2 Abstract Sialic acid (Sia) conjugated nanocarriers have emerged as

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Biological and Medical Applications of Materials and Interfaces

Imaging and Targeting of the #(2-6) and #(2-3) Linked Sialic Acid Quantum Dots in Zebrafish and Mouse Models Rohan Yadav, Preeti Madhukar Chaudhary, Balamurugan Subramani, Suraj Toraskar, Harikrishna Bavireddi, Raghavendra Vasudeva Murthy, Sivakoti Sangabathuni, and Raghavendra Kikkeri ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07668 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on August 4, 2018

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ACS Applied Materials & Interfaces

Abstract Sialic acid (Sia) conjugated nanocarriers have emerged as attractive biomarkers with promising biomedical applications. The translation of these nanocarriers into clinical applications requires indepth assessment in animal models. However, due to the complexity, ethical concerns, and cost of the high-order animal system, there is an immediate need of information rich simple animal models to decipher the biological significance. Herein, we performed in-vivo head-to-head comparison of Neu5Acα(2-6) and α(2-3)Gal conjugated quantum dots (QDs) toxicity, biodistribution and sequestration in wild-type zebrafish (Danio rerio) and mouse model (C57BL). The fluorescent properties and cadmium composition of quantum dots was used to map the blood clearance, biodistribution and sequestration of the sialylated-QDs in major organs of both the models. We observed that α(2-6) sialylated-QDs preferentially have prolonged circulating half-life and broader bio-distribution in both the models. On the contrary, α(2-3) sialic acid and galactose conjugatedQDs have shortened blood circulation time and sequestered in the liver, kidney and cleared after several hours in both models. These results demonstrate the applicability of the zebrafish and sialylated-QDs to target specific organs, as well as drug delivery and biomedical diagnostics.

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Introduction Sialic acids (Sia) are α-keto acid backbone monosaccharides, typically present at the terminals of N- and O-glycans chain of glycoproteins and glycolipids. Over the decades, Sias have been reported in various pathological and physiological processes.1,2 Hence, the functionalization of Sia on nanocarriers will undoubtedly assist in designing smart probes for targeting and inhibiting the specific biological events.3-6 In addition to its targeting ability, Sias are non-immunogenic in nature. Consequently, sialylation of the biomolecules such as proteins, enzymes, nano-carriers mask the active sites of the immune system and prolong their lifetime.7-8 Recently, Sia residue in the form of N-glycan, GM1 and GM3 were conjugated to liposomes, dendrimers, nanoparticles to establish drug delivery system.9-15 However, the ability to predict the clinical significance of these nanoconjugates is based on the in-depth understanding of in-vivo studies. To date, mice are the most widely and the most efficient model for preclinical studies.16 However, the biological system of mice model is complex and requires a large number of materials to quantify the biological significance. Hence, there is an urge of the alternative in-vivo system which is simple and consumes fewer materials and resembles biological results of the higher animal model. Previously, fish, insects and worms are used as simple in-vivo models. However, all these models have their own advantages and disadvantages. Among them, zebrafish has several advantages as compared to Caenorhabditis elegans, Drosophila melanogaster and Hydra, which includes quick lifespan from embryo to larva. Easy to maintain a large pool of fishes in a single container and ease of producing transparent zebrafish mutant to improve the detection sensitivity and display vertebrate organs such as cardiovascular, nervous and digestive systems similar to the human system.17-18 The zebrafish has been described as “the canonical vertebrate” due to the similarities between mammalian and zebrafish biology19 and used as a prominent model to study


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the diseases such as cancer, liver, cardiovascular and neurological disorder.20-25 Furthermore, the fish model has been extensively used to investigate the pharmacokinetics of nanoparticles and carbon nanorods.26-29 Hence, deciphering the carbohydrate-mediated bio-distribution and sequestration in zebrafish will give preliminary information to target specific human diseases. In this study, to find a simple, active animal model for glyco-nanotechnology research, we have compared in vivo efficacy of sialylated-QDs in zebrafish and mouse model. Although these two models are independently targeted in glycobiology and glyconanotechnology research, the head-tohead comparison at multiple stages will provide a broader profile for pharmacology and clinical research.

Results and discussions. Commercial PEG-COOH conjugated quantum dot was used to the synthesis of glyco-QDs. The sugar functionalization of QDs was carried out by using water soluble coupling reagents and Nhydroxysuccinimide, followed by capping the unreacted carboxylic acid terminals by using ethanolamine (Scheme 1).30, 31 The size and shape of the glyco-QDs were characterized by using TEM (Fig 1). The QDs were well dispersed and shown homogenous size and spherical shape. The diameter of Q-1 to Q-4 was around 12-14 nm. After glycan conjugation, the surface potential of Q1 charged from -24.1 mV to -12.5 mV, indicating that negative charge of the native QDs was replaced by Sia moiety and remaining carboxylic acid groups of the QDs were neutralized with ethanolamine linker. Similarly, Q-2 and Q-3 displayed zeta potential of -10.7 and -13.3 mV respectively. While the Q-4 displayed 0.78 mV, due to the non-ionic nature of the S-4. The quantum yield of QDs was recorded to characterize the sugar conjugation. It was observed that the quantum yield was slightly increased, indicating that the carbohydrate conjugation stabilizes of 4 ACS Paragon Plus Environment


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CdSe core and enhance the fluorescence intensity. Finally, the number of Sia or galactose moieties on each QDs was quantified by resorcinol

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and phenol-sulfuric acid method. It was observed that

each QDs carried around 45-55 sialic acid moieties, demonstrating the multivalent display of sugar on a fluorescent template (Table 1). After synthesizing and characterizing the glyco-QDs, we first performed the mortality of adult zebrafish and mice after i.p injection of various QDs. LD50 values of the 24h acute toxicity test was summarized in Table 2. As dosage varied from 1 to 20 nmol/kg, a significant change in the mortality was observed in both models. This dosage is similar to that of the concentration used by different research groups to perform in vivo experiments with QDs.33-38 Among the QDs, Q-1 showed less toxicity compared to other glyco-QDs. It was observed that doses below 5 nmol/kg, no animal death occurred. For Q-1, maximum animal death took place at 20 nmol/kg in both animal models. LC50 value was 13.6 nmol/kg and 14.7 nmol/kg in the mice and zebrafish model respectively. While Q-2 to Q-4 showed maximum animal death at 15 nmol/kg in both models and the LD50 value were 11.7, 9.6 and 9.3 nmol/kg in the mice model and 11.4, 9.51 and 9.6 nmol/kg in zebrafish model respectively (Fig 2a, 2b and Table 2). Previously, it has been shown that acute toxicity of the QDs in mice and zebrafish varies with the surface functionalization.39-41 Tang et al. studied the acute toxicity of QDs in mice model using anionic, cation and non-ionic surfactants conjugated QDs.42 It was shown that the cationic-QDs are more sensitive compared to anionic and non-ionic surfactant-QDs. Vaughan et al. compared the toxicity of six chemicals in zebrafish embryos and adult fish.43 They found that the toxicity of chemicals in embryos are comparable to the acute toxicity in adult zebrafish system. Similarly, the Kovrižnych et al. showed the similarity in toxicity effect of 31 different types of nanoparticles in zebrafish egg and adult fishes.26 However, the comparative toxicity studies with two animal model have rarely been investigated. Our results


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demonstrate that surface functionalization of different Sia linkage alters the in vivo acute toxicity in both animal models, probably due to the differences in the biodistribution and sequestration level. The above toxicity studies clearly showed the QDs of different Sia glycans alter the mortality in both animal model. Next, the half-life of the blood clearance of glyco-QDs was elucidated to support the mechanism of acute toxicity. The concentration of the CdSe/ZnS quantum dots in blood was measured by quantifying the cadmium concentration by using ICP-MS. We set 7 nmol/kg (weight of the fish or mouse) of QDs as an ideal concentration for head-to-head in vivo experiments, as they displayed less toxicity and ideal concentration for the detection of biodistribution of QDs as reported in the literature.39-42 QDs were intraperitoneally injected to respective models and blood was drawn after 0, 30, 60, 90, 120 and 180 mins. Table 2 represents the half-life of glyco-QDs. It could be seen that QDs coated with galactose exhibited a rapid elimination from the bloodstream in both models, while the sialylated QDs exhibited prolonged circulation in the bloodstream. This result correlates with pharmacokinetic studies of polysialic acid and other Sia conjugated therapeutic molecules blood circulation results.44-48 The half-life (t1/2) calculated for Q-1 was 105 and 82 mins in mouse and zebrafish model respectively (Fig 2c, 2d & Table 2). The prolong blood circulation of Q-1 attributes to the fact that Sia moiety on the QDs binds to siglecs receptors to immune cells and increases circulation time and slowly enter in organs.49 while, Q-2 to Q-4 cleared from the bloodstream much faster in both the models. Next, we investigated the biodistribution of glyco-QDs in the zebrafish and mice model. QDs were intraperitoneally injected into the zebrafish and mouse model and after 1, 3 and 24 h, fishes and mice were sacrificed, dissected and organs were collected. In mice, six vital organs such as liver, kidney, lung, heart, brain were collected, whereas in zebrafish, we managed to collect four organs such as liver, heart, brain and spleen, as the size of fish is tiny to dissect. In the mouse model, Q-4



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rapidly cleared from the blood and sequestered in liver and kidney after 1h, due to asialoglycoprotein receptors-galactose interactions.50 ICP-MS analysis after 3h revealed the substantial amount of Q-4 accumulation in the liver compared to other organs. These results are correlated to previous galactose-QDs results in the mouse model.38 In case of zebrafish model, similar biodistribution mechanism was observed in liver, and after 24 h, Q-4 was completely cleared from most of the organs. Sialylated-QDs have longer blood circulation half-life compared to Q-4, found to be sequestered in several organs. Among them, α(2-6) conjugated QDs (Q-1) showed significantly broad biodistribution compared to Q-2 and Q-3 (Fig 3). The probable reason behind the broad biodistribution is may be due to the binding affinity of S-1 in the blood components. In the zebrafish model, a similar trend was observed, although it was less pronounced compared to the mouse model. Q-1 treated zebrafish showed the nearly 1.3-fold maximum QDs sequestration after 3 h compared to that of 1 h. While, in the mice model, nearly 2-fold increase the Q-1 sequestration was observed at different organs after 3 h. After 24 h, Q-1 sequestration was found in liver and traces of QDs were in other organs. In contrast, Q-2 was readily uptake by the liver, compared to other organs and slowly cleared by both the system. While Q-3 was biodistributed in the kidney and traces of it was found in other organs. The sequestration of the sialylated-QDs in the spleen is correlated to the binding affinity of QDs with blood components, as spleen is considered to be a reservoir for blood compositions. While the sequestration in liver illustrates the stability of Sia on QDs to form galactose residue which binds to asialoglycoprotein receptor in hepatocyte cells in the liver.38 The least sequestration of galactose-QDs in heart and lungs in mice demonstrated the absence of asialoglycan receptors. The slow and steady brain sequestration of Q-1 and Q-2 illustrated the potential property of Sias to penetrate the blood-brain barrier (BBB) compared to Q-3 and Q-4.51-53 All mice results correlates to literature evaluation. For


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example, Tanak et al. tested the Gd(III) complexes of α(2-6) and α(2-3) sialylated N-glycans. They found the multivalency and α(2-6) exhibited broad biodistribution.54 By using sialyl-lewis

X

conjugated QDs, Ohyanagi et al. showed that α(2-3) sialylated-nanoparticles appeared to be broad biodistribution at initial hours and later sequestered in the liver.55 Thus, the majority of the mice results seem to be comparable to literature results, and the zebrafish results can be considered a suitable alternative to the mice model. After quantitatively analyzing glyco-QDs biodistribution in both models, it was qualitatively supported by confocal images of the tissue sections. We performed paraffin embedded tissue sectioning and QDs sequestration Q-1 and Q-2, as they have distinct biodistribution after 3 h in both the models (Fig 4). The inherent optical property of QDs was used to visualize the sequestration in the tissue section. As seen in Fig 4, the Q-1 was widely sequestered in the cardiac muscle tissues in the heart, hepatocyte and vacuoles of the liver region, the periarteriolar lymphoid sheath of spleen region and cortex of brain region in both models respectively. In addition, pulmonary fibrosis tissue of lung region and glomerulus structure of kidney region in mice model. While, Q-2 sequestration was observed in liver, and brain regions in both model and kidney region in mice model, which further support the ICP-MS data. Overall, all these results support that the zebrafish can be used as a simple and alternative in-vivo model to study the carbohydrate-mediated interactions. Based on the in vivo screening experiments, we examined the long-term toxicity of QDs by measuring the fluctuating body weight in the mice and zebrafish model. QDs (2.5 nmol/kg) were i.p. Injected and the fluctuation in the body weight was monitor over a period of 20 days (Fig 5a & 5b). The body weight of the control and QDs injected mice were approximately identical, illustrating that the QDs had no side effect on the mice model at low concentration. Overall, the



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comparative studies displayed the zebrafish could be a potential in-vivo model for glyconanotechnology research. Conclusion In this study, for the first time, we have reported the head-to-head comparison of two distinct sialylated-QDs toxicity, biodistribution, and sequestration in mouse and zebrafish model. The serum analysis of the α(2-6) Sia-conjugated QD showed long-term blood circulation in both the models compared to other glyco-QDs. Biodistribution results of both the model systems revealed that most of the QDs were sequestered in the liver, with quick clearance was observed in zebrafish compared to that of the mouse model. Furthermore, Q-1 and Q-2 in both the model are capable to sequester in brain by passing the BBB. These results advocate zebrafish as an ideal simple in vivo system for preliminary screening carbohydrate-mediated interactions and thus opening up the immense possibility for the successful application of carbohydrate-based drug delivery and imaging studies in quick time intervals. Materials. Synthesis of Q-1 to Q-4. Glyco-conjugation of QD has been done via peptide coupling method, following Thermo Fisher Scientific procedure.30 In brief, in borate buffer (50 mM, pH- 7.4, 0.5 ml) carboxylic acid-quantum dots (0.1 µM) were dissolved. To this solution EDC (10 µM) and Nhydroxyl succinimide (12 µM) was added and stirred at RT for 2 h, followed by the addition of oligosaccharides (S-1 to S-4) (10 µM) at room temperature. After overnight stirring, the reaction mixture was quenched with ethanolamine, followed by dialysis of the mixture with 10,000 cut-off membranes in water. Finally, the product was lyophilized and diluted to desired concentration. The concentration of the QDs was established using a previously published procedure.31 QDs were


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dissolved in phosphate buffer (50 mM at pH 7.4) and used as such for further biological experiments. Estimation of the concentration of sialic acid per QDs. Sugar capped QDs (20 nmol) was dissolved in 100 µL of doubled distilled water. To this solution, resorcinol reagent (100 µL) was added and heated to 80 °C on a water bath for 15min.32 The reaction mixture was extracted with nbutyl acetate and n-butanol (85:15) mixture. The organic layer separated and

quantify by

measuring the absorption intensity at 580 nm by UV-spectrometer. The sialic acid and galactose concentration was determined by plotting a calibration curve of commercial sialic acid-resorcinol derivatives obtained by the same method. The ratio of the concentration of sialic acid ligand to concentration of the QD was use to evaluate the number of sialic acid moeity per QD. Q-1 to Q-3 showed approximately 53, 47 and 56 sialic acid residues per each particle respectively. Phenolsulfuric acid method was use to quantify the concentration of galactose in case of Q-4.33 Q-4 showed 78 galactose residue per each particle. Characterization of Glyco-QDs. UV-visible spectra was recorded by Evolution 300 UV-visible spectrophotometer (Thermo Fisher Scientific, USA). Fluorescence spectra of glyco-QDs were quantified by FluoroMax-4 spectrofluorometer (Horiba Scientific, U.S.A.). Transmission electron microscopy (TEM) images were collected by CM 200, operated voltage 100 kV resolution 2.4 Å. Zeta potential analysis was quantified by applying unit field strength (1 Volt per meter) to the QD solution. Mouse model. All animal studies were performed in accordance with INTOX toxicology service, Pune ethical committee regulation. Male and female C57BL mice (3-4 weeks old) were collected from Reliance life science, Bombay. Prior to the experiment, the mice were maintained in the



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animal house with proper food and water supply. The surgical procedures were performed as per Animal Ethical Committee regulation, set up by CPCSEA, Govt. of India. Zebrafish model. Local wild-type zebrafish strain of 2-3 months old were maintained under standard IISER, Pune laboratory conditions of the water maintained at pH 7.2- 7.4. Acute toxicity determination. Mice were first anesthetized with ketamine and glyco-QDs were intraperitonially injected at different concentration with 4 fish per glyco-QDs. The highest dose of glyco-QDs used was 20 nmol/kg. LD50 value was calculated according to the equation. LD50 = lg-1{Xm-i × Ʃp-0.5} --1 where Xm represents logarithm of the maximal dose, i corresponds to the difference between the logarithm values of two adjacent doses, and P is the mortality of each group. In case of zebrafish, fishes were first anesthetized with 2-phenoxyethanol and glyco-QDs were intraperitonially injected. The number of zebrafish in each experimental and control group was four per sample.

Blood sample analysis. Mice were anesthetized with ketamine and received 7 nmol/Kg of quantum dots (Q-1 to Q-4). The number of mice for each experimental and control group was 3 per sample. After 0, 30, 60, 90, 120 and 180 mins. Mice were sacrificed, and 0.5 ml of blood was drawn. Collected blood samples were digested with 70% nitric acid (500 µl) at 90°C for 30 min. Then each samples were diluted to 6 ml milliQ water. The concentration of QDs in the blood samples was determined by ICP-MS (Thermo-Fisher Scientific, Germany) by quantifying the cadmium concentration. Finally, the concentration of cadmium was converted into µg/mg. Zebrafish were anesthetized and injected 7 nmol/Kg (Q-1 to Q-4) via intraperitoneal injection. The number of fish for each experiment was 3 per sample. After 0, 30, 60, 90, 120 and 180 mins zebrafish were sacrificed and its tail was cut to collect the blood sample (~0.1ml). Collected blood 11 ACS Paragon Plus Environment

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samples were digested, diluated with milliQ water and finally QDs concentration of quantified by ICP-MS. Finally, the concentration of cadmium was converted into µg/mg. Mouse dissection. Mice were received 7 nmol/Kg of quantum dots (Q-1 to Q-4) via i.p injection. The number of mice for each experiment was 3 per sample. After 1h, 3h and 24 h mice were sacrificed and organs were collected. Before dissection, mice were anesthetized using ketamine, followed by pinning down with belly facing up and washed with ethanol. Then dissection was done along the ventral midline starting from the groin up to the chin to expose the organs. Different organs (liver, spleen, lung, kidney, heart and brain) were collected and washed with PBS buffer (100 mM, pH 7.4) followed by fixation in formalin (5%) solution. These samples were taken as such for further analysis (ICP-MS and confocal imaging). ICP-MS analysis. Organs were dried and weighed before digesting it in 3 ml of 70% nitric acid at 90 °C for 3 h. Further samples were diluted with 6 ml using milliQ water and filtered through the membrane filter before subjected to ICP-MS analysis. Confocal imaging.

In case of confocal imaging studies, organs were subjected gradient

dehydration with increasing ethanol concentration (75%, 95%, 100%) for 30 min each. Finally washed with xylene for 1 h followed by fixing in paraplast. Blocks are stored in -10 °C for 12 h before proceeding to sections. Leica microtome used for sectioning and sections were collected on PLL-coated glass plates. Before imaging excess paraplast was washed with xylene. Sequestration of glyco-QDs in different organs was analyzed by confocal fluorescence microscopy. The excitation wavelength 480 nm, emission wavelength 680 nm was employed to collect images. Zebrafish dissection. For dissection of organs, zebrafish were anesthetized with 2-phenoxy ethanol in water (1: 2000). The number of zebrafish for each experiment was 3 per sample. Both head and tail of the fish were fixed using needles and body wall was cut at the abdomen and cut until the



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operculum to expose all internal organs. Different organs such as heart, brain, liver and spleen were collected. All organs were washed with Millipore water, dried and weighed before following the above procedure for ICP-MS and confocal slides preparation. Long-term toxicity evaluation. After i.p. administration of Q-1 and Q-2 at 2.5 nmol/kg, the longterm toxicity test was performed by measuring body weight of the male mice and zebrafish. The number of mice and zebrafish used for each experiment and control is 3 per sample. Body weights of the mice and zebrafish weight in both groups were recorded for next 20 days. Statistical analysis. All statistical analysis was done using the Student t test or one-way ANOVA. The p < 0.05 is considered to be statistical significance. AUTHOR INFORMATION Corresponding Authors [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Acknowledgements. ‡ Financial support from the IISER, Pune, Max-planck partner group and DST-Nano is gratefully acknowledged. P.M.C acknowledgment DBT Bio-CARe for research grant, S.T and B.S thanks CSIR-SRF for supporting their fellowships. Special thanks to Prof. N. K. Subhedar and Dr. Aurnab Ghosh for helping to setup zebrafish experiments. Supporting Information. Experimental details, synthesis, 1H- and 13C-NMR, mass spectra of all new compounds and representative dark field images, fluorescent confocal and ICP-MS analysis.


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COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

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Gene Expression and Biochemical Responses in Brain of Zebrafish Danio Rerio



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Scheme 1. Synthesis of Q-1 to Q-4 nanoparticles. Reagents and Conditions: (l) S-1 or S-2 or S-3 or S-4, EDC, N-hydroxysuccinimide, ethanolamine, H2O.



(a)

(c)

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(b)

(d)

Figure 1. TEM images of carbohydrate conjugated quantum dots: Q-1 (a); Q-2 (b); Q-3 (c); Q-4 (d). Scale bar = 100 nm.


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QDs Q-1 Q-2 Q-3 Q-4 QDCOOH

Quantum Yield (ϕ) 0.37 0.36 0.39 0.44 0.32

Number of Sugar unit per QD 53 ± 4 47 ± 6 56 ± 3 78 ± 4 -

Zeta potential (mV) -12.5 ± 1.8 -10.7 ± 1.9 -13.3 ± 2.4 +0.78 ± 0.3 -24.1 ± 3.2

Table 1. Physical characteristics of QDs.



150

150

(a)

Q-1

Q-2

Q-1

(b)

Q-3

100

50

Q-3

100

50

0

0 0

5

10

15

0

20

5

10

15

20

Conc (nmol)

Conc (nmol)

(c)

Q-2 Q-4 Control

Control

Mortality %

Q-4 Mortality %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(d)

Figure 2. Mortality of adult zebrafish (Danio rerio) (a) and mice (b) after 24 h of glyco-QDs i. p injection; blood clearance profile of glyco-QDs in zebrafish (c) and mice (d).


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QDs Q-1 Q-2 Q-3 Q-4

LD50 (nmol/kg) Mice 13.6 11.7 9.6 9.3

LD50 (nmol/kg) Zebrafish 14.7 11.4 9.51 9.6

t1/2 (mins) Mice 105 ± 2 51 ± 3 48 ± 3 21 ± 2

t1/2 (mins) Zebrafish 35.5± 3 25.2 ± 4 21.6 ± 6 17 ± 4

Table 2. LD50 and t1/2 of the QDs in animal model.



40

Liver

Kidney

Lung

Heart

Brain

Spleen

(b) Conc of Cadmium (µ g/mg)

Conc of Cadmium (µ g/mg)

(a)

30 20 10 0 Q-1

Q-2

Q-3

Kidney

Heart

100

Lung

Brain

Spleen

80 60 40 20 0 Q-1

Q-2

Q-3

Q-4

(d) 8

Heart

Lung

Kidney Brain


Liver

Spleen

6 4 2 0 Q-1

(e)

Q-2

Liver 60

Q-3

Spleen

20

0 Q-2

Q-3

Heart

Brain

Spleen

20 10 0 Q-1

40

Q-1

Liver

30

(f)

Heart

Brain

40

Q-4



Liver

Q-4

(c)


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Q-4

5

Q-2

Liver

Heart

Brain

Spleen

Q-3

Q-4

4 3 2 1 0 Q-1

Q-2

Q-3

Q-4

Figure 3. Statistical analysis of ICP-MS biodistribution data of quantum dots at different time intervals. (a) Mouse-1 h; (b) Mouse-3 h; (c) Mouse-24 h; (d) Zebrafish-1 h; (e) Zebrafish-3 h; (f) Zebrafish- 24 h. Results are expressed as mean ±SD.


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(a)

(b)

Figure 4. Confocal images of QD sequestration at different organs of (a) mouse and (b) zebrafish model.



600

Control

Q-1

(b)

Q-2

550 500 450 400 350

50

Mouse weight (g)

(a) Zebra fish weight (mg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0

5

10

15

20

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Control

Q-1

Q-2

40 30 20 10 0

0

Time (day)

5

10

15

20

Time (day)

Figure 5. Changes in the body weight of the zebrafish (a) and mice (b) after i.p. administration of Q-1 and Q-2 at 2.5 nmol/kg for 20 days.


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Table of Contents Graphic


2 Abstract Sialic acid (Sia) conjugated nanocarriers have emerged as

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