Tissue Plasminogen Activator-Porous Magnetic Microrods for

Subscriber access provided by UNIV OF WESTERN ONTARIO

Biological and Medical Applications of Materials and Interfaces

Tissue Plasminogen Activator-Porous Magnetic Microrods for Targeted Thrombolytic Therapy after Ischemic Stroke Jiangnan Hu, Shengwei Huang, Lu Zhu, Weijie Huang, Yiping Zhao, Kunlin Jin, and Qichuan Zhuge ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09423 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 8, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 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

ACS Applied Materials & Interfaces

1

Tissue Plasminogen Activator-Porous Magnetic Microrods for Targeted

2

Thrombolytic Therapy after Ischemic Stroke

3 4 5 6

Jiangnan Hu1,2*, Shengwei Huang1*, Lu Zhu4, Weijie Huang5, Yiping Zhao5†, Kunlin Jin1,2,3†, Qichuan ZhuGe1† 1

7 8 9 10 11 12 13 14 15 16 17

Zhejiang Provincial Key Laboratory of Aging and Neurological Disorder Research, Department of Neurosurgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000, China 2 Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas 76107, USA 3 Beijing Key Laboratory of Hypoxic Conditioning Translational Medicine, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China. 4 College of Engineering, University of Georgia, Athens, Georgia 30602, USA 5 Department of Physics and Astronomy, Nanoscale Science and Engineering Center, University of Georgia, Athens, Georgia 30602, USA

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

*

These authors contributed to the work equally.

†

Corresponding Author:

Dr. Yiping Zhao, Department of Physics and Astronomy, University of Georgia, Athens, GA 30602, USA. Tel: 706 542 7792; Fax: 706 542 2492; Email: [email protected] Dr. Kunlin Jin, M.D., Ph.D., Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas 76107, USA Tel: 817-735-2579, Fax: 817-735-0408. E-mail: [email protected] Dr. Zhuge Qichuan, Zhejiang Provincial Key Laboratory of Aging and Neurological Disorder Research, Department of Neurosurgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, 325000, China, Tel: 8655755578085; Fax: 86 577 88069607; Email: [email protected]

17 18

KEY WORDS: Tissue plasminogen activator, microrods, stroke, ischemia, thrombolysis

19

1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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

1 2

ABSTRACT

3 4

Tissue plasminogen activator (tPA) is the only FDA approved thrombolytic drug for acute

5

ischemic stroke but concerns regarding its limitations remain. Here, we developed a new strategy

6

by incorporating tPA into porous magnetic iron oxide (Fe3O4)-microrods (tPA-MRs) for targeted

7

thrombolytic therapy in ischemic stroke induced by distal middle cerebral artery occlusion. We

8

showed that intra-arterial injection of tPA-MRs could target the cerebral blood clot in vivo under

9

the guidance of an external magnet, where tPA was subsequently released at the site of embolism.

10

When applied with an external rotating magnetic field, rotating MRs significantly improved not

11

only the mass transport of the tPA-clot reaction, but also mechanically disrupted the clot network,

12

which thus increased clot interaction and penetration of tPA. Importantly, intravenously injected

13

MRs could be discharged from the kidney, and the function of liver and kidney were not damaged

14

at different durations after administration of tPA-MRs. Our data suggest that tPA-MRs overcome

15

the limitations of thrombolytic therapy with tPA alone, which may be not only just for the

16

treatment of ischemic stroke but also have majorly impact on other thrombotic diseases.

17 18 19 20 21 22

2


Page 2 of 36

Page 3 of 36 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

1


INTRODUCTION

2 3

Tissue plasminogen activator (tPA) is the only FDA approved thrombolytic drug for patients

4

with acute ischemic stroke for over a decade 1-2. However, because of its short therapeutic window,

5

low specificity and high risk of intracranial hemorrhage, only small population of stroke patients

6

(1-2%) can benefit from tPA. Therefore, stroke remains the leading cause of disability in the world

7

3-5

.

8

To further improve thrombolysis and recanalization rates, several controlled delivery methods

9

have been developed recently 6. For example, highly biocompatible and efficient encapsulation

10

liposomes have been used for tPA delivery, which can prolong circulation of tPA. However, the

11

poor stability of liposome in the blood flow as well as inefficient targeting limited their application

12

in clinical setting

13

targeted therapies 9-10, as nanocarriers allow the drug to reach a desired target at a relatively high

14

concentration, and thus improve the specificity of drug. This technology has been used for

15

therapeutics in cancer 11-13, neurodegeneration diseases 14-15 and central nervous system infection

16

16-17

17

mediated thrombolysis in femoral embolism mouse model 18. However, nickel is toxic, and thus

18

limited its clinical application. Superparamagnetic iron oxide (Fe3O4) is a promising candidate for

19

biomedical applications due to its biocompatible property and strong magnetic response, and has

20

been widely used as contrast enhancing agents for magnetic resonance imaging

21

diagnosis 21, and magnetically guided drug delivery

22

can be covalently loaded onto the Fe3O4 nanomaterials for target delivery

23

vitro study showed that tPA-loaded Fe3O4 nanorods significantly enhanced thrombolysis

7-8

. Recent developed magnetic nanocarriers have received much attention in

. Using similar technology, we developed the magnetic nickel nanorods to enhance tPA-

3

22-24

19-20

, magnetic

. Several studies have reported that tPA


25-27

. Our previous in


Page 4 of 36

1

efficiency in comparison with high-dose tPA 22. However, the challenge is that these ferromagnetic

2

Fe3O4 nanorods could aggregate in vitro, limiting its application in vivo. In this study, we developed new nanoporous, superparamagnetic-like Fe3O4-C microrods (MRs)

3

22, 28-29

4

that encapsulated with tPA more efficiency, compared with our previous protocol

. The

5

resulting tPA-Fe3O4-C MRs (tPA-MRs) could target the blood clot occluded in the middle cerebral

6

artery (an ischemic stroke model) in mice under the guidance of an external magnet, which

7

dissolved clots via both tPA (chemical lysis) and rotating MRs (mechanical lysis) with aid of an

8

external rotating magnetic field. With much less concentration of tPA on tPA-MR injection (0.13

9

mg/kg) in a mouse stroke model, it took less than 1/3 time to lyse the blood clot, compared to that

10

of tPA injection alone (10 mg/kg). Importantly, injected tPA-MRs were not harmful to liver and

11

kidney function, excreted from major organs, and detected in urine. Our data suggest that tPA-

12

MRs overcome the limitations of thrombolytic therapy with tPA alone, which may be a promising

13

approach for the treatment of ischemic stroke and other thrombotic diseases.

14 15

MATERIALS AND METHODS

16 17

Fabrication of the Fe3O4-C MRs

18

All materials were used as received without any further purification. The MRs were fabricated

19

through a solvothermal method

20

glucose were dissolved in 75 ml ethylene glycol (EG) firstly. Then the mixture was transferred

21

into a 100 ml Teflon-lined stainless-steel autoclave and kept at 220 °C for 12 h. After cooling

22

down to room temperature, the precipitates were washed with absolute ethanol twice then dried in

23

an oven overnight. The final product Fe3O4-C microrods were then obtained by annealing the dried

29

. In a typical synthesis, 0.7575 g Fe(NO3)3·9H2O and 0.5 g

4


Page 5 of 36 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

1


powders in N2 flow carried ethanol at 350 °C for 1 h.

2 3

Bioconjugation of tPA on MRs

4

The Fe3O4-C MRs were dispersed in ethanol/water mixture at a volume ratio of 4:1. Then, 3-

5

aminopropyltriethoxysilane (APTES) and dimethyl- formamide were added into the solution until

6

the final concentration of each was 5% by volume. The solution was shaken for 2 h at room

7

temperature in order to functionalize the surface of the Fe3O4-C MRs with amine groups. The

8

amine-derived Fe3O4-C MRs were then separated from the solution using a magnet and washed

9

with phosphate-buffered saline (PBS) solution 3 times. After washing, the MRs were re-dispersed

10

in PBS solution, mixed with 0.5% glutaraldehyde and shaken at 30 °C for 30 min, then followed

11

by washing 3 times with PBS. Finally, the glutaraldehyde-modified MRs were mixed with tPA

12

solution with a concentration of 0.5 mg/ml at 4 °C for 12 h to obtain the tPA-immobilized Fe3O4-

13

C MRs. The loading ratio of tPA onto the MRs was determined by a PierceTM BCA protein assay

14

kit from Thermal Scientific to measure the concentrations of unbound tPA in the supernatants.

15

Both of the physical and chemical tPA loadings were measured. The amidolytic activity of tPA

16

was measured with the chromogenic subtract S-2288TM (Chromogenix, no. 82085239). Three

17

types of solutions were tested: different concentrations of tPA-MRs (stored at -20 ℃ for 6 months)

18

and washed three times by normal saline; the supernatants of different concentrations of tPA-MRs

19

dispersed in normal saline, rotated by a magnetic field for 60 min at room temperature, and then

20

collected; and freshly prepared tPA solutions. All specimens were diluted to 1:50 with water. Each

21

solution (50 µl) was pipetted in triplicates into a 96-well cell culture plate and mixed with 1 mM

22

assay buffer (S-2288TM in 0.1mM Tris-HCl pH8.4). The absorption A was recorded at 405 nm in

23

a spectrophotometer (FilterMax F5, Moleccular Devices, Sunnyvale, CA, USA) and the kinetic

5



Page 6 of 36

∆ %

1

activity η was determined as the linear slop of ΔA = A(t) – A(0) versus test time t, η =

2

The activity retention for tPA-MRs and the supernatants was determined as the ratio of their kinetic

3

activity to the kinetic activity of corresponding concentration of free tPA solution. For instance,

4

the activity retention for 5 mg/ml tPA-MRs was determined as the ratio of the kinetic activity of 5

5

mg/ml tPA-MRs in normal saline (tPA loading ratio = 12.9%) divided by the activity of 0.65

6

mg/ml free tPA. The experiments were performed in triplicate, and the results were averaged.

&

.

7 8

Characterizations of the magnetic Fe3O4-C MRs

9

Morphologies of the samples were investigated by a field-emission scanning electron microscope

10

(FESEM) equipped with an energy dispersive X-ray spectroscopy (FEI Inspect F). The crystal

11

structures of all the as-prepared samples were characterized by an X-ray diffractometer (XRD;

12

PANalytical X’Pert PRO MRD) with a Cu Kα source (λ = 1.5405980 Å) at 45 kV and 40 mA.

13

Transmission electron microscopy (TEM) analysis was carried out using a Hitachi HF-3300

14

TEM/STEM at 300 kV to further investigate the morphologies of Fe3O4–C microrods. Magnetic

15

properties were measured at room temperature by a vibrating sample magnetometer (VSM, Model

16

EZ7; MicroSense, LLC, Lowell, MA, USA) with a 2.15 T electromagnet. The magnetization of

17

the sample was measured over a range of applied fields from −15 to +15 kOe.

18 19

Sonication of the tPA-MRs

20

After sonication, magnetic tPA-MRs homogeneously dispersed in normal saline solution and

21

remained for at least 2 h (the longest time examined). To test the effect of sonication on their ability

22

to pass through the small capillaries, mice were injected with sonicated or unsonicated tPA-MRs

23

separately, and then sacrificed at 2 h later after injection and lung samples were collected for

6


Page 7 of 36 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


1

histology. Magnetic clusters were observed blocking the lung capillaries in the unsonicated tPA-

2

MRs injection group unlike the sonicated group (Fig. S4).

3 4

FeCl3-induced distal middle cerebral artery occlusion mouse model

5

Animal experiments were conducted according to National Institutes of Health guidelines after

6

approval by the University of North Texas Health Science Center Animal Care and Use

7

Committee. Enhanced thrombolysis by active tPA-MRs was demonstrated in FeCl3 induced

8

dMCAO mouse mode (CD1-IGS mouse, male, 2 months old) 30. Each CD1 mouse was initially

9

anesthetized with 2-3% isoflurane and maintained with 1% isoflurane in a mixed air of 70% N2O

10

and 30% O2 by inhalation. Mice were placed in a stereotaxic apparatus, and a craniotomy was

11

carried out over the left hemisphere. The scalp was opened, and the temporal muscle was bluntly

12

dissected until the squamous part of the temporal bone was exposed. The skull area above the

13

junction between the zygomatic arch and the squamous bone was thinned using a high-speed drill

14

and cooled with saline. The MCA was visualized through the thinned skull. Attention was paid not

15

to damage the MCA and the overlying dura mater while the remaining thin bony film was lifted

16

up by forceps. At the same side of the ischemia brain, separated and blocked the blood flow of the

17

common carotid artery (CCA), followed with observation of the blood flow of the distal middle

18

cerebral artery (dMCA) under a microscope, a small piece of 10% FeCl3-saturated filter paper

19

(sterile, thickness ≈ 0.18 mm, surface ≈ 0.5 x 0.3 mm2) was placed over the intact dura mater along

20

the trace of the dMCA right after the zygomatic arch for 1 min. Then the strip was removed and

21

washed with normal saline. After formation of the visible thrombus in dMCA, recovered the blood

22

flow of both sides of CCA immediately. The infarct area was established by 2,3,5-

23

triphenyltetrazolium hydrochloride (TTC) staining at 24 h later after induction of ischemic stroke.

7



1

To further confirm the stability of this dMCAO mouse model, the quantitation analysis of blood

2

flow was measured by using Laser-Doppler Flowmetry (measure time period was 15 min) before

3

and after the formation of thrombus (Fig. S6).

4 5

tPA-MRs and tPA injection in vivo

6

To determine whether tPA-MRs can accelerate thrombolysis in vivo, animals under anesthesia

7

were treated with tPA-MRs in normal saline (1 mg/kg), MRs in normal saline (1 mg/kg), tPA

8

solution (10 mg/kg) or normal saline via internal carotid artery injection (50 µl in total) at 30 min

9

after ischemic stroke. The rationality for dose selection of tPA, MRs and tPA-MRs for this study

10

was discussed in Supporting information (Part S8). Animals were randomly assigned to four

11

groups: 1) dMCAO group treated with NS, 2) dMCAO group treated with tPA, 3) dMCAO group

12

treated with MRs, 4) dMCAO group treated with tPA-MRs. The sham-operated mice involved

13

same surgeries, without vessel occlusion. tPA-MRs were stored in 0.5 mg/ml tPA solution. Right

14

before injection, the tPA-MRs were centrifuged and washed with normal saline three times to

15

completely remove the free tPA. To inject the drugs, the right external carotid artery (ECA) and

16

internal carotid artery (ICA) were separated; the blood flow of distal ECA was blocked with 8-0

17

string, slipknots were put on ECA, ICA and CCA, then a catheter containing drugs (Infusion

18

Technologies, MTV-02) was inserted into ECA followed by opening a small window on the

19

surface of the artery (Fig. 1D). Then, blocks of magnets (K&J Magnetics, BX8C4-N52) were

20

placed besides the dMCA on the ischemic side of mouse, the distance between the edge of magnets

21

and the blood clot site should be less than 1 cm (magnetic field strength = 0.24 T), the plane of

22

magnets should be perpendicular to dMCA (Fig. 1A, B). The magnetic field strength of the

23

magnets at different distances was measured by a Gauss meter (Fig. 1C). After making sure the

8


Page 8 of 36

Page 9 of 36 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


1

magnets are on the right position, injected solution via ICA. After injection, pulled out the catheter

2

and tied up ECA. Once MRs were targeted to thrombus site successfully, magnets were removed.

3

The mouse was then placed into a magnetic field (Fig. 1E), a custom-made rotational magnetic

4

field (20 Hz) with strength of 40 mT was applied above the head of each mouse (near infarction

5

region) for 120 min under anesthesia, and the thrombolysis process was observed through

6

microscope every 5 min until to 120 min. Laser-Doppler test of dMCA blood flow was recorded

7

before and after formation of thrombus in dMCA, and every 30 min until to 120 min during the

8

recanalization process. After surgery, the skin was closed with non-absorbable sutures. Brains

9

were removed 24 h after ischemic stroke and 2-mm coronal sections were stained with TTC.

10

Infarct area and total brain area were measured by a blinded observer using the NIH Image program,

11

and areas were multiplied by the distance between sections to obtain the respective volumes.

12

Infarct volume was calculated as a percentage of the volume of the contralateral side of brain, as

13

described previously 31.

14

9



1

Figure 1. The schematic images of tPA-MRs injection and surgical procedure. (A) Magnets

2

were placed on the right side of mouse’s head. After injection of solution, magnets were put close

3

to blood clot, keeping the distance between blood clot and the edge of magnets less than 1 cm. (B)

4

Schematic representation of tPA-MRs travel through ICA to MCA under the guidance of magnets.

5

(C) The magnetic field strength of the magnets used for MRs or tPA-MRs guidance at different

6

distances. (D) Injection route of ICA in dMCAO mouse model. Explosion of CCA, ECA and ICA

7

(left panel). Occlusion of ECA blood flow (middle panel). Injection of tPA-MRs solution through

8

ICA (right panel). (E) During thrombolysis process, the head of mouse was placed in an extra

9

rotational magnetic field (20 Hz, 40 mT). CCA: Common carotid artery; ECA: external carotid

10

artery; ICA: internal carotid artery; PPA: pterygopalatine artery; MCA: middle cerebral artery.

11 12

Toxicity analysis of the tPA-MRs in vivo

13

The impacts of tPA-MRs on liver and kidney function was evaluated in this assay. Four biomarkers,

14

serum alanine transaminase (ALT), aspartate aminotransferase (AST), albumin (ALB) and serum

15

malondialdehyde (MDA), were quantified by using enzyme-linked immunosorbent assay (ELISA)

16

kits (Boyun, Shanghai, China). Two biomarkers, nitrogen (BUN) and creatinine (Cr), for kidney

17

function were examined respectively by using BUN and Cr assay kits (Jiancheng Bioengineering,

18

Nanjing, China). All the procedures were operated according to the manufacturer's instructions.

19

The blood samples were collected at seven different time-points (1h, 3h, 6h, 12h, 24h, 3d and 7d)

20

after tPA-MRs (10 mg/kg, i.v.) administration, and NS groups were set as a parallel control at each

21

corresponding time.

22 23

Distribution and clearance of the tPA-MRs in vivo

10


Page 10 of 36

Page 11 of 36 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


1

1) Distribution of the tPA-MRs in major organs. Twelve CD-1 mice were randomly divided

2

into three groups (control group, 1 h and 24 h groups, each group n = 4) and intravenously injected

3

50 µl of normal saline (control group) or tPA-MRs solution (1 mg/kg). At t = 1 and 24 h after the

4

injection, the mice were sacrificed by decapitation after isoflurane 4% inhalation. Brain, liver,

5

spleen, kidney, and lung samples were collected for tPA-MRs distribution study. Tissues were

6

embedded in optimal cutting temperature compound (OCT), section at 20 µm and stained with

7

methyl green solution (Vector, H-3402) using standard techniques for histopathological

8

examination. Slides were rinsed in tap water, then stained with Methyl Green solution and

9

incubated slides at 60 oC for 3 minutes; After incubation, slides were rinsed with deionized water

10

until rinse water was clear. The slides were then immediately dehydrated through 95% and 100%

11

ethanol, air dried, and permanently mounted. Images were acquired by using Nikon Eclipse Ti-U

12

microscope.

13

2) Urinalysis examination of the tPA-MRs. tPA-MRs (1mg/kg) were intravenously injected into

14

mice. After injection, mice were placed into metabolic cage and 24-hour urine was collected.

15

Drops of urine were then pipetted on a glass slide and observed under the microscope for the

16

presence of MRs. The secreted MRs were further confirmed via magnetic separation by a

17

permanent magnet.

18 19

Statistical analyses

20

Prism statistical analysis software was used. Continuous variables were expressed as mean ± SEM.

21

Statistical analysis was performed by the two-tailed Student’s t-tests. Multiple groups were

22

compared using the analysis of variance (ANOVA) with pairwise multiple comparison where

23

appropriate. Significance was declared as P < 0.05.

11



1 2

RESULTS

3 4

Characterizations of the tPA-loaded Fe3O4-C MRs

5

The size and morphology of the Fe3O4-C MR microstructure were determined using scanning

6

electron microscope (SEM), scanning transmission electron microscope (STEM) and transmission

7

electron microscope (TEM). As shown in Fig. 2A, an individual Fe3O4-C MR featured rod-like

8

nanostructures with an average length of L = 1.3 ± 0.2 µm, and an average diameter of D = 0.5 ±

9

0.1 µm, which were assembled by smaller particles with nanoscale pores (Fig. 2B). TEM image

10

(Fig. 2C) displays that these small particles were formed by clusters of small grains with a size of

11

15 ± 3 nm. The X-ray diffractometer (XRD) pattern indicates the cubic crystalline structures of

12

Fe3O4 (PDF reference code: 01-089-0688) (Fig. 2D). The MRs had a saturated magnetization of

13

Ms = 39 emu/g and a remanent magnetization of Mr = 3 emg/g (Fig. 2E). Thus, the coefficient of

14

squareness, Kp ( K p =

15

that the MRs have a superparamagnetic-like property. The micro-hematocrit capillary tube

16

containing normal saline, which mimics in vivo capillary condition, was used to validate the

17

magnetic guided movements of tPA-MRs in vitro. We found that magnetic tPA-MRs were rapidly

18

moved to the targeted site, when the magnets were applied (Fig. S2). After chemical

19

immobilization, tPA was able to conjugate to the MRs with 12.9% loading ratio, which was better

20

than physical immobilization (Fig. 2F). The remaining activity of immobilized tPA on the MRs

21

were determined by the S-2288TM assay using equation (see Methods). Compared to pure and

22

diafiltrated tPA formation, the measurements of tPA-MRs (5 mg/ml, 2.5 mg/ml) showed an

Mr ), of the hysteresis loop was ~0.08, which was close to 0, suggesting Ms

12


Page 12 of 36

Page 13 of 36 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


1

activity retention of about 71% to 74% for initially added tPA concentration of 0.5 and 0.25 mg/ml

2

respectively (Fig. 2G, Fig. S3). Interestingly, the supernatant also exhibited a minimal activity

3

(1.2% ~ 4.4%), which further confirmed that immobilized tPA can be released from MRs under a

4

rotational magnetic field, and remains the enzymatic activity (Fig. 2G, Fig. S3). Meanwhile, the

5

morphology and crystallinity of MRs did not alter before and after tPA immobilization since the

6

immobilization only occurred at the surface of Fe3O4 MRs (Fig. S1). The in vitro cytotoxicity of

7

tPA-MRs was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)

8

cell viability assay, and no cell toxicity was found in HT-22 mouse hippocampal neuronal cell line

9

treated with the different concentration of tPA-MRs (20, 50, 100 and 200 µg/ml) (Fig. S5).

10

11 12

Figure 2. Characterizations of magnetic Fe3O4-C MRs. (A) SEM images of the Fe3O4-C

13

MRs. (B) STEM image of the Fe3O4-C MRs. (C) TEM image of the edge of a Fe3O4-C MR. (D)

14

The XRD pattern of the MRs. (E) The vibrating sample magnetometer curve of the MRs. (F)

13



1

UV-Vis spectra of tPA concentrations after binding. (G) Activity retention of immobilized tPA

2

on the Fe3O4-C MRs and supernatants in relation to corresponding free tPA measured with the S-

3

2288TM substrate. Shown are the mean values of n =3.

4 5

tPA-MRs-mediated thrombolysis after ischemic stroke

6

After craniotomy and ICA exposure, the blood flow of dMCA of the sham-operated group were

7

normal (Fig. S7), compared with the preoperative results (Fig. 3F). The tPA-MRs mediated

8

thrombolysis was assessed by using the distal middle cerebral artery occlusion (dMCAO) model

9

induced by topical FeCl3 application (Fig. S6), which blood clot was formed in the middle cerebral

10

artery. The blood flow of dMCA was significantly decreased due to the formation of blood clot

11

(Fig. 3F). As expected, no thrombolysis was observed in 120 min in the control group injected

12

with saline (Fig. 3A). When tPA at the concentration of 10 mg/kg was applied, the recanalization

13

occurred, and the blood flow was restored in 85 min (Fig. 3B, F). However, tPA could not

14

completely lysis the blood clot located in large arteries. The clot partially dissolved and traveled

15

to distal vessels and re-occluded small arteries (data not shown). We found that MRs were able to

16

rapidly target to the blood clot under the guidance of magnets. The MRs could penetrate the blood

17

clot when an external rotating magnetic field was applied, but could not achieve recanalization in

18

120 min (Fig. 3C, F). Yet, when the mice were treated with tPA-MRs (1 mg/kg; equal to 0.13

19

mg/kg tPA) after dMCAO, the blood flow was restored in 25 min (Fig. 3D, F), moreover, no

20

occlusion was observed in the distal bifurcation. The infarct volume of dMCAO mice treated with

21

tPA-MRs were significantly decreased compared with the tPA treated group (Fig. 3G, Fig. S9).

22

Our data suggest that much less tPA dosage (~ two orders of magnitude less) is required to achieve

14


Page 14 of 36

Page 15 of 36 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


1

high effective thrombolysis. In addition, the time for recanalization is significantly less, compared

2

to tPA-treated groups (Fig. 3E, F).

15



1

16


Page 16 of 36

Page 17 of 36 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


1

Figure 3. The tPA-MRs-mediated thrombolysis in a mouse model of stroke. (A) The

2

representative images of thrombolysis in dMCAO mouse treated with normal saline (NS). (B)

3

With the treatment of tPA (10 mg/kg), blood clot was lysed in 85 min after injection. (C) The

4

representative images of thrombolysis in dMCAO mouse treated with MRs (1 mg/kg) under a

5

rotational magnetic field (RMF, 20 Hz, 40 mT). (D) In tPA-MRs solution (1 mg/kg) group,

6

thrombus could be lysed in 25 min under a rotational magnetic field (RMF, 20 Hz, 40 mT). (E)

7

The plot of lysis time for recanalization in dMCAO mouse model with a 120 min observation

8

period under a rotational magnetic field (RMF, 20 Hz, 40 mT): Sham, NS, 1 mg/kg MRs solution

9

(MRs), 10 mg/kg tPA solution (tPA), and 1 mg/kg tPA-MRs in NS (tPA-MRs). *** P < 0.001 by

10

two-tailed Student’s t-test (n = 5). (F) Laser-Doppler test of dMCA blood flow in Sham-operated

11

mice and before and after formation of thrombus in dMCA, as well as the one during thrombolysis

12

with a 120 min observation period: NS, 1 mg/kg MRs solution (MRs), 10 mg/kg tPA solution

13

(tPA), and 1 mg/kg tPA-MRs in NS (tPA-MRs). The blood flow of dMCA was significantly

14

decreased due to the formation of blood clot, recanalization of dMCA blood flow was achieved by

15

tPA and tPA-MRs in NS groups. PU: perfusion units. *** P < 0.001, ** P < 0.01 and *** P < 0.05,

16

tPA-MRs in NS group vs tPA group, by two-tailed Student’s t-test (n = 4). (G) Ischemic infarct

17

volumes were quantified at 24 h after stroke by TTC staining. Data are expressed as mean ± SD,

18

analyzed by one-way analysis of variance (ANOVA) followed by individual comparisons of

19

means (n = 4~7 mice/group; ****P < 0.0001; compared with stroke mice treated with tPA-MRs

20

in NS group). (H) Scheme of how magnetic tPA-MRs target to the cerebral blood clot on site.

21

With the guidance of magnets, tPA-MRs could precisely move to the thrombus site. (I) Scheme to

22

show the mechanism of magnetic tPA-MR-mediated thrombolysis. The magnetic tPA-MRs could

17



1

rotate under an extra rotational magnetic field and release tPA to sufficiently enhance thrombolysis.

2

Scare bar = 300 µm.

3 4

Toxicity of the tPA-MRs in vivo

5

Serum alanine transaminase (ALT), aspartate aminotransferase (AST) levels, and albumin (ALB)

6

are common markers for hepatic toxicity, of which levels are rapidly increased when the liver is

7

damaged by any cause. In the NS treated-control group, the minimum and maximum values of

8

these biomarkers which found in our study are in the ranges of the standardized values of mice

9

based on the literature reports32-34. Importantly, we found that the levels of these protein values

10

were not significantly altered up to 7 days after intravenous administration of tPA-MRs (10 mg/kg),

11

compared with the control injected with saline (Fig. 4A-C). The levels of nitrogen (BUN) and

12

creatinine (Cr) in mice blood serum were tested as a measure of kidney. A detailed analysis of all

13

these metabolites in serum of animals treated with tPA-MRs (10 mg/kg, i.v.) at durations from 1

14

h to 7 days as compared to controls showed no statistically significant differences in any of the

15

parameters tested (Fig. 4E, F). Moreover, the levels of serum malondialdehyde (MDA) were

16

detected to evaluate lipid peroxidation, no significant difference was discovered between tPA-

17

MRs treatment group and the control group (Fig. 4D). Histological study shows that no significant

18

cellular damage including vacuolar degeneration, hyaline droplets and casts, karyorrhexis and

19

karyolysi was observed in the liver and kidney (data not shown). After the application of tPA-MRs,

20

no significant leukocyte infiltration was observed in the wall of middle cerebral arteries based on

21

the Wright/Giemsa staining (Fig. S10).

22

18


Page 18 of 36

Page 19 of 36 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


1 2

Figure 4. Quantitation of blood biomarkers of liver and kidney function. 10 mg/kg tPA-MRs

3

suspension or NS (as control) was injected through the mouse jugular vein. The concentrations of

4

biomarkers either for liver (A-C), kidney (E-F) functions or lipid peroxidation (D) were

5

determined at seven different time points after injection. The lines of “Max” and “Min” represent

19



1

the maximum and minimum level of individual biomarker detected in the control groups. Data are

2

presented as mean ± SEM at each time point, n = 3 per group. Two-tailed Student’s t-tests was

3

used to compare the difference between the treatment and control group at the indicated time points

4

in A-F. As data show, in each biomarker, no significant difference was found between NS and the

5

tPA-MRs treatment groups during the whole test period.

6 7

Distribution and clearance of the tPA-MRs in vivo

8

The concentrations of tPA-MRs in all major tissues, including liver, spleen, kidney, lung and brain,

9

were measured after administration over a period of time until the elimination phase (the longest

10

observation period is 12 weeks after administration of tPA-MRs). We found that the maximum

11

concentration (Cmax) in these tissues was within 1 h after MR injection (Fig. 5), which most

12

presented in the spleen. MRs were dramatically decreased in the spleen, liver and kidney 24 h after

13

administration (Fig. 5) and were even barely detected in these organs at 12 weeks later after

14

administration of tPA-MRs (data not shown). At t = 24 h and 12 weeks after the injection, no

15

damaged cells were observed in cresyl violet stained histopathological slices of spleen. And, all of

16

the cells in the spleen at different time points (24 h or 12 weeks) after the injection of MRs are

17

basically at the same status compared with healthy mice of the same age (Fig. S11). To determine

18

whether MRs could be discharged from the kidney, a 24-hour urine collection was performed using

19

metabolic cages after MR injection. After concentration, a large of number of black particles were

20

found in the collected urine specimen (Fig. 5P, Q). These particles could be attracted to a

21

permanent magnet, suggesting that they are the aggregated MRs. In addition, MRs were observed

22

in the biliary tract, including gall bladder, bile tracts and common hepatic duct, indicating that

23

MRs could also be discharged into the biliary system (data not shown).

20


Page 20 of 36

Page 21 of 36 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


1 2

Figure 5. Distribution and clearance of the tPA-MRs in vivo. Representative methyl green

3

stained histological images of liver (A-C), spleen (D-F), kidney (G-I), lung (J-L) and brain (M-

4

O) of CD-1 mice after administration of tPA-MRs (1 mg/kg) for 1 and 24 h. Scale bars (A-L) =

5

10 µm. Black arrows indicate tPA-MRs. Representative images of urinalysis examination of 24 h

6

urine after injection of tPA-MRs (P, Q). Right panel has higher magnification from the insert of

7

left one. Scale bars (P, Q), 50 µm. Black arrows indicate tPA-MRs detected in the urine, white

8

arrows indicate fodder residues from metabolic cage during urine collecting process. (R)

9

Quantitation analysis of the distribution of tPA-MRs at 1 and 24 h. *** P < 0.001, ** P < 0.01,

10

and * P < 0.05 by two-tailed Student’s t-test (n = 4).

11 12

DISCUSSION

21



Page 22 of 36

1 2

In this study, we demonstrate that intra-arterial injected tPA-MRs could target to the distal

3

cerebral artery under a magnetic guidance, and significantly improved tPA-mediated thrombolysis

4

in a mouse model of stroke. As a result, usage of the total dose of tPA is lower, and tPA-mediated

5

hemorrhagic complications are thus dramatically reduced. In addition, we also found that the tPA-

6

MRs are safe for use in vivo, as the injected tPA-MRs did not cause liver and kidney damage and

7

could be discharged from kidney.

8 9

Successful thrombolysis depends on the joint effects of conversion of activated plasminogen to

10

plasmin and effective exposure of both the substrate and the plasminogen activator to the entire

11

blood clot. The delivery of tPA into a blood clot is either dependent on passive diffusion

12

reliant on pressure facilitated (bulk) flow 36, as the rate of lysis is increased up to 100-times when

13

plasminogen activator and plasminogen are introduced into cylindrical clots by pressure-induced

14

bulk flow in comparison with diffusion alone

15

weakened by inadequate collateral circulation or systemic hemodynamic compromise. In addition,

16

progression of tPA-mediated clot lysis in the blood vessel proceeds gradually and stepwise,

17

allowing the thrombolytic zone to only move slowly, layer-by-layer through the clot, progressively

18

restricting the amounts of tPA available and, thus, limiting the lysis efficiency at deeper thrombus

19

sections

20

physical adsorption and chemical adsorption 22. Although the tPA was immobilized on the MRs,

21

it still remains the enzymatic activity. For instance, as tPA storage concentration (0.5 mg/ml), the

22

bounded tPA on MRs can have a remaining activity of about 74.08%. Further more, when the tPA-

23

MRs were suspended into the normal saline, they started to release free tPA and play the enzymatic

24

thrombolysis function. Since the tPA-MRs can target the clot in the brain by a magnetic guidance

37-38

36

35

or

. However, pressure-induced bulk flow is often

. In our study, the tPA was immobilized on MRs by two kinds of immobilizations:

22


Page 23 of 36 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


1

and be spanned under a rotational magnetic field, two effects could significantly enhance the

2

thrombolysis efficiency. First, the rotation of the tPA-MRs could increase the tPA release rate

3

from the tPA-MRs and improve the mass transport of the tPA and tPA-clot reaction products

4

during the thrombolysis 18, 22. Second, the cross-linked fibrin network in the clots can be disrupted

5

by mechanical force and tPA can be delivered into the clot, which allow plasminogen to reach the

6

new binding sites and enhances the susceptibility of clots to lysis. Our previous in vitro study

7

demonstrated that the rotating MRs in normal saline could achieve a lysis efficiency of 15% 22,

8

magnetic MRs have a rod-like shape and as a results they can be spanned as beaters to disrupt the

9

cross-linked fibrin mesh. Thus, the clot was loosened with mechanical force and the tPA could be

10

delivered and released into the center of clot. In addition, our study showed that no re-occlusion

11

phenomenon was observed on site at 24 h after thrombolysis, which could be due to the fact that

12

magnetite coated by thrombolytic enzymes can also cover the clotting surface to form a thrombin-

13

inhibiting coating that could theoretically prevent re-thrombosis or further new thrombus

14

formation as the tPA-MRs continue to bind on the thrombin

15

prevention of re-occlusion could be achieved by a single bolus dose with minimal systemic

16

hemorrhagic incidence. As tPA-MRs can be delivered at the site of embolism at high concentration,

17

so that lower doses of tPA can be applied in ischemic stroke, which, in turn, dramatically reduce

18

tPA-mediated hemorrhagic complications. Meanwhile, it is important to mention that 1 mg/kg was

19

optimal dosage in our study, higher amount of tPA-MRs and MRs (5, 10, 15 mg/ml) could cause

20

occlusion in other untargeted branches of cerebral arteries and other organs in circulation system.

21

On the other hand, lower dose of tPA-MRs (0.5 mg/kg) were not sufficient enough to achieve

22

advanced thrombolysis (Fig S8). More importantly, based on our in vitro cytotoxicity test, the

23

optimal dosage of tPA-MRs (1mg/kg) used in our mouse model (body weight ≈ 0.03 kg, totally

23


4, 39

. Overall, both reperfusion and


Page 24 of 36

1

injected tPA-MRs ≈ 30 µg/ml) did not show any significant cellular toxicity (Fig. S5), suggesting

2

that the optimal therapeutic dosage of tPA-MRs did not cause cytotoxicity in the body, which is

3

consistent with our in vivo toxicity results tested in the organ functions (Fig. 4).

4 5

tPA-MRs belong to iron oxide-based nanomaterials (IONPs), and thus is safe to use in vivo.

6

Supportively, previous studies have shown that mitochondrial respiratory chain complexes (I, II,

7

III, and IV) activities remained unchanged in brain, heart, lung, liver and kidneys when exposed

8

to Fe3O4 NPs (from 100 to 500 µg/ml) 40. Unlike ZnO, CuO and MgO nanoparticles, IONPs do

9

not significantly cause cytotoxicity, permeability and inflammation response in human cardiac 41

10

microvascular endothelial cells (HCMECs)

11

ischemia through up-regulation of serum superoxide dismutase (SOD) and down-regulation of

12

serum MDA, lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase isoenzyme-

13

MB (CK-MB) 42. Consistently, our data show that tPA-MRs had no toxic effects on liver cells. In

14

addition, the levels of blood BUN and Cr in tPA-MRs groups were not increased significantly

15

compared with the control group, indicating that tPA-MRs did not damage renal function, although

16

IONPs might lead to vascular dysfunction and oxidative stress

17

filtration of particles is highly dependent on the filtration-size threshold

18

with a hydrodynamic diameter (HD) < 6 nm are typically filtered, while those > 8 nm are not

19

typically capable of glomerular filtration

20

urine (Fig. 5P, Q). Taken together, the nanoporous MRs may be potentially dissociated into

21

smaller Fe3O4 nanocrystals (Fig. 2B, C), which would be able to travel through the glomerular

22

capillaries, and secreted in the urine.

47

. In turn, IONPs could protect myocardium from

. As we know, glomerular 46

. Molecules/particles

. We observed the large amount of MR secretion in

23

43-45

24


Page 25 of 36 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


1

Fe3O4 are biodegradable, and thus participate in the iron homeostasis in the body 48. Magnetite

2

(Fe3O4) uptake by immune cells may occur both in the bloodstream by monocytes, platelets,

3

leukocytes, and dendritic cells (DC) and in tissues by resident phagocytes (e.g., Kupffer cells in

4

liver, alveolar macrophages in lung, DC in lymph nodes, macrophages and B cells in spleen) 49. It

5

has been reported that the reticular-endothelial system is able to capture most of the magnetite of

6

similar size in circulation, which is then rapidly retained primarily in liver and spleen 50. We found

7

that, although little, MRs remain in the liver and kidney up to 12 weeks after injection. It could be

8

possible that the MRs are uptake by immune cells in these tissues. Interestingly, no obvious MRs

9

distribution were found in the brain tissue, it may mainly due to blood-brain barrier (BBB), the

10

tightest endothelium in the body, which is a unique membranous barrier that tightly controls the

11

transport of nanoparticles 51. Yet, many factors such as the size, the surface charge and the presence

12

or absence of a polymer coating, can affect the clearance and the distribution of MRs, which should

13

be further investigated before any clinical trials are conducted.

14 15

In summary, this study provides a proof of concept for developing novel, biocompatible,

16

magnetically guided tPA-MR delivery system to enhance thrombolysis after ischemic stroke. This

17

delivery system could deliver tPA at the site of embolism at high concentration, so that lower doses

18

of tPA can be applied in ischemic stroke and more tPA could be released under a rotating magnetic

19

field at the target site, which would maximize drug accumulation on clot site, enhance mass

20

transport, and introduce a mechanical disruption to the clot, to achieve a significantly enhanced

21

recanalization rate and minimize tPA side effects. This approach could revolutionize not only just

22

for the treatment of ischemic stroke but also have majorly impact on other deadly thrombotic

23

diseases such as myocardial infarction and pulmonary embolism.

24

25



1

AUTHOR CONTRIBUTIONS

2

J.H. conceived and designed the experiments. J.H., S.H., L.Z. and W.H., performed, analyzed,

3

and interpreted experiments. J.H., S.H. and L.Z. wrote and edited the manuscript. K.J. Q.Z. and

4

Y.Z. supervised the project and designed and interpreted experiments. All authors provided

5

feedback and agreed on the final version of the manuscript.

6 7

ACKNOWLEDGMENTS

8

This work is supported by National Science Foundation under the contract ECCS-1303134,

9

National Institutes of Health under the contact of R21 NS084148-01A1, National Natural Science

10

Foundation of China (No. 81771262), Zhejiang Provincial Key Research and Development

11

Program (2017C03027), National Science Foundation of Beijing (No. 7161014) and Sigma Xi

12

Grants-in-Aid of Research Program Fellowship for Jiangnan Hu (G2017100192773410).

13 14 15 16 17 18 19 20 21 22 23

26


Page 26 of 36

Page 27 of 36 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

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


References (1) Stricker, R. B.; Wong, D.; Shiu, D. T.; Reyes, P. T.; Shuman, M. A. Activation of plasminogen by tissue plasminogen activator on normal and thrombasthenic platelets: effects on surface proteins and platelet aggregation. Blood 1986, 68 (1), 275-280. (2) Xu, X.; Wang, B.; Ren, C.; Hu, J.; Greenberg, D. A.; Chen, T.; Xie, L.; Jin, K. Age-related Impairment of Vascular Structure and Functions. Aging Dis 2017, 8 (5), 590-610, DOI: 10.14336/AD.2017.0430. (3) Clark, W. M.; Wissman, S.; Albers, G. W.; Jhamandas, J. H.; Madden, K. P.; Hamilton, S. Recombinant tissue-type plasminogen activator (Alteplase) for ischemic stroke 3 to 5 hours after symptom onset. The ATLANTIS Study: a randomized controlled trial. Alteplase Thrombolysis for Acute Noninterventional Therapy in Ischemic Stroke. Jama 1999, 282 (21), 2019-26. (4) Adams, H. P., Jr.; del Zoppo, G.; Alberts, M. J.; Bhatt, D. L.; Brass, L.; Furlan, A.; Grubb, R. L.; Higashida, R. T.; Jauch, E. C.; Kidwell, C.; Lyden, P. D.; Morgenstern, L. B.; Qureshi, A. I.; Rosenwasser, R. H.; Scott, P. A.; Wijdicks, E. F. Guidelines for the early management of adults with ischemic stroke: a guideline from the American Heart Association/American Stroke Association Stroke Council, Clinical Cardiology Council, Cardiovascular Radiology and Intervention Council, and the Atherosclerotic Peripheral Vascular Disease and Quality of Care Outcomes in Research Interdisciplinary Working Groups: the American Academy of Neurology affirms the value of this guideline as an educational tool for neurologists. Stroke 2007, 38 (5), 1655-711, DOI: 10.1161/strokeaha.107.181486. (5) Xu, X.; Wang, B.; Ren, C.; Hu, J.; Greenberg, D. A.; Chen, T.; Xie, L.; Jin, K. Recent Progress in Vascular Aging: Mechanisms and Its Role in Age-related Diseases. Aging Dis 2017, 8 (4), 486-505, DOI: 10.14336/AD.2017.0507. (6) Huang, L.; Hu, J.; Huang, S.; Wang, B.; Siaw-Debrah, F.; Nyanzu, M.; Zhang, Y.; Zhuge, Q. Nanomaterial Applications for Neurological Diseases and Central Nervous System Injury. Prog Neurobiol 2017, DOI: 10.1016/j.pneurobio.2017.07.003. (7) Psarros, C.; Lee, R.; Margaritis, M.; Antoniades, C. Nanomedicine for the prevention, treatment and imaging of atherosclerosis. Maturitas 2012, 73 (1), 52-60. (8) Kim, J.-Y.; Kim, J.-K.; Park, J.-S.; Byun, Y.; Kim, C.-K. The use of PEGylated liposomes to prolong circulation lifetimes of tissue plasminogen activator. Biomaterials 2009, 30 (29), 5751-5756. (9) Torno, M. D.; Kaminski, M. D.; Xie, Y.; Meyers, R. E.; Mertz, C. J.; Liu, X.; O'Brien, W. D.; Rosengart, A. J. Improvement of in vitro thrombolysis employing magnetically-guided microspheres. Thrombosis research 2008, 121 (6), 799-811. (10) Horák, D.; Babič, M.; Mackova, H.; Beneš, M. J. Preparation and properties of magnetic nano-and microsized particles for biological and environmental separations. Journal of separation science 2007, 30 (11), 1751-1772. (11) Chiang, C. S.; Shen, Y. S.; Liu, J. J.; Shyu, W. C.; Chen, S. Y. Synergistic Combination of Multistage Magnetic Guidance and Optimized Ligand Density in Targeting a Nanoplatform for Enhanced Cancer Therapy. Adv Healthc Mater 2016, 5 (16), 2131-41, DOI: 10.1002/adhm.201600479. (12) Taghavi Pourianazar, N.; Gunduz, U. CpG oligodeoxynucleotide-loaded PAMAM dendrimer-coated magnetic nanoparticles promote apoptosis in breast cancer cells. Biomed Pharmacother 2016, 78, 81-91, DOI: 10.1016/j.biopha.2016.01.002. (13) Choi, W. I.; Lee, J. H.; Kim, J. Y.; Heo, S. U.; Jeong, Y. Y.; Kim, Y. H.; Tae, G. Targeted antitumor efficacy and imaging via multifunctional nano-carrier conjugated with anti-HER2 trastuzumab. Nanomedicine 2015, 11 (2), 359-68, DOI: 10.1016/j.nano.2014.09.009. (14) Niu, S.; Zhang, L. K.; Zhang, L.; Zhuang, S.; Zhan, X.; Chen, W. Y.; Du, S.; Yin, L.; You, R.; Li, C. H.; Guan, Y. Q. Inhibition by Multifunctional Magnetic Nanoparticles Loaded with Alpha-Synuclein RNAi Plasmid in a Parkinson's Disease Model. Theranostics 2017, 7 (2), 344-356, DOI: 10.7150/thno.16562.

27



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

(15) Dehvari, K.; Lin, K. S. Synthesis, characterization and potential applications of multifunctional PEOPPOPEO- magnetic drug delivery system. Curr Med Chem 2012, 19 (30), 5199-204. (16) Sagar, V.; Pilakka-Kanthikeel, S.; Atluri, V. S.; Ding, H.; Arias, A. Y.; Jayant, R. D.; Kaushik, A.; Nair, M. Therapeutical Neurotargeting via Magnetic Nanocarrier: Implications to Opiate-Induced Neuropathogenesis and NeuroAIDS. J Biomed Nanotechnol 2015, 11 (10), 1722-33. (17) Raymond, A. D.; Diaz, P.; Chevelon, S.; Agudelo, M.; Yndart-Arias, A.; Ding, H.; Kaushik, A.; Jayant, R. D.; Nikkhah-Moshaie, R.; Roy, U.; Pilakka-Kanthikeel, S.; Nair, M. P. Microglia-derived HIV Nef+ exosome impairment of the blood-brain barrier is treatable by nanomedicine-based delivery of Nef peptides. J Neurovirol 2016, 22 (2), 129-39, DOI: 10.1007/s13365-015-0397-0. (18) Cheng, R.; Huang, W.; Huang, L.; Yang, B.; Mao, L.; Jin, K.; ZhuGe, Q.; Zhao, Y. Acceleration of tissue plasminogen activator-mediated thrombolysis by magnetically powered nanomotors. ACS nano 2014, 8 (8), 7746-54, DOI: 10.1021/nn5029955. (19) Gamarra, L.; Brito, G.; Pontuschka, W.; Amaro, E.; Parma, A.; Goya, G. Biocompatible superparamagnetic iron oxide nanoparticles used for contrast agents: a structural and magnetic study. Journal of Magnetism and Magnetic Materials 2005, 289, 439-441. (20) Cheng, F.-Y.; Su, C.-H.; Yang, Y.-S.; Yeh, C.-S.; Tsai, C.-Y.; Wu, C.-L.; Wu, M.-T.; Shieh, D.-B. Characterization of aqueous dispersions of Fe 3 O 4 nanoparticles and their biomedical applications. Biomaterials 2005, 26 (7), 729-738. (21) Liberti, P. A.; Rao, C. G.; Terstappen, L. W. Optimization of ferrofluids and protocols for the enrichment of breast tumor cells in blood. Journal of magnetism and magnetic materials 2001, 225 (1), 301-307. (22) Hu, J.; Huang, W.; Huang, S.; ZhuGe, Q.; Jin, K.; Zhao, Y. Magnetically active Fe3O4 nanorods loaded with tissue plasminogen activator for enhanced thrombolysis. Nano Research 2016, 9 (9), 2652-2661, DOI: 10.1007/s12274-016-1152-4. (23) Ma, Y. H.; Wu, S. Y.; Wu, T.; Chang, Y. J.; Hua, M. Y.; Chen, J. P. Magnetically targeted thrombolysis with recombinant tissue plasminogen activator bound to polyacrylic acid-coated nanoparticles. Biomaterials 2009, 30 (19), 3343-51, DOI: 10.1016/j.biomaterials.2009.02.034. (24) Hua, M.-Y.; Yang, H.-W.; Liu, H.-L.; Tsai, R.-Y.; Pang, S.-T.; Chuang, K.-L.; Chang, Y.-S.; Hwang, T.-L.; Chang, Y.-H.; Chuang, H.-C. Superhigh-magnetization nanocarrier as a doxorubicin delivery platform for magnetic targeting therapy. Biomaterials 2011, 32 (34), 8999-9010. (25) Zhou, J.; Guo, D.; Zhang, Y.; Wu, W.; Ran, H.; Wang, Z. Construction and evaluation of Fe3O4-based PLGA nanoparticles carrying rtPA used in the detection of thrombosis and in targeted thrombolysis. ACS applied materials & interfaces 2014, 6 (8), 5566-5576. (26) Xie, Y.; Kaminski, M. D.; Torno, M. D.; Finck, M. R.; Liu, X.; Rosengart, A. J. Physicochemical characteristics of magnetic microspheres containing tissue plasminogen activator. Journal of Magnetism and Magnetic Materials 2007, 311 (1), 376-378. (27) Chen, J.-P.; Yang, P.-C.; Ma, Y.-H.; Tu, S.-J.; Lu, Y.-J. Targeted delivery of tissue plasminogen activator by binding to silica-coated magnetic nanoparticle. International journal of nanomedicine 2012, 7, 5137. (28) Chung, T. W.; Wang, S. S.; Tsai, W. J. Accelerating thrombolysis with chitosan-coated plasminogen activators encapsulated in poly-(lactide-co-glycolide) (PLGA) nanoparticles. Biomaterials 2008, 29 (2), 228-37, DOI: 10.1016/j.biomaterials.2007.09.027. (29) Zhu, L.; Huang, W.; Rinehart, Z. S.; Tam, J.; Zhao, Y. Multifunctional iron oxide-carbon hybrid microrods. RSC Advances 2016, 6 (101), 98845-98853, DOI: 10.1039/C6RA19489C. (30) Karatas, H.; Erdener, S. E.; Gursoy-Ozdemir, Y.; Gurer, G.; Soylemezoglu, F.; Dunn, A. K.; Dalkara, T. Thrombotic distal middle cerebral artery occlusion produced by topical FeCl(3) application: a novel model suitable for intravital microscopy and thrombolysis studies. J Cereb Blood Flow Metab 2011, 31 (6), 1452-60, DOI: 10.1038/jcbfm.2011.8.

28


Page 28 of 36

Page 29 of 36 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

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


(31) Swanson, R. A.; Morton, M. T.; Tsao-Wu, G.; Savalos, R. A.; Davidson, C.; Sharp, F. R. A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 1990, 10 (2), 290-3, DOI: 10.1038/jcbfm.1990.47. (32) Al-Sayed, E.; Abdel-Daim, M. M.; Kilany, O. E.; Karonen, M.; Sinkkonen, J. Protective role of polyphenols from Bauhinia hookeri against carbon tetrachloride-induced hepato- and nephrotoxicity in mice. Ren Fail 2015, 37 (7), 1198-207, DOI: 10.3109/0886022X.2015.1061886. (33) Orsolic, N.; Sirovina, D.; Koncic, M. Z.; Lackovic, G.; Gregorovic, G. Effect of Croatian propolis on diabetic nephropathy and liver toxicity in mice. BMC Complement Altern Med 2012, 12, 117, DOI: 10.1186/1472-6882-12-117. (34) Singh, D. P.; Mani, D. Protective effect of Triphala Rasayana against paracetamol-induced hepatorenal toxicity in mice. J Ayurveda Integr Med 2015, 6 (3), 181-6, DOI: 10.4103/0975-9476.146553. (35) Francis, C. W. Ultrasound-enhanced thrombolysis. Echocardiography 2001, 18 (3), 239-46. (36) Sabovic, M.; Blinc, A. Biochemical and biophysical conditions for blood clot lysis. Pflugers Arch 2000, 440 (5 Suppl), R134-6. (37) Devcic-Kuhar, B.; Pfaffenberger, S.; Gherardini, L.; Mayer, C.; Groschl, M.; Kaun, C.; Benes, E.; Tschachler, E.; Huber, K.; Maurer, G.; Wojta, J.; Gottsauner-Wolf, M. Ultrasound affects distribution of plasminogen and tissue-type plasminogen activator in whole blood clots in vitro. Thromb Haemost 2004, 92 (5), 980-5, DOI: 10.1267/THRO04050980. (38) Diamond, S. L. Engineering design of optimal strategies for blood clot dissolution. Annu Rev Biomed Eng 1999, 1, 427-62, DOI: 10.1146/annurev.bioeng.1.1.427. (39) Myerson, J.; He, L.; Lanza, G.; Tollefsen, D.; Wickline, S. Thrombin-inhibiting perfluorocarbon nanoparticles provide a novel strategy for the treatment and magnetic resonance imaging of acute thrombosis. J Thromb Haemost 2011, 9 (7), 1292-300, DOI: 10.1111/j.1538-7836.2011.04339.x. (40) Baratli, Y.; Charles, A. L.; Wolff, V.; Ben Tahar, L.; Smiri, L.; Bouitbir, J.; Zoll, J.; Piquard, F.; Tebourbi, O.; Sakly, M.; Abdelmelek, H.; Geny, B. Impact of iron oxide nanoparticles on brain, heart, lung, liver and kidneys mitochondrial respiratory chain complexes activities and coupling. Toxicol In Vitro 2013, 27 (8), 2142-8, DOI: 10.1016/j.tiv.2013.09.006. (41) Sun, J.; Wang, S.; Zhao, D.; Hun, F. H.; Weng, L.; Liu, H. Cytotoxicity, permeability, and inflammation of metal oxide nanoparticles in human cardiac microvascular endothelial cells: cytotoxicity, permeability, and inflammation of metal oxide nanoparticles. Cell Biol Toxicol 2011, 27 (5), 333-42, DOI: 10.1007/s10565-011-9191-9. (42) Xiong, F.; Wang, H.; Feng, Y.; Li, Y.; Hua, X.; Pang, X.; Zhang, S.; Song, L.; Zhang, Y.; Gu, N. Cardioprotective activity of iron oxide nanoparticles. Sci Rep 2015, 5, 8579, DOI: 10.1038/srep08579. (43) Abukabda, A. B.; Stapleton, P. A.; Nurkiewicz, T. R. Metal Nanomaterial Toxicity Variations Within the Vascular System. Curr Environ Health Rep 2016, 3 (4), 379-391, DOI: 10.1007/s40572-016-0112-1. (44) Nemmar, A.; Beegam, S.; Yuvaraju, P.; Yasin, J.; Tariq, S.; Attoub, S.; Ali, B. H. Ultrasmall superparamagnetic iron oxide nanoparticles acutely promote thrombosis and cardiac oxidative stress and DNA damage in mice. Part Fibre Toxicol 2016, 13 (1), 22, DOI: 10.1186/s12989-016-0132-x. (45) Wu, X.; Tan, Y.; Mao, H.; Zhang, M. Toxic effects of iron oxide nanoparticles on human umbilical vein endothelial cells. Int J Nanomedicine 2010, 5, 385-99. (46) Deen, W. M.; Lazzara, M. J.; Myers, B. D. Structural determinants of glomerular permeability. Am J Physiol Renal Physiol 2001, 281 (4), F579-96, DOI: 10.1152/ajprenal.2001.281.4.F579. (47) Longmire, M.; Choyke, P. L.; Kobayashi, H. Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats. Nanomedicine (Lond) 2008, 3 (5), 703-17, DOI: 10.2217/17435889.3.5.703. (48) Tassa, C.; Shaw, S. Y.; Weissleder, R. Dextran-coated iron oxide nanoparticles: a versatile platform for targeted molecular imaging, molecular diagnostics, and therapy. Accounts of chemical research 2011, 44 (10), 842-852.

29



1 2 3 4 5 6 7 8

(49) Dobrovolskaia, M. A.; Aggarwal, P.; Hall, J. B.; McNeil, S. E. Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects on nanoparticle biodistribution. Molecular pharmaceutics 2008, 5 (4), 487-495. (50) Wu, T.; Hua, M.-Y.; Chen, J.-p.; Wei, K.-C.; Jung, S.-M.; Chang, Y.-J.; Jou, M.-J.; Ma, Y.-H. Effects of external magnetic field on biodistribution of nanoparticles: A histological study. Journal of Magnetism and Magnetic Materials 2007, 311 (1), 372-375. (51) Wong, A. D.; Ye, M.; Levy, A. F.; Rothstein, J. D.; Bergles, D. E.; Searson, P. C. The blood-brain barrier: an engineering perspective. Front Neuroeng 2013, 6, 7, DOI: 10.3389/fneng.2013.00007.

9 10

TABLE OF CONTENTS GRAPHIC

11

12 13 14 15 16 17 18 19

30


Page 30 of 36

Page 31 of 36 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


441x194mm (300 x 300 DPI)



337x195mm (300 x 300 DPI)


Page 32 of 36

Page 33 of 36 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


515x294mm (300 x 300 DPI)



291x385mm (300 x 300 DPI)


Page 34 of 36

Page 35 of 36 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


277x297mm (300 x 300 DPI)



443x305mm (300 x 300 DPI)


Page 36 of 36

Tissue Plasminogen Activator-Porous Magnetic Microrods for

Recommend Documents