Multimodal Nanoprobes Evaluating ... - Wiley Online Library

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Shuyan Zheng, Ying-Ying Bai, Yinzhi Changyi, Xihui Gao, Wenqing Zhang, Yuancheng Wang, Lu Zhou, Shenghong Ju,* and Cong Li*

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Multimodal Nanoprobes Evaluating Physiological Pore Size of Brain Vasculatures in Ischemic Stroke Models

and it originates from a lasting or transient reduction in cerebral blood flow (CBF) that arises from the occlusion of a cerebral artery either by embolus or by other particle matters.[2] In the management of ischemic stroke, the first line of action is to restore the CBF by intravenous injection of thrombolytic agent: recombinant tissue-type plasminogen activator (rt-PA). As the only clinically approved drug for acute stroke treatment, rt-PA has been proved to dredge the occlusive vasculatures, increase perfusion of ischemic regions, and preserve salvageable ischemic tissues.[3] Although rt-PA benefits clinical outcome,[4] its application in clinic is limited by short therapeutic time window (within 3.0 h after ischemic symptoms)[5] and potential risk of brain hemorrhage.[6] Therefore, alternative therapeutic strategies with extended intervention time window and minimized side effects are urgently needed. Ischemic cascade, starting with a severe focal reduction in CBF triggers a cascade of events in the ischemic region including depletion of energy compounds, excessive activation of glutamate receptors, accumulation of intracellular calcium cations, acidosis of intracellular environment, excessive production of free radicals, abnormal recruitment of inflammatory cells, and initiation of pathological apoptosis,[7] which are actively involved in ischemic damage.[8] The interruption of these cascades holds potential to protect the neurological tissues from above ischemic damages. Neuroprotection aims to prevent the ischemic insults by blocking the deleterious ischemic cascade. Even though neuroprotective agents such as glutamate antagonists,[9] ion channel modulators,[10] antiinflammatory agents,[11] and free radical scavenges[12] have been developed and showed pronounced therapeutic effects in preclinical studies, their clinical transformations were disappointing.[13] One of the major reasons is the low brain delivery efficiency of the neuroprotectants due to their short circulation lifetime. For example, compared with the native superoxide dismutase (SOD) with short resident time (≈6 min) in blood pool,[14] SOD encapsulated in biodegradable poly(D,L-lactide coglycolide, PLGA) demonstrated a higher reduction in infarct volume and increased survival rate by eliminating the free

Ischemic stroke accounts for 80% strokes and originates from a reduction of cerebral blood flow (CBF) after vascular occlusion. For treatment, the first action is to restore CBF by thrombolytic agent recombinant tissue-type plasminogen activator (rt-PA). Although rt-PA benefits clinical outcome, its application is limited by short therapeutic time window and risk of brain hemorrhage. Different to thrombolytic agents, neuroprotectants reduce neurological injuries by blocking ischemic cascade events such as excitotoxicity and oxidative stress. Nano-neuroprotectants demonstrate higher therapeutic effect than small molecular analogues due to their prolonged circulation lifetime and disrupted blood–brain barrier (BBB) in ischemic region. Even enhanced BBB permeability in ischemic territories is verified, the pore size of ischemic vasculatures determining how large and how efficient the therapeutics can pass is barely studied. In this work, nanoprobes (NPs) with different diameters are developed. In vivo multimodal imaging indicates that NP uptakes in ischemic region depended on their diameters and the pore size upper limit of ischemic vasculatures is determined as 10–11 nm. Additionally, penumbra defined as salvageable ischemic tissues performed a higher BBB permeability than infarct core. This work provides a guideline for developing nano-neuroprotectants by taking advantage of the locally enhanced BBB permeability in ischemic brain tissues.

1. Introduction Stroke is the second most frequent cause of death and a leading cause of disability and cognitive impairment worldwide.[1] Ischemic stroke accounts for approximately 80% of strokes S. Zheng, Y. Changyi, X. Gao, W. Zhang, Dr. L. Zhou, Dr. C. Li Key Laboratory of Smart Drug Delivery Ministry of Education School of Pharmacy Fudan University Shanghai 201203, China E-mail: [email protected] Y.-Y. Bai, Y. Wang, Prof. S. Ju Jiangsu Key Laboratory of Molecular and Functional Imaging Department of Radiology Zhongda Hospital Medical School of Southeast University Nanjing 210009, China E-mail: [email protected]

DOI: 10.1002/adhm.201400159

Adv. Healthcare Mater. 2014, 3, 1909–1918

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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oxidative radicals.[15] Therefore, the development of nanosized neuroprotective agents or drug delivery vectors holds promise to achieve improved therapeutic efficiency. As a unique physiological structure of the blood vessels in the brain, blood–brain barrier (BBB) precisely regulates the movement of molecules between blood and brain and maintains a precisely controlled microenvironment for neuronal circuits, synaptic transmission, and neurogenesis.[16] Due to the low permeability of BBB, overwhelming majority of small molecular drugs and almost all macromolecular pharmaceutics cannot reach brain tissues.[17] The acute ischemia results in local BBB permeability enhancement, which can be explained by the disruption of tight junctions (TJs)[18] that are zipper-like seals between adjacent endothelial cells[19] and restrict the entry of blood-borne substances into brain. The compromised BBB tightness in ischemic region results in brain damages such as edema and hemorrhagic transformation.[20] On the other hand, the leaky BBB provides the opportunity for delivery of the drugs, especially the nanoparticle-based therapeutics. Even though the BBB opening dynamics in ischemic regions were systemically studied,[21] as far as we know, the physiologic pore size of ischemic vasculatures that determines how large and how efficient the nanoparticles can pass is barely studied.[22] Elucidating the vascular pore size up-limit in ischemic regions, especially in penumbra that is referred to the “salvageable” tissue[23] will provide a guideline for development of nanoparticle-based neuroprotective agents by taking advantage of the enhanced BBB permeability in the ischemic regions. Magnetic resonance imaging is widely used for ischemic stroke diagnosis due to its noninvasiveness, the ability to offer anatomic information with high spatial resolution and the feasibility to characterize the etiology and pathophysiology.[20] Meanwhile, the unrivaled advantages of optical imaging stem from its ability to monitor the biological events with detection sensitivity at molecular or cellular level for providing functional information.[24] To determine the physiologic pore size of vasculatures in the ischemic regions, a series of dendrimer-based nanoprobes (NPs) with diameters in a range of 3.3–14.8 nm was developed. The conjugation of both paramagnetic chelators and fluorophores on the NPs helps to elucidate the pore sizes of vasculatures in infarct core as well as penumbra area by noninvasively quantifying their brain uptake efficiencies after their extravasation from the leaky vasculatures (Figure 1).

doubles and the overall diameter increases by 1–2 nm.[26] Previous reports demonstrated that the pore size upper limit in brain tumor vasculatures is about 12 nm.[27] By hypothesizing the similar magnitude of vascular pore sizes between ischemic lesion and brain tumor, NPs were prepared by using PAMAM dendrimers with generation from 2 to 6. The modification of polyethylene glycols (PEGs) not only improves the biocompatibility of the NPs but also minimizes the nonspecific tissue binding by shielding the surface charge on the NPs. To improve imaging sensitivity, multiple copies of paramagnetic Gd-DTPA chelators were labeled on the NPs via physiologically inert amide bonds. Meanwhile, near-infrared (NIR) fluorophore IR783 was conjugated to track the NP uptakes in vivo because tissue absorption and autofluorescence in NIR wavelength region (650–900 nm) are low, which allows the NIR light to penetrate into deep tissues.[28] Additionally, the labeled rhodamine helps to track the NPs in excised brain tissues under fluorescence microscope because the NIR fluorophore cannot be excited well by conventional fluorescence microscope. The general synthetic procedure of the NPs is described in Scheme 1. mPEG2K-NHS ester (3.2, 6.4, 12.8, 25.6, and 51.2 molar equivalences to G2, G3, G4, G5, and G6 dendrimer, respectively) dissolved in anhydrous DMF was added dropwise to the dendrimer (1.0 mmol) in 2.0 mL PBS (pH 7.4). The mixture was allowed to react for 1.0 h at r.t. and purified by gel column chromatography (GCC, Sephadex G25) using deionized water as mobile phase to give compound 1a–e. 1a–e (0.9 mmol) in 3.0 mL sodium bicarbonate buffered solution (0.1 M, pH 9.5) reacted with DTPA dianhydride (12, 24, 48, 96, 192 equiv.). The pH of the reaction solution was maintained at pH 9.5 by adding NaOH solution (0.1 M). The product labeled with multiple DTPA chelators (2a–e) was purified by GCC. Compound 2a–e (0.8 mmol) in 2.0 mL HEPES buffer (0.1 M, pH 8.3) was added with Gd2(CO3)3 powder (24, 48, 96, 192, 384 equiv.) and stirred for overnight at 60 °C. At the end of reaction, excess Gd2(CO3)3 was filtrated and the clear supernatant was further purified by a centrifugal filter (MW 3000 and 10 000 cutoff, 4000 rpm) to give compound 3a–e. Home-made reactive IR783-NHS ester (4.0 equiv.) in 50 µL anhydrous DMF was added dropwise to

2. Results 2.1. Design, Synthesis, and Characterization of the NPs Poly(amidoamine) (PAMAM) dendrimers as multigenerational polymers were chosen as scaffolds of NPs due to their globular architecture, identical molecular weight, and welldefined reactive groups on surface.[25] With each successive generation, the number of reactive groups on dendrimer surface

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Figure 1. Determination of physiological pore size upper limit of vasculatures in brain ischemic regions. The pore size upper limit of vasculatures in ischemic territories was determined by measuring the extravasation efficiencies of NPs with different hydrodynamic diameters via in vivo MRI/NIR fluorescence imaging and ex vivo fluorescence microscopic imaging.




FULL PAPER Scheme 1. Development of multimodal NPs with different particle size. A) Synthetic procedure of the PAMAM dendrimer-based NPs. i) mPEG2KNHS, pH 7.4; ii) DTPA-dianhydride, pH 9.5; iii) Gd2(CO3)3, pH 8.3; iv) IR783-NHS, pH 8.3; v) Rho-NHS, pH 8.3. B) Schematics of the NPs based on G2 –G6 PAMAM dendrimer.

3a–e (0.7 mmol) in 2.0 mL HEPES (pH 8.3) solution. After stirring 1.0 h at r.t., rhodamine-NHS ester (3.0 equiv.) in 50 µL anhydrous DMF was added. The dual-fluorophore conjugated dendrimers were purified by GCC respectively to offer the final products 5a–e. The physical parameters of the NPs were demonstrated in Table 1. The hydrodynamic diameters of the NPs correlated with the dendrimer generation and increased from 3.3 nm of G2-P to 14.8 nm of G6-P (Figure S1A, Supporting Information). Every NP migrated as a single band in fluorescence images of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGEs), which indicated their purities (Figure S1B, Supporting Information). Notably, the migration distances of the NP bands decreased with dendrimer generation number, which can be interpreted by the increased molecular weights. All the migration bands in NIR and rhodamine fluorescence images colocalized well with each other, which verified the dual-fluorophore conjugation on the Table 1. Physical properties of dendrimer based NPs. d [mm]a)

PDIa)

ζ [mV]a)

Den/PEG/ Gdb)

r1p [mM−1 s−1]c)

G2-P

3.3

0.306

3.6

1/10/3.6

2.71

G3-P

3.9

0.294

8.8

1/10/8.5

4.10

G4-P

6.5

0.348

10.8

1/10/20

5.48

G5-P

10.7

0.313

8.7

1/10/28.0

6.20

G6-P

14.8

0.233

20.5

1/10/50.2

8.80

NP

polydispersity index (PDI), and zeta potential (ζ) of the NPs were measured by dynamic light scattering (DLS) in PBS (pH 7.4). b)The Gd concentrations were measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES). c)T1-weighted relaxivities (r1p) of the NPs were determined on 7.0 T MRI scanner at 25 °C. a)Diameter,


NPs. 1H NMR spectroscopy indicated that averagely 10% primary amines on the NPs were modified with PEG. Meanwhile, the molar ratios between Gd3+-DTPA chelators and primary amines on the NPs were measured in a range of 20%–30%. Moreover, the average longitudinal relaxivity r1p of the labeled Gd3+ chelators enhanced with the generation and the values increased from 2.7 mM−1 s−1/Gd3+ of G2-P to 8.8 mM−1 s−1/Gd3+ of G6-P. Averagely 2–4 IR783 and rhodamine fluorophores were labeled on the NPs, which makes them sensitive enough to be visualized by in vivo optical imaging (Figure S2, Table S1, Supporting Information).

2.2. The Particle-Size-Dependent NP Uptakes in Ischemic Regions The ischemic stroke models were developed in nude mice underwent a permanent middle cerebral artery occlusion (MCAO) via photochemically induced strategy.[29] The ischemic lesion defined by T2W-MRI distributed in large part of cortex (from primary motor and sensory cortices to the end), striatum and nucleus accumbens of the right brain hemisphere. Remarkably, the ischemic location and volume were highly reproducible in the mouse models, which benefited to accurately evaluate vascular pore size in ischemic regions. Figure 2A demonstrated the representative T1W MR images of the mouse brains before and at selected time post-injection (PI) of NP via intravenous (i.v.) injection. Although no obvious MR enhancement was observed after the administration of G6-P, other NPs performed obvious uptakes in the ischemic territories. In the first 2 h PI, MR signals were observed in ventral pallidum and nucleuses below the striatum in the ipsilateral hemisphere. Compared with the rapidly reduced G2-P signal, G3, G4, and



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Figure 2. MRI indicated the particle-size-dependent NP uptakes in the ischemic region. A) In vivo T1W MR images of ischemic mouse brains before and selected time points after the administration of NPs (150 µL, T1 = 60 ms) via i.v.. The ischemia territories were indicated by the T2W MRI. The color code (C.C.) MR images presented the T1W signal enhancement of the NPs at 24 h PI. B) Time-dependent T1W MR signal intensity ratio between the ipsilateral and contralateral hemisphere after i.v. injection of NP. C) Contrast to noise ratio (CNR) in the ipsilateral hemisphere (Is) at 2 and 24 h PI of NPs. The data were presented as mean±SD (n = 4).

G5-P were detected in the boundary between the cortex and striatum including claustrum, dorsal endopiriform nucleus, and ventral endoporiform nucleus at 12 h PI. At 24 h, MR signals of G3, G4, and G5-P were found in the cortex and striatum of the ipsilateral hemisphere. The color-coded T1W-MR images showed that G4-P not only offered the highest signal enhancements but also distributed in the largest volume in the ischemic territories. Figure 2B demonstrated the time-dependent T1W-MR signal ratio between the ipsilateral and contralateral hemispheres after administration of NPs. Significantly, while the target to background (T/B) ratios of G4, G5, and G6-Ps increased consistently, the ratios of G2 and G3-P reached their maximal values at 2 and 12 h, respectively. The normalized T/B ratios at 24 h PI of the NPs were demonstrated in Figure 2C, and they decreased with a sequence of G4-P> G3-P> G2-P > G5-P > G6-P (mean ± SD, n = 4). The NP delivery efficiency into ischemic regions was further investigated by in vivo NIR fluorescence imaging. Figure 3A demonstrated representative in vivo NIR fluorescence images of ischemic mouse models at selected time points after the injection of NP via i.v.. All NPs visualized the ischemic region located at the ipsilateral hemisphere. While the T/B ratios of G4, G5,

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and G6-Ps increased consistently with time, the values of G2 and G3-P reached their maximal values at 4 h PI (Figure 3B). Above experimental result was verified by ex vivo NIR fluorescence images of brains that were excised at 2 and 24 h PI (Figure 3C). While strong fluorescence signals of G2, G3, and G4-P in the ischemic regions were detected at 2 h PI of NPs, there were little signals of G5 and G6-P to be observed. In contrast, when the signals of G2 and G3-P remarkably decreased at 24 h PI, the fluorescence signals of G4 and G5-P increased substantially compared to the values at 2 h PI. Quantitative studies demonstrated that all of the NPs, except G2-P, showed higher T/B ratio at 24 h PI compared to the values at 2 h PI (Figure 3D). G4-P demonstrated the highest T/B ratio at 24 h PI.

2.3. Pore Size Upper Limit of BBB in Infarct Core and Penumbra Due to the heterogeneity of CBF within the ischemic regions, tissues far away from collateral supply and exposed to the most severe reduction of CBF are referred to the infarct core where the damages are irreversible. Tissue at the periphery of the infarct core with a less severe reduction in CBF is defined




FULL PAPER Figure 3. NIR fluorescence imaging indicated particle-size-dependent NP uptakes in the ischemic region. A) In vivo NIR fluorescence imaging of mouse brain area at selected time-points PI of NPs. B) Ex vivo NIR fluorescence imaging of the excised mouse brain at selected time-points PI of NPs. C) Time-dependent NIR fluorescence intensity ratio between the ipsilateral and contralateral hemisphere after injection of NP (10 nmol/mouse) via i.v. (mean ± SD, n = 4). D) Average NIR fluorescence intensities on ipsilateral hemisphere at 2 and 24 h PI of NPs via i.v. (n = 4).

as ischemic penumbra where the prompt restoration of CBF can recover its neurological function. Saving the penumbra shows crucial clinical relevance because its salvage correlates to the therapeutic efficiency.[30] The location of infarct core and penumbra in ischemic mouse brain was delineated by triphenyl tetrazolium chloride (TTC) staining, Nissl staining, and immunohistochemical staining, respectively. The location of infarct core in the cortex of ipsilateral hemisphere was first indicated by TTC staining that distinguished the metabolically inactive/necrotic tissues as pale color (Figure S3A, Supporting Information). Nissl staining further verified the infarct core location with characteristic triangularly shaped and deep blue stained neurons due to the condensation of cytoplasm and karyoplasm[31] (Figure 4A, Figure S3B, Supporting Information). Meanwhile, the inducible heat shock protein HSP70 was widely used as a marker for penumbra due to its restrict expression in the peri-lesional neurons.[32] The HSP70 positive cells shown as brown color and shuttle shaped morphology were observed in the striatum, hippocampus, thalamus, and hypothalamus of the ipsilateral hemisphere that were indicated as the ischemic penumbra (Figure 4B, Figure S3C, Supporting Information). NIR fluorescence microscopic images of the ischemic brain sections demonstrated that: 1) higher NP uptakes in the ipsilateral hemisphere compared to the contralateral hemisphere; 2) higher NP uptakes in penumbra than the infarct core; 3) particle-size-dependent NP uptakes in both infarct core and penumbra (Figure 4C). Quantitative studies demonstrated that


while the normalized NIR fluorescence intensities of NPs were determined with a sequence of G4-P > G3-P > G2-P >> G5-P > G6-P in the penumbra, the sequence was changed to G3-P > G4-P > G2-P > G5-P >> G6-P in the infarct core (Figure 4D). Moreover, the fluorescence signals of G2, G3 and G4-P in the penumbra were higher than in the infarct core. Notably, there was not any NP signal detected in the contralateral hemisphere regardless of their particle size, which confirms the extremely low permeability in the nonischemic brain tissues (Figure S3D, Supporting Information).

2.4. Higher BBB Permeability in Penumbra Compared to Infarct Core To investigate the BBB permeability in the ischemic regions, the brain capillary endothelial cells (BCECs) were immunohistochemically stained by CD31 antibody at 24 h PI of the NPs. Confocal fluorescence microscopic images indicated the remarkable rhodamine fluorescence of G3, G4, and G5-NP in the penumbra areas (Figure 5A, upper panel). While most of the G3-P dispersedly distributed in the extravascular space, G5-P predominately located in the vessels. Notably, G4-P was found in both intra- and extravascular spaces. In the infarct core, even the fluorescence of G3, G4, and G5 was evident, most of them colocalized well with vasculatures, which indicates the low extravasation degree of the NPs (Figure 5A, low



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Figure 4. Higher NP permeability in the ischemic penumbra than that in infarct core. A) Nissl staining delineated infarct core in ispilateral hemisphere. B) HSP70 immunostaining defined the location of penumbra in ispilateral hemisphere. C) Fluorescence microscopic images of penumbra and infarct core at 24 h PI of NPs. Scale bar: 100 µm. D) Average rhodamine fluorescence intensities of penumbra (upper panel) and infarct core (low panel) at 24 h PI of NPs. Data were presented as mean ± SD (n = 4).

panel). G2-P and G6-P were hardly found in the ischemic regions, which can be explained by the fast excretion rate of

G2-P and larger diameter of G6-P than that of pore size upper limit in ischemic vasculatures. As expected, no obvious brain

Figure 5. NPs demonstrating higher transvascular efficiency in penumbra than infarct core. A) Fluorescence microscopic images of penumbra and infarct core in ipsilateral hemisphere at 24 h PI of NPs via i.v. CD31 immunofluorescence presenting vasculatures, rhodamine fluorescence presenting the NPs and DAPI fluorescence presenting nuclei were displayed in green, red, and blue, respectively. Scale bar: 100 µm. B) The colocalization coefficients between NP and brain vasculatures in penumbra and infarct core were quantified at 24 h PI. The values were quantified from six randomly selected images. Data were presented as mean ± SD (n = 6).

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3. Discussion Understanding of the physiological pore size of vasculatures is important to optimize the delivery of imaging/therapeutic agents. During the past years, the permeability of vasculatures to blood borne molecules has been systemically studied.[33] For example, the average pore size of capillaries in normal brain, skeletal muscles, and kidney glomerulus were measured as 0.5, 5, and 15 nm, respectively.[33] The leakage of vasculatures is widely observed under pathological conditions due to the disorganized endothelial cells and the degradation of the basal membrane.[34] For example, the pore size in human brain tumor vasculatures was determined around 12 nm[27a] and the values of peripheral tumors even reached micrometer range. The enhanced permeability of tumor vasculatures has been widely used to delivery macromolecular agents for cancer therapy.[35] Similarly, the increased BBB permeability in the ischemic lesions will offer the opportunity to specifically deliver the neuroprotective agents. However, as far as we know, the pore size upper limit of BBB in ischemic territories, especially the “salvageable” penumbra has not been investigated. The elucidation of above question will pave the way for the development of nanosized neuroprotective agents with improved delivery efficiency. PAMAM dendrimers are branched, multilayer, and synthetic polymers with uniform size and molecular weight for each generation. By choosing the dendrimer with suitable size and precisely manipulating the functional groups, it is possible to tune the biocompatibility and pharmacokinetics of resulting NPs.[25b] Compared with native dendrimers that usually increase 1–2 nm in diameter with each successive generation,[27a] the hydrodynamic diameters of the NPs actually increased 1–4 nm, which can be explained by the multiple water molecular spheres surrounding the labeled Gd3+ center.[36] Notably, only 50%–60% labeled DTPA chelators were complexed with Gd3+ ions even a large excess of Gd2(CO3)3 was treated. The phenomenon was reported previously and can be explained by the coordination between a single Gd3+ ion and two or more adjacent DTPA chelators.[37] The T1 relaxivity of a Gd3+complex is primarily determined by: 1) the number of water molecules q directly coordinated with Gd3+ ion; 2) the rotational correlation time τR of the agent (how fast the chelator rotates) and 3) the residence lifetime of Gd3+ bound water molecule τM (how fast the exchange rate).[38] The generation-dependent relaxivity enhancement of the NPs (from 2.7 to 8.8 mM−1 s−1/Gd3+) can be attributed to the increased τR values of the bulky NPs. Moreover,


the lower relaxivites of Gd3+ chelators in the NPs (maximally 8.8 mM−1 s−1/Gd3+) compared to the reported dendrimer-based MR agents (13.9–26.9 mM−1 s−1)[37,39] could be interpreted by 1) the measurements under 7.0 T because of the magneticfield-dependent relaxivity according to the nuclear magnetic relaxation dispersion (NMRD) profile[40]; 2) the replacement of the Gd3+bound water molecules by negatively charged carboxylate group from an adjacent uncomplexed DTPA chelators.[39b] Middle cerebral artery (MCA) is the most commonly affected blood vessels in human ischemic stroke and MCAO animal model is the most widely used in preclinical studies.[41] In this work, photothrombosis strategy was used to develop the MCAO model because of its advantages including minimized mechanical damage to cerebral blood vessels, the flexibility to modulate lesion location, the low mortality of animals (≈5%), and high ratio of model establishment (100%). Additionally, the good reproducibility in infarct size and functional deficit benefit to accurately determine the pore size of ischemic BBB. In vivo MR and NIR optical imaging studies showed a close relationship between the particle size and NP uptake efficiency in the ischemic regions. Both G6-P with the largest diameter (14.8 nm) and G2-P with the smallest diameter (3.3 nm) showed the low uptakes, which can be explained by the bulky size of G6-P larger than the pore size upper limit of ischemic vasculatures and the rapid excretion of G2-P via renal filtration.[42] Remarkable uptakes of G3, G4, and G5-P in the ischemic regions attributed to their prolonged circulation lifetime and their diameters below the pore size upper limit of ischemic BBB. According to the in vivo and ex vivo experimental results, the pore size upper limit in ischemic regions should be between the diameters of G5-P and G6-P and in a range of 10–11 nm. Even though numerous studies have been made to assess the vascular permeability of tumors,[27a] as far as we know, there is very limited investigations evaluating BBB permeability in the ischemic stroke. Loss of blood supply in brain initiates a cascade of events including the increased production of reactive oxygen species (ROS),[43] overexpression of proteases,[44] and down-regulation of TJ-associated proteins,[18] which jointly increase BBB permeability through the promotion of vascular inflammation, compromise of TJ tightness, degradation of basal membrane, and loss of the vasoregulatory capability. Previous work indicated the highest vascular permeability was within the infarct core because it suffers the greatest degree of TJ dysregulation and disassembly.[18] However, in this work, even infarct core and penumbra demonstrated a similar vascular pore size upper limit, penumbra indeed showed a higher BBB permeability than that in infarct core with the evidence of the earlier MR signal enhancement and higher NP uptake efficiency. Above phenomenon could be explained by high CFB, active angiogenesis, and overexpression of caspases in penumbra. High CFB leads to the increased mass transfer rate through vascular wall,[45] active angiogenesis generates the neovascultures with enhanced permeability,[46] and the caspases actively cleave TJ-related proteins in maintaining BBB tightness.[47] Taking advantage of the higher vascular permeability in penumbra than that of normal neurological tissues and infarct core, it is possible to specifically deliver the nanosized neuroprotective therapeutics into the “salvageable” penumbra by finely tuning their particle size.



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uptake of the NPs was observed in normal brain tissues regardless of their particle sizes (Figure S4, Supporting Information). Figure 5B shows the colocalization coefficients between the NPs and brain vasculatures in the penumbra and infarct core at 24 h PI. Interestingly, while the colocalization coefficients of G3, G4, and G5-P in penumbra increased consistently from 0.57 ± 0.06 to 0.86 ± 0.07, the data of all these three NPs were above 80% in the infarct core. The colocalization coefficients of G2-P and G6-P kept below 30% in both penumbra and infarct core, which can be explained by their low uptakes in ischemic region.

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Even the disrupted BBB in the ischemic regions offers an opportunity for the entry of the nanosized therapeutics, the compromised drug delivery efficiency is still the main obstacle for neuroprotective therapy because the drug concentration in the lesion is still less than the therapeutic threshold. How to specifically enhance BBB permeability in the ischemic regions, especially the penumbra is the next challenge to be overcome. In our previous work, a two-order targeted brain tumor imaging strategy was put forward.[48] The NP labeled with cyclic [RGDyK] peptides targeting αVβ3 integrin and angiopep-2 peptides targeting the lipoprotein receptor-related protein (LRP) was developed. This NP first targets the tumor vasculatures and its increased concentration in endothelial cells facilitates its BBB traverse via LRP-mediated transcytosis.[49] The BBB penetrated NP further targeted brain cancer cells. In this way, brain tumor with a diameter less than 1.0 mm was visualized with high target to background ratio. Enlightening by the two-order targeting strategy, it is possible to label penumbra targeting ligands and BBB opening ligands on the NAs. The dual-labeled NP will first accumulate in penumbra and then specifically upregulate local BBB permeability. The targeted BBB opening in the ischemic site will benefit to maximize the delivery efficiency of neuroprotective agents and minimize the off-targeted side-effects.

4. Conclusions Since the first thrombolytic agent rt-PA trial in 1995, more than 160 clinical trials of the neuroprotective drugs have been tested, but none has shown clinical efficacy.[50] Although multiple reasons lead to the failures, the low drug delivery efficiency in the ischemic tissues is the main challenge faced by researchers. Nanoparticle-based neuroprotective agents with prolonged circulation lifetime but smaller than the pore size upper limit of ischemic vasculatures hold the promise to improve the therapeutic efficacy. In this work, by developing NPs with different diameters and utilizing multimodal MR/optical imaging, the upper limit pore sizes of ischemic vasculatures were determined in a range of 10–11 nm and the higher permeability in the penumbra than that in the infarct core was verified. Importantly, this work provides a guideline for development of nanosized neuroprotectants with the capability to specifically deliver into ischemic lesions, especially the salvageable penumbra. This guideline holds the promise to improve the therapeutic efficiency of ischemic stroke.

5. Experimental Section Materials: All chemical reagents were obtained from Aladdin Reagent (Shanghai, China) unless otherwise specified. PAMAM dendrimers were purchased from Weihai CY Dendrimer Technology Co., Ltd (Weihai, China). Rhodamine-NHS was purchased from Thermofisher Scientific (New York, USA). Rabbit anti-mouse CD31 primary antibody was purchased from Abcam (Cambridge, UK). Alexa Fluo488-labeled goat anti-rabbit secondary antibody was purchased from Cell Signaling Technology (Danvers, USA). Rabbit anti-mouse HSP70 primary antibody was purchased from Enzo Life Sciences, Inc. (Switzerland). PEG–NHS was purchased from JenKem Technology Co. Ltd (Beijing, China). DTPA– dianhydride was obtained from TCI (Shanghai, China). IR783-NHS was

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prepared according to our previous work.[51] Amicon ultra-15 centrifugal filter tubes (3000 and 10 000 MW cutoff) were from Millipore (Bedford, USA). Characterization: The purity of NPs was determined by SDS-PAGE. NP (50 µg) in treatment buffer was loaded on a 12% polyacrylamide gel. Photographic and fluorescent images of the resolved SDS-PAGE gels were acquired under IVIS Spectrum (Caliper PerkinElemer, USA) Imaging System (FOV = 13.6 cm, F/Stop = 2, bin = medium resolution, exposure time = auto). The molar ratios between dendrimer, PEG, and DTPA chelators in NPs were quantified by integrating the characteristic proton of dendrimer (3.3–2.2 ppm), PEG (3.6–3.7 ppm), and DTPA chelator (2.37 and 3.30 ppm) in 1H NMR (Varian Mercury400 spectrometer, USA). The fluorophore labeling degree was calculated according to standard curve method on UV-2401PC UV–vis spectrophotometer (Shimadzu, Japan). The absorbance of rhodamine and IR783 was measured at 548 and 775 nm. The hydrodynamic size distribution and Zeta potentials of NPs were determined by Malvern Zetasizer (Malvern Instruments Inc., USA) dynamic light scattering (DLS) instrument after instrumental calibration. Gd3+ concentrations of NPs were determined by a Hitachi P-4010 inductively coupled plasma atomic emission spectroscopy (ICP-AES, Tokyo, Japan) with RF power at 1100 W and nebulizer gas flow at 0.9 L min−1. The standard solutions with the Gd3+ concentration of 1, 5, 10, 20, 50, 100, 200 ppm were prepared and a calibration curve was made by plotting the corresponding signal peaks versus the Gd3+ concentrations. The Gd3+ concentrations of the NPs were determined by fitting the Gd3+ peak intensity against the calibration curve. The longitudinal relaxivities of the NPs were determined at 7.0 T MRI coilaccording to the equation of r1p = (1/Tsample – 1/TPBS)/[Gd]. NP with five different concentrations was measured respectively. Plotting the (1/Tsample – 1/TPBS) values with corresponding Gd3+ concentrations measured by ICP-AES offered the T1-weighted relaxivities. Animal Model of Ischemic Stroke: Male nude mice weighing 18–22 g were anesthetized with 1% pentobarbital sodium solution (0.03 mg kg−1) via intraperitoneal (i.p.) injection. The anesthetized mouse was positioned on a plate. A 5-mm incision was made between the orbit and ear. Under an operating microscope, an incision was made by dividing the temporal muscle, and the right lateral aspect of the skull was exposed. The main trunk of the middle central artery (MCA) was visualized without a craniotomy. Rose bengal dissolved in saline with a concentration of 10 mg mL−1 was injected via caudal vein (0.5 mL). At 2 min post dye injection, a laser beam of 0.1 mm diameter (Shanghai Laser & Optics Century Co., Ltd. Shanghai, China) and 532 nm wavelength was stereotactically positioned onto the MCA for 3 min. Afterwards, the skin was sutured and T2-weighted images were acquired at 2 h after surgery to verify the establishment of ischemic stroke models. In Vivo MRI Studies: All MRI experiments were conducted on Bruker Pharmascan 7.0 T micro-MRI scanner (Bruker, Germany) using a transmit/receive quadrature volume radio frequency (RF) coil of an inner diameter of 38 mm. The mice were used for in vivo imaging at 8 h after ischemic stroke. In vivo MR images of mouse brain were acquired before and at selected time points after i.v. injection of NP in 150 µL PBS with T1 value of 60 ms via caudal vein. Animals were anesthetized with isoflurane (1.5–2% in 20% oxygen) and then positioned prostrate with head snugly inserted into a nose cone centered within the bore of the magnet. Respiration rate was monitored and mice were kept warm in heating pad. Fast spin echo T1-weighted anatomical scans using a 2 × 2 cm field of view for 24 slices of 0.5 mm thickness through the brain were performed with TR = 574.9 ms and TE = 15.0 ms. ROIs contained the ischemic lesions and a corresponding region in the contralateral hemisphere. Contralateral ROIs were exactly matched in size and shape with respect to their ipsilateral counterparts. The color-coded MR images were processed by ImageJ 1.48a software. T1-weighted images were first flippedhorizontally followed by registering with the original MR images. After subtracting the MR signals in flipped images from the original images, the resulting images were pseudo-colored with signal intensity ranged from 0 to 5000. The color-coded images were finally overlaid on the original MR images after deleting the signals in contralateral hemisphere.




Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgement S.Z. and Y.B. contributed equally to this work. This work was supported by the National Basic Research Program of China (973 Program,


2013CB733801, 2011CB910404), the National Natural Science Foundation of China (Nos. 81371624, 81171384), New Century Excellent Talents in University Award and the Shanghai Foundation for Development of Science and Technology (Nos. 13NM1400400, 12NM0501400). The authors thank the helpful discussions with Prof. W. Lu and Dr. X. Gu. Received: March 21, 2014 Revised: April 29, 2014 Published online: June 4, 2014

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In Vivo Optical Imaging Studies: Fluorescence images of a whole mouse body and close-up image of brain areas were obtained using Maestro In Vivo Imaging System (CRi Inc., Woburn, MA, USA). A serial of images were acquired before and at selected time points after administration of NP via i.v. In vivo NIR fluorescence images were acquired by using a band-pass excitation filter from 745 to 785 nm and a long-pass emission filter over 760 nm. The regions of interest were defined and quantified by using the instrument-equipped software (Maestro software, CRi). At 2 h and 24 h after administration of NPs, the mice were scarified and were perfused transcardially with PBS followed by 4% paraformaldehyde (PFA). The whole brains were excised carefully and ex vivo NIR fluorescence images were performed using the CRi imaging system with similar instrumental setting to the in vivo studies. Triphenyl Tetrazolium Chloride Staining: At 24 h after NP injection via i.v., mouse brains were carefully isolated and then frozen at −40 °C for 30 min. Thereafter, the brains were sectioned into coronal slices with thickness of 2 mm by using a brain-sectioning block. The sections were stained with 0.1% TTC (Beyotime, China) solution at 37 °C for 30 min and then imaged by SONY DSC-W120 camera (SONY, Japan). Nissl Staining: The brains at 24 h after NP injection were harvested and preserved at 4% PFA for overnight and then transferred to 30% sucrose for 3 d at 4 °C. The brains were embedded in wax and sectioned coronally with a thickness of 4 µm. The slides were rehydrated and then incubated in the Nissl–thionin staining solution for 4 min and maintained under agitation. The slides were rinsed in distilled water for 2 min, progressively dehydrated in 70% alcohol (10 min), 95% alcohol (2–3 min), and finally 100% alcohol (10 min). After cleared in xylene for 5 min, sections were mounted on glass slides using Aqua-Mount mounting media (Thermo Scientific) and cover-slipped. Photographic and rhodamine fluorescence images of the contralateral and ipsilateral hemispheres of brain sections were collected and then quantified by ImageJ software. Immunohistochemistry Staining: The paraffin-embedded brain cryosections were incubated in warm water bath (55 °C) for 5–10 min and in a mixture of 5% H2O2 and 10% methanol (v/v) in PBS for 20 min and then blocked with IHC blocking solution for 30 min at room temperature (r.t.). After incubation with 100 µL HSP70 primary antibody (1:500 dilution) overnight at 4 °C, the slides were washed and incubated with100 µL Alexa Fluor 488 secondary antibody (1: 500 dilution). Last, incubated each section with 100 µL of DAB-chromogen solution for 1–5 min and immersed the slide in PBS and mounted with a coverslip and predried before the microscopic imaging. For CD31 antibody immunostainning, free floating sections were fixed with 4% formaldehyde for 30 min and washed with PBS. Specimens were treated with 0.3% Triton X-100 and 3% BSA for 1.5 h to block against nonspecific binding and to permeabilize the cell membranes to enable staining. Then, BSA/Triton X-100 was removed, and the brain sections were incubated with anti-CD 31 primary antibody (1:100 dilution, Abcam) with supplemental 0.3% BSA overnight at 4 °C. After washing, the sections were incubated with goat anti-rabbitAlexa Fluor 488 secondary antibody (1:500 dilution) with supplemental 1% BSA for 1 h at room temperature; and the nuclear DNA was stained with DAPI (1:500 dilution). The fluorescence images were processed by ZEN 2012 software (Carl Zeiss, Germany) and colocalization of NPs and blood vessel was performed by Image-Pro Plus 6.0 software. Statistical Analysis: Values are presented as mean ± SD when the sample number was above 4 (n > 4).

[1] A. S. Go, D. Mozaffarian, V. L. Roger, E. J. Benjamin, J. D. Berry, W. B. Borden, D. M. Bravata, S. Dai, E. S. Ford, C. S. Fox, S. Franco, H. J. Fullerton, C. Gillespie, S. M. Hailpern, J. A. Heit, V. J. Howard, M. D. Huffman, B. M. Kissela, S. J. Kittner, D. T. Lackland, J. H. Lichtman, L. D. Lisabeth, D. Magid, G. M. Marcus, A. Marelli, D. B. Matchar, D. K. McGuire, E. R. Mohler, C. S. Moy, M. E. Mussolino, G. Nichol, N. P. Paynter, P. J. Schreiner, P. D. Sorlie, J. Stein, T. N. Turan, S. S. Virani, N. D. Wong, D. Woo, M. B. Turner, C. American Heart Association Statistics, S. Stroke Statistics, Circulation 2013, 127, e6. [2] L. B. Goldstein, Circulation 2007, 116, 1504. [3] A. Ciccone, L. Valvassori, M. Nichelatti, A. Sgoifo, M. Ponzio, R. Sterzi, E. Boccardi, S. E. Investigators, N. Engl. J. Med. 2013, 368, 904. [4] O. Adeoye, R. Hornung, P. Khatri, D. Kleindorfer, Stroke 2011, 42, 1952. [5] W. M. Clark, S. Wissman, G. W. Albers, J. H. Jhamandas, K. P. Madden, S. Hamilton, JAMA 1999, 282, 2019. [6] C. M. Maier, L. Hsieh, T. Crandall, P. Narasimhan, P. H. Chan, Ann. Neurol. 2006, 59, 929. [7] P. Deb, S. Sharma, K. M. Hassan, Pathophysiology 2010, 17, 197. [8] G. J. del Zoppo, N. Engl. J. Med. 2006, 354, 553. [9] A. Lau, M. Tymianski, Pflugers Arch. 2010, 460, 525. [10] Z. G. Xiong, X. P. Chu, R. P. Simon, J. Membr. Biol. 2006, 209, 59. [11] H. J. Kim, M. Rowe, M. Ren, J. S. Hong, P. S. Chen, D. M. Chuang, J. Pharmacol. Exp. Ther. 2007, 321, 892. [12] J. E. Slemmer, J. J. Shacka, M. I. Sweeney, J. T. Weber, Curr. Med. Chem. 2008, 15, 404. [13] Y. D. Cheng, L. Al-Khoury, J. A. Zivin, NeuroRx 2004, 1, 36. [14] D. S. Warner, H. Sheng, I. Batinic-Haberle, J. Exp. Biol. 2004, 207, 3221. [15] M. K. Reddy, V. Labhasetwar, FASEB J. 2009, 23, 1384. [16] N. J. Abbott, A. A. Patabendige, D. E. Dolman, S. R. Yusof, D. J. Begley, Neurobiol. Dis. 2010, 37, 13. [17] a) W. M. Pardridge, NeuroRx 2005, 2, 3; b) C. A. Lipinski, J. Pharmacol. Toxicol. Methods 2000, 44, 235. [18] K. E. Sandoval, K. A. Witt, Neurobiol. Dis. 2008, 32, 200. [19] J. D. Huber, R. D. Egleton, T. P. Davis, Trends Neurosci. 2001, 24, 719. [20] A. Kastrup, T. Engelhorn, C. Beaulieu, A. de Crespigny, M. E. Moseley, J. Neurol. Sci. 1999, 166, 91. [21] a) T. Kuroiwa, P. Ting, H. Martinez, I. Klatzo, Acta Neuropathol. 1985, 68, 122; b) Z. G. Huang, D. Xue, E. Preston, H. Karbalai, A. M. Buchan, Can. J. Neurol. Sci. 1999, 26, 298; c) A. Durukan, I. Marinkovic, D. Strbian, M. Pitkonen, E. Pedrono, L. Soinne, U. Abo-Ramadan, T. Tatlisumak, Brain Res. 2009, 1280, 158. [22] A. Abulrob, E. Brunette, J. Slinn, E. Baumann, D. Stanimirovic, In Vivo Optical Imaging of Ischemic Blood-Brain Barrier Disruption, Springer, NY, USA 2011. [23] W. D. Heiss, Int. J. Stroke 2010, 5, 290. [24] K. Licha, C. Olbrich, Adv. Drug Delivery Rev. 2005, 57, 1087.



1917

www.advhealthmat.de

FULL PAPER


1918

[25] a) E. R. Gillies, J. M. Frechet, Drug Discovery Today 2005, 10, 35; b) C. C. Lee, J. A. MacKay, J. M. Frechet, F. C. Szoka, Nat. Biotechnol. 2005, 23, 1517. [26] J. B. Wolinsky, M. W. Grinstaff, Adv. Drug Delivery Rev. 2008, 60, 1037. [27] a) H. Sarin, A. S. Kanevsky, H. Wu, K. R. Brimacombe, S. H. Fung, A. A. Sousa, S. Auh, C. M. Wilson, K. Sharma, M. A. Aronova, R. D. Leapman, G. L. Griffiths, M. D. Hall, J. Transl. Med. 2008, 6, 80; b) X. Gao, C. Li, Small 2014, 10, 426. [28] X. He, J. Gao, S. S. Gambhir, Z. Cheng, Trends Mol. Med. 2010, 16, 574. [29] A. Schmidt, M. Hoppen, J. K. Strecker, K. Diederich, W. R. Schabitz, M. Schilling, J. Minnerup, Exp. Transl. Stroke Med. 2012, 4, 13. [30] P. Ramos-Cabrer, F. Campos, T. Sobrino, J. Castillo, Stroke 2011, 42, S7. [31] M. Oechmichen, C. Meissner, J. Forensic Sci. 2006, 51, 880. [32] A. Popp, N. Jaenisch, O. W. Witte, C. Frahm, PLoS One 2009, 4, e4764. [33] H. Sarin, J. Angiogenes Res. 2010, 2, 14. [34] D. M. McDonald, P. Baluk, Cancer Res. 2002, 62, 5381. [35] V. Torchilin, Adv. Drug Delivery Rev. 2011, 63, 131. [36] P. Caravan, J. J. Ellison, T. J. McMurry, R. B. Lauffer, Chem. Rev. 1999, 99, 2293. [37] H. Xu, C. A. Regino, M. Bernardo, Y. Koyama, H. Kobayashi, P. L. Choyke, M. W. Brechbiel, J. Med. Chem. 2007, 50, 3185. [38] L. M. De Leon-Rodriguez, A. J. Lubag, C. R. Malloy, G. V. Martinez, R. J. Gillies, A. D. Sherry, Acc. Chem. Res. 2009, 42, 948.


[39] a) H. Xu, C. A. Regino, Y. Koyama, Y. Hama, A. J. Gunn, M. Bernardo, H. Kobayashi, P. L. Choyke, M. W. Brechbiel, Bioconjug. Chem. 2007, 18, 1474; b) K. Nwe, H. Xu, C. A. Regino, M. Bernardo, L. Ileva, L. Riffle, K. J. Wong, M. W. Brechbiel, Bioconjug. Chem. 2009, 20, 1412. [40] P. Caravan, Chem. Soc. Rev. 2006, 35, 512. [41] I. M. Macrae, Br. J. Pharmacol. 2011, 164, 1062. [42] H. Kobayashi, M. W. Brechbiel, Adv. Drug Delivery Rev. 2005, 57, 2271. [43] P. H. Chan, J. Cereb Blood Flow Metab. 2001, 21, 2. [44] Y. Yang, E. Y. Estrada, J. F. Thompson, W. Liu, G. A. Rosenberg, J. Cereb Blood Flow Metab. 2007, 27, 697. [45] R. B. Huang, S. Mocherla, M. J. Heslinga, P. Charoenphol, O. Eniola-Adefeso, Mol. Membr. Biol. 2010, 27, 312. [46] Z. Kovacs, K. Ikezaki, K. Samoto, T. Inamura, M. Fukui, Stroke 1996, 27, 1865. [47] C. Manabat, B. H. Han, M. Wendland, N. Derugin, C. K. Fox, J. Choi, D. M. Holtzman, D. M. Ferriero, Z. S. Vexler, Stroke 2003, 34, 207. [48] H. Yan, L. Wang, J. Wang, X. Weng, H. Lei, X. Wang, L. Jiang, J. Zhu, W. Lu, X. Wei, C. Li, ACS Nano 2012, 6, 410. [49] H. Yan, J. Wang, P. Yi, H. Lei, C. Zhan, C. Xie, L. Feng, J. Qian, J. Zhu, W. Lu, C. Li, Chem. Commun. 2011, 47, 8130. [50] M. D. Ginsberg, Neuropharmacology 2008, 55, 363. [51] J. Du, W.-L. Lu, X. Ying, Y. Liu, P. Du, W. Tian, Y. Men, J. Guo, Y. Zhang, R.-J. Li, J. Zhou, J.-N. Lou, J.-C. Wang, X. Zhang, Q. Zhang, Mol. Pharm. 2009, 6, 905.

