and prioritize shared success over personal recognition. The challenge is that, if you manage a diverse team like myself, youll need to translate these objectives in a way that resonates with everyone. This is the key to cultivating coherence and a teamwork mindset between people from different functions, skill sets, and roles. For example, if you clearly align the technical details of your product with the real-world business requirements and outcomes, you can contextualize the full impact of the work for your technologists and engineers, beyond their individual subject matter areas. This helps synthesize efforts, and can also dissuade informational and cultural silos from forming.

In addition, make it a point to give credit where credit is due. This can go a long way in helping team members feel seen, and acknowledging the value of their hard work and contributions. It also helps take the pressure off of those who may feel like they constantly need to prove themselves to the group.

Structured, efficient, effective communicationOne reason swarms are able to achieve unparalleled scalability is by relying primarily on local, robot-to-robot communication to share information, which helps eliminate the dependence on a centralized infrastructure. This is especially useful if you are operating swarms in dangerous or otherwise inaccessible and unfamiliar areas, where you cant necessarily have a human operator within the same environment to work alongside and monitor the swarm. When the swarm does need to transmit critical information to a remote location (e.g., wherever the humans reside), it can tap special individual membersso-called gateway robotscapable of aggregating the local data and then relaying it via global communication.

Understanding how critical information is shared within the swarm and to external sources presents interesting parallels for human teams as well. For example, when is one-to-one communication more effective than one-to-many, and vice versa? And how frequently should these touch points happen? Perhaps specialty groups need to huddle more frequently and less formally, but the whole company would benefit from more structured communications at regular intervals. Perhaps you decide that team leaders should also act as gateway communicators in streamlining information flow across the company, and that this affects how you evaluate who is best suited for that role.

Whether robot or human, we all need to identify the specific challenges we each face to efficient and effective communication, and devise ways to overcome them. For robots, these might include technical issues and real-world limitationsthings like latency, bandwidth, asynchrony, topological changes, etc. Humans, meanwhile, need to devise a clear plan for unifying information across disparate tools and systems, managing handoffs, and addressing work-culture differences.

Build robustness from the ground upAnother remarkable quality of robot swarms is their robustness. They are designed such that, if any one component were to fail or malfunction, the system can continue performing its tasks as normal. Its like how the newer Christmas lights have fail protection in their circuitryif one bulb goes out, the rest of the string keeps shining. In this sense, swarms have built-in safety nets. By distributing and decentralizing the system across multiple units, you reduce the overall risk that something goes wrong.

From a leadership perspective, robustness can be addressed in two key ways: in the development of the technology and in the approach to team culture. On the technology side, for example, we focus on higher quality, modular code with less interdependencies, putting processes in place for continuous assessment and validation, and ensuring aggressive requirements for testing from start to finish. (This is where autonomous robots differ dramatically from other types of software-based tech. You cant just test in a sandbox or a simulated environmentonly real-world field testing will ensure you deliver the best product!)

On the team culture side, we build different kinds of safety nets. We focus on leading with a foundation of trust and respect, and put protocols in place to help affirm a positive work environment. For example, we set mutual expectations among teams or individuals for reaching certain milestones, to encourage a sense of shared responsibility and ownership instead of making people feel like they are being assigned a task list. Both the CEO and myself have standing open-door policies, in which we welcome anyone to approach us and raise concerns or discuss important issues. We have regular team building and team wellness days, where we dedicate time for meaningful, non-work activities to boost well-being and morale. As a recent example, we held a company offsite to hear a former NASA astronaut share his perspective on the value of teamwork in solving seemingly impossible challengeswhich we followed up with an escape room exercise. Its important to us to create a workplace that makes everyone feel included and valued, and helps each team member be at their very best.As a bonus benefit, these three principlesorganizational alignment, effective communication, and robustnessare also what help swarms to be flexible in quickly adapting to unexpected changes in their environment. In the face of unpredictability, uncertainty about the future, and every-evolving needs and requirements, technology-driven businesses need to think about fortifying themselves to weather the storm. Why not take inspiration from technology itself?

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In vivo Comparison of the Biodistribution and Toxicity of InP/ZnS Quan | IJN – Dove Medical Press

Li Li,1,2 Yajing Chen,1 Gaixia Xu,2,3 Dongmeng Liu,1 Zhiwen Yang,1 Tingting Chen,1 Xiaomei Wang,1 Wenxiao Jiang,1 Dahui Xue,1 Guimiao Lin1

1Base for International Science and Technology Cooperation: Carson Cancer Stem Cell Vaccines R&D Center, Shenzhen Key Laboratory of Synthetic Biology, Department of Physiology, School of Basic Medical Sciences, Shenzhen University, Shenzhen 518055, Peoples Republic of China; 2Key Laboratory of Optoelectronics Devices and Systems of Ministry of Education/Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, Peoples Republic of China; 3Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen 518055, Peoples Republic of China

Correspondence: Guimiao LinSchool of Basic Medical Sciences, Shenzhen University Health Sciences Center, Shenzhen 518060, Peoples Republic of ChinaTel/ Fax +86-755-86671903Email

Introduction: Indium phosphide (InP) quantum dots (QDs) have shown a broad application prospect in the fields of biophotonics and nanomedicine. However, the potential toxicity of InP QDs has not been systematically evaluated. In particular, the effects of different surface modifications on the biodistribution and toxicity of InP QDs are still unknown, which hinders their further developments. The present study aims to investigate the biodistribution and in vivo toxicity of InP/ZnS QDs.Methods: Three kinds of InP/ZnS QDs with different surface modifications, hQDs (QDs-OH), aQDs (QDs-NH2), and cQDs (QDs-COOH) were intravenously injected into BALB/c mice at the dosage of 2.5 mg/kg BW or 25 mg/kg BW, respectively. Biodistribution of three QDs was determined through cryosection fluorescence microscopy and ICP-MS analysis. The subsequent effects of InP/ZnS QDs on histopathology, hematology and blood biochemistry were evaluated at 1, 3, 7, 14 and 28 days post-injection.Results: These types of InP/ZnS QDs were rapidly distributed in the major organs of mice, mainly in the liver and spleen, and lasted for 28 days. No abnormal behavior, weight change or organ index were observed during the whole observation period, except that 2 mice died on Day 1 after 25 mg/kg BW hQDs treatment. The results of H&E staining showed that no obvious histopathological abnormalities were observed in the main organs (including heart, liver, spleen, lung, kidney, and brain) of all mice injected with different surface-functionalized QDs. Low concentration exposure of three QDs hardly caused obvious toxicity, while high concentration exposure of the three QDs could cause some changes in hematological parameters or biochemical parameters related to liver function or cardiac function. More attention needs to be paid on cQDs as high-dose exposure of cQDs induced death, acute inflammatory reaction and slight changes in liver function in mice.Conclusion: The surface modification and exposure dose can influence the biological behavior and in vivo toxicity of QDs. The surface chemistry should be fully considered in the design of InP-based QDs for their biomedical applications.

Keywords: InP/ZnS quantum dots, surface chemistry, in vivo, biodistribution, nanotoxicology

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License.By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.

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In vivo Comparison of the Biodistribution and Toxicity of InP/ZnS Quan | IJN - Dove Medical Press

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Functionalized Gold and Silver Bimetallic Nanoparticles Using Deinococ | IJN – Dove Medical Press

Yulan Weng, 1 Jiulong Li, 1 Xingcheng Ding, 2 Binqiang Wang, 1 Shang Dai, 1 Yulong Zhou, 3 Renjiang Pang, 1 Ye Zhao, 1 Hong Xu, 1 Bing Tian, 1, 3 Yuejin Hua 1

1MOE Key Laboratory of Biosystems Homeostasis & Protection, Zhejiang University, Hangzhou, Peoples Republic of China; 2Zhejiang Runtu Chemical Research Institute, Shaoxing, Peoples Republic of China; 3Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, School of Chemistry and Chemical Engineering, Shihezi University, Shihezi, Xinjiang, Peoples Republic of China

Correspondence: Bing TianZhejiang University Zijingang Campus West Part, A403 Biophysics Building, 866 Yuhangtang Road, Hangzhou 310058, Peoples Republic of ChinaTel/Fax +86-571-86971215Email

Background: Biodegradation of toxic organic dye using nanomaterial-based microbial biocatalyst is an ecofriendly and promising technique.Materials and Methods: Here, we have investigated the novel properties of functionalized Au-Ag bimetallic nanoparticles using extremophilic Deinococcus radiodurans proteins (Drp-Au-AgNPs) and their degradation efficiency on the toxic triphenylmethane dye malachite green (MG).Results and Discussion: The prepared Drp-Au-AgNPs with an average particle size of 149.8 nm were capped by proteins through groups including hydroxyl and amide. Drp-Au-AgNPs demonstrated greater degradation ability (83.68%) of MG than D. radiodurans cells and monometallic AuNPs. The major degradation product was identified as 4-(dimethylamino) benzophenone, which is less toxic than MG. The degradation of MG was mainly attributed to the capping proteins on Drp-Au-AgNPs. The bimetallic NPs could be reused and maintained MG degradation ability (> 64%) after 2 cycles.Conclusion: These results suggest that the easilyprepared Drp-Au-AgNPs have potential applications as novel nanomedicine for MG detoxification, and nanomaterial for biotreatment of a toxic polyphenyl dye-containing wastewater.

Keywords: bimetallic nanoparticles, Deinococcus radiodurans, biodegradation, toxic triphenylmethane dye, malachite green, detoxification

This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License.By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.

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Functionalized Gold and Silver Bimetallic Nanoparticles Using Deinococ | IJN - Dove Medical Press

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Children’s cancer researcher named Woman of the Year – UNSW Newsroom

Professor Maria Kavallaris, a leading childhood cancer researcher and a pioneer of nanomedicine in Australia, is the 2020 NSW Premier's Woman of the Year.

Professor Kavallaris is Head of Translational Cancer Nanomedicine at Childrens Cancer Institute and Founding Director of the Australian Centre for NanoMedicine at UNSW Sydney.

The prestigious award, announced a ceremony in Sydney this morning,recognises NSW women who have excelled in their chosen career, field or passion; are exceptional achievers who have made a significant contribution to NSW; and whose accomplishments make them a strong role model for other women.

I am truly honoured to have received this award and I hope it inspires young women to do what they love, grow and learn, and to lead with generosity and respect, Professor Kavallaris said.

Professor Kavallaris is internationally renowned for her research in cancer biology and therapeutics. She has been widely recognised for the innovation and impact of her research, her leadership as well as her mentoring of talented young scientists. She is passionate about training the next generation of research leaders.

Her personal journey with cancer began at the age of 21 and has driven her research to develop effective and less toxic cancer treatments.

As one of the original three scientists appointed at the Childrens Cancer Institute when its laboratories first opened in 1984, she has made important discoveries in relation to the mechanisms of clinical drug resistance and tumour aggressiveness in childhood cancer.

Her studies have not only identied how some tumours can grow and spread;she has also applied this knowledge to develop eective, less toxic cancer therapies using nanotechnology.

To be able to make a difference to the lives of children with cancer and their families by developing better treatments and improving survival rates is very humbling. Even if you can save one childs life, thats an incredible feat, Professor Kavallaris said.

As a conjoint professor in the UNSW Faculty of Medicine, Professor Kavallaris relishes her role of mentor and has supervised many Honours and PhD students, several of whom have become research leaders.

Professor Kavallariss extensive research and leadership contributions have been recognised withnumerous awards including the NSW Premiers Prize for Science and Engineering (Leadership in Innovation in NSW) in 2017, the Australian Society for Biochemistry and Molecular Biologys Lemberg Medal in 2019 and she was made aMember of the Order of Australia (AM) for significant service to medicine, and to medical research, in the field of childhood and adult cancerson Australia Day 2019.

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Children's cancer researcher named Woman of the Year - UNSW Newsroom

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Here’s how nanoparticles could help us get closer to a treatment for COVID-19 – News@Northeastern

There is no vaccine or specific treatment for COVID-19, the disease caused by the severe acute respiratory syndrome coronavirus 2, or SARS-CoV-2.

Since the outbreak began in late 2019, researchers have been racing to learn more about SARS-CoV-2, which is a strain from a family of viruses known as coronavirus for their crown-like shape.

Northeastern chemical engineer Thomas Webster, who specializes in developing nano-scale medicine and technology to treat diseases, is part of a contingency of scientists that are contributing ideas and technology to the Centers for Disease Control and Prevention to fight the COVID-19 outbreak.

Professor and chair of the Department of Chemical Engineering Tom Webster. Photo by Adam Glanzman/Northeastern University

The idea of using nanoparticles, Webster says, is that the virus behind COVID-19 consists of a structure of a similar scale as his nanoparticles. At that scale, matter is ultra-small, about ten thousand times smaller than the width of a single strand of hair.

Webster is proposing particles of similar sizes that could attach to SARS-CoV-2 viruses, disrupting their structure with a combination of infrared light treatment. That structural change would then halt the ability of the virus to survive and reproduce in the body.

You have to think in this size range, says Webster, Art Zafiropoulo Chair of chemical engineering at Northeastern. In the nanoscale size range, if you want to detect viruses, if you want to deactivate them.

Finding and neutralizing viruses with nanomedicine is at the core of what Webster and other researchers call theranostics, which focuses on combining therapy and diagnosis. Using that approach, his lab has specialized in nanoparticles to fight the microbes that cause influenza and tuberculosis.

Its not just having one approach to detect whether you have a virus and another approach to use it as a therapy, he says, but having the same particle, the same approach, for both your detection and therapy.

SARS-CoV-2 spreads mostly through tiny droplets of viral particlesfrom breathing, talking, sneezing, coughingthat enter the body through the eyes, mouth, or nose. Preliminary research also suggests that those germs may survive for days when they attach themselves to countertops, handrails, and other hard surfaces.

Thats one reason to make theranostics with nanoparticles the focus of the COVID-19 outbreak, Webster says.

Nanoparticles can disable these pathogens even before they break into the body, as they hold on to different objects and surfaces. His lab has developed materials that can be sprayed on objects to form nanoparticles and attack viruses.

Even if it was on a surface, on someones countertop, or an iPhone, he says. It doesnt mean anything because its not the active form of that virus.

That same technology can be fine-tuned and tweaked to target a wide range of viruses, bacteria, and other pathogens. Unlike other novel drugs with large molecular structures, nanoparticles are so small that they can move through our body without disrupting other functions, such as those of the immune system.

Almost like a surveyor, they can go around your bloodstream, Webster says. They can survey your body much easier and under much longer times and try and detect viruses.

To do all that, the CDC needs to know the specifics about what kind of structure is needed to neutralize SARS-CoV-2, Webster says. That information isnt public yet.

You have to identify what we need to put in our nanoparticle to attract it to that virus, he says. The CDC must know that, because theyve developed a kit that can determine if you have [COVID-19], versus influenza, or something else.

An alternative to nanomedicine is producing synthetic molecules. But Webster says that tactic presents some challenges. In the case of chemotherapies used to treat cancer cells, such synthetic drugs can cause severe side effects that kill cancer cells, as well as other cells in the body.

The same thing could be happening with synthetic chemistry to treat a virus, where molecules are killing a lot more than just that virus, Webster says.

Still, Webster acknowledges that there arent many researchers focusing on nanoparticles to kill viruses.

One of the main reasons for the lack of those solutions is that the same benefits that make nanoparticles ideal to fight infectious diseases also make them a concern for the U.S. Federal Drug Administration.

Because of their size, nanoparticles are pervasive (too pervasive, maybe) to seep through other parts of the body. To reduce that risk, Websters lab has focused on using iron oxide. Particles of that make up entail chemistry that is already natural to our bodies and diets.

Even if you have a viral infection, you need more iron, because you could be anemic depending on how bad the infection is, Webster says. Were actually developing these nanoparticles out of chemistries that can help your health.

And, he says, iron-based nanoparticles could be directed with magnetic fields to target specific organs in the body, such as lungs and other areas susceptible to respiratory complications after contracting viral infections. That too, Webster says, is something that you couldnt do with a novel synthetic molecule.

Really, what this all means is that we just have to do the studies to show those iron nanoparticles are not going into the brain or the kidney, Webster says, that these nanoparticles are going exactly where you want them to go to the virus.

For media inquiries, please contact Shannon Nargi at or 617-373-5718.

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Here's how nanoparticles could help us get closer to a treatment for COVID-19 - News@Northeastern

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Nanowires, due to properties, such as, high crystallinity, high surface to volume to ratio, and high resistance, nanowires are increasingly being used in nanomedicines, biomedicine, consumer electronics, bioelectronics, etc. Their use has helped to propel the field of nanomedicine. Nanomedicine is defined as the application of nanotechnology for diagnosis, monitoring, drug delivery, treatment, and control of biological systems. In the United States, the Food & Drug Administrator (FDA) approved over 100 nanodrugs for use in medicinal trials and clinical treatment. Also, nanowires are now increasingly being used in LEDs. They allows for faster communication between devices and microchips. These emerging applications of nanowires in LEDs, consumer electronics, and nano medicines are expected to contribute to the growth of the global nanowires market during the forecast period.

Metal Nanowires to Dominate the Market

Metals have unique thermal, mechanical, electrical, and catalytic properties. On the other hand, metallic nanowires are promising materials for a variety of applications, such as, transparent conductive films for photovoltaic devices, electrodes for batteries, and nano-reinforcement for composite materials. Electronics is one of the major end-user applications of nanowires. They are used in transistors as they are very good conductors or semiconductors. They are also expected to play a significant role in quantum computers in the near future. Moreover, new applications of metal nanowires are also being developed in the field of energy.

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