Thrombin-Responsive, Brain-Targeting Nanoparticles for Improved

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Thrombin-Responsive, Brain-Targeting Nanoparticles for Improved Stroke Therapy Xing Guo, Gang Deng, Jun Liu, Pan Zou, Fengyi Du, Fuyao Liu, Ann T. Chen, Rui Hu, Miao Li, Shenqi Zhang, Zhishu Tang, Liang Han, Jie Liu, Kevin N. Sheth, Qianxue Chen, Xingchun Gou, and Jiangbing Zhou ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b04787 • Publication Date (Web): 14 Aug 2018 Downloaded from http://pubs.acs.org on August 14, 2018

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Thrombin-Responsive, Brain-Targeting Nanoparticles for Improved Stroke Therapy Xing Guo,†,# Gang Deng,†,※ # Jun Liu,† Pan Zou,† Fengyi Du,† Fuyao Liu,† Ann T. Chen,†, Rui ,

⊥

Hu,‡ Miao Li,† Shenqi Zhang,†,※ Zhishu Tang,§ Liang Han,† Jie Liu,∆ Kevin N. Sheth,£ Qianxue Chen,※ Xingchun Gou,§,* and Jiangbing Zhou†,⊥,*

†

Department of Neurosurgery, ‡Cardiovascular Research Center, £Department of Neurology,

⊥

Department of Biomedical Engineering, Yale University, New Haven, CT 06510, USA.

※

Department of Neurosurgery, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060,

China §

Shaanxi Key Laboratory of Brain Disorders & Institute of Basic and Translational Medicine,

Xi’an Medical University, Xi’an, Shanxi 710021, China, ∆

Department of Biomedical Engineering, School of Engineering, Sun Yat-sen University,

Guangzhou, Guangdong 510006, China.

KEYWORDS: drug delivery, ischemic stroke, CXCR4, glyburide, protease-responsive

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ABSTRACT: Current treatments for ischemic stroke are insufficient. The lack of effective pharmacological approaches can be mainly attributed to the difficulty in overcoming the bloodbrain barrier. Here, we report a simple strategy to synthesize protease-responsive, brain-targeting nanoparticles for improved treatment of stroke. The resulting nanoparticles respond to proteases enriched in the ischemic microenvironment, including thrombin or matrix metalloproteinase-9, by shrinking or expanding their size. Targeted delivery was achieved using surface conjugation of ligands that bind to proteins that were identified to enrich in the ischemic brain using protein arrays. By screening a variety of formulations, we found that AMD3100-conjugated, size shrinkable nanoparticles (ASNPs) exhibited the greatest delivery efficiency. The brain targeting effect is mainly mediated by AMD3100, which interacts with CXCR4 that is enriched in the ischemic brain tissue. We showed that ASNPs significantly enhanced the efficacy of glyburide, a promising stroke therapeutic drug whose efficacy is limited by its toxicity. Due to their high efficiency in penetrating the ischemic brain and low toxicity, we anticipate that ASNPs have the potential to be translated into clinical applications for improved treatment of stroke patients.

Stroke is one of the leading causes of death and disability worldwide. Intravenous administration of recombinant human tissue-type plasminogen activator within three hours of symptom onset remains the only Food and Drug Administration (FDA)-approved pharmacotherapy for clinical management of this disease.1 With such a narrow therapeutic window, only around 7% of patients are eligible for this treatment.2 The lack of therapeutic approaches is primarily due to the inability of current treatments to overcome the blood-brain barrier (BBB) and deliver therapeutic agents to the brain. The BBB protects the brain from toxins


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and infections in normal physiological conditions. However, it also prevents penetration of most drugs into the brain for medical intervention.3 Although the BBB may be compromised after stroke insults, the degree of disruption is often not enough for delivery of pharmacologically significant quantities of drugs for effective treatment.4,5 Thus, improved treatment of stroke requires developing new approaches for enhancing drug delivery to the brain. Enhanced drug delivery to the brain is possible utilizing emerging nanotechnology approaches.6-9 In addition to providing protection to encapsulated cargos that are normally exposed to the circulatory system, nanoparticles can be engineered for brain-targeted delivery through surface conjugation of ligands that interact with receptors highly expressed in the BBB, such as transferrin receptors (TfR),10 or molecules enriched in the stroke microenvironment, such as fibrin fibrils.11 We recently demonstrated that the ligand-mediated, brain-targeted delivery can be further enhanced through internal encapsulation of BBB modulators that enable autocatalysis.7 By using poly(lactic-co-glycolic acid) (PLGA) nanoparticles as an example, we showed that a combination of surface conjugation of chlorotoxin (CTX) and internal encapsulation of lexiscan (LEX) increased delivery efficiency of nanoparticles to the ischemic brain significantly greater than either therapy alone. CTX is a 36-amino acid peptide with high specificity and affinity for matrix metalloproteinase 2 (MMP-2), which is up-regulated in the ischemic brain.12 LEX is a small molecule approved by the FDA for myocardial perfusion imaging and was found to transiently enhance the BBB permeability.13 Despite their potential, the existing engineering approaches may not be optimal. First, a number of ligands have been explored for potential brain-targeted drug delivery. However, none were selected based on an unbiased screen.7,10,11 Second, accumulating evidence suggests that traditional ligand-mediation approaches are insufficient for disease management in human patients and that the development


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of next generation nanoparticles requires incorporation of dynamic properties that respond to biological cues in the diseased microenvironment.14 Here, we report a simple strategy to synthesize ligand-conjugated, enzyme-responsive polymeric micellar nanoparticles for targeted drug delivery to the ischemic brain. Nanoparticles were fabricated using block copolymers consisting of polyethylene (PEG), poly(ε-caprolactone) (PCL), and enzyme-cleavable peptides. Targeted delivery was achieved using surface conjugation of ligands that target molecules enriched in the ischemic brain. We hypothesized that these nanoparticles would respond to enzymes enriched in the stroke microenvironment by expanding or shrinking their size. Nanoparticles in various formulations were evaluated for delivery efficiency in mice bearing ischemic stroke. Among all of the tested formulations, AMD3100-conjugated, size shrinkable nanoparticles (ASNPs) demonstrated the greatest efficiency. AMD3100 is a well-characterized antagonist of CXCR4 and has been previously employed as a ligand for targeting drug delivery to diseases in which CXCR4 is highly expressed.15,16 We further evaluated ASNPs for delivery of therapeutic molecules for stroke treatment. Glyburide, a diabetes medication that we recently found to be effective for human stroke patients, was used as a model drug.17 Due to the risk of hypoglycemia, the dose of glyburide used in patients is limited to 3 mg/day. Therefore, increased delivery of glyburide to the ischemic brain is required for improved glyburide therapy. We demonstrate that ASNPs significantly enhance the efficacy of glyburide for stroke treatment. RESULTS AND DISCUSSION In addition to surface conjugation of targeting ligands, improved delivery of nanoparticles for stroke therapy can also be achieved by engineering nanoparticles that respond to the ischemic microenvironment and subsequently alter their size in response. In one scenario, nanoparticles


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may shrink their size after reaching the ischemic brain, resulting in enhanced brain penetration (Figure 1a). In another scenario, nanoparticles may expand their size in the ischemic brain. As a result, reverse transcytosis from the brain to the blood can be prevented (Figure 1b). It was previously shown that the prevention of reverse transcytosis after brain penetration can efficiently increase drug delivery to the brain.18 Size-changeable nanoparticles can be synthesized using block copolymers bearing peptides that are cleavable by enzymes enriched in the ischemic microenvironment. Specifically, shrinkable nanoparticles can be synthesized using polymers consisting of four blocks, including a hydrophobic polymer, such as PCL, an enzymecleavable peptide, and two PEG segments. Upon enzyme cleavage, the outer hydrophilic corona formed by the peptide-linked PEG is shielded, resulting in stable, small nanoparticles (Figure 1c). The expandable nanoparticles can be synthesized using polymers that are synthesized simply by conjugating PCL to PEG using an enzyme-cleavable peptide. Upon enzyme cleavage, the balance of hydrophobicity and hydrophilicity is disrupted, resulting in collapse of the micellar structure and nanoparticle aggregation (Figure 1d).


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Figure 1. Schematic diagrams of shrinkable (a, c) and expandable (b, d) micellar nanoparticles in response to proteases enriched in the ischemic microenvironment.

Thrombin is highly expressed in the ischemic brain and has been previously used for targeted drug delivery for stroke treatment.19 In addition to thrombin, matrix metalloproteinase 9 (MMP9) is another protease that was reported to be highly enriched in the ischemic brain.20 Therefore, MMP-9 is considered as an alternative protease that can be targeted for protease-triggered drug delivery for stroke treatment. We confirmed that the expression of thrombin and MMP-9 in the ischemic brain was significantly elevated 3-8 h after ischemic insult (Figure S1a). To identify which protease mediates the delivery of nanoparticles with greater efficiency, we synthesized expandable nanoparticles using block copolymers containing either NH2-norleucine-TPRSFL-CSH, a thrombin-cleavable peptide,19 or NH2-LGRMGLPGK-C-SH, a MMP-9-cleavable peptide (Figure 2a,b).21 Block polymers were synthesized using a two-step reaction. First, PEGylated peptides were obtained by reacting maleimide-terminated PEG with cysteine-terminated peptides. Next, the PEGylated peptides were conjugated to carboxy-terminated PCL that was pre-activated by N,N’-carbonyldiimidazole (CDI). The resulting block copolymers bearing the thrombin- or MMP-9-cleavable peptide are designated as PEG-T-PCL or PEG-M-PCL, respectively. Using 1H NMR, the molecular weight of PEG-T-PCL and PEG-M-PCL was found to be 12,100 Da and 12,400 Da, respectively (Figure S1b, c). PEG-T-PCL and PEG-M-PCL micellar nanoparticles were synthesized by the standard nanoprecipitation method (Figure S2). We characterized the responsiveness of nanoparticles by incubating nanoparticles in PBS, PBS with 100 nM thrombin, or PBS with 100 nM MMP-9. Nanoparticle size change was monitored by dynamic light scattering (DLS). After 24 h in PBS without proteases, PEG-T-PCL nanoparticles and PEG-M-PCL nanoparticles, which had an average diameter of 105 nm and 107


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nm, respectively, did not change in size (Figure 2c,d). In contrast, in the presence of proteases, both nanoparticles responded within 5 minutes, expanded gradually over time and precipitated out from the PBS. By the end of 24 h, the average sizes of the remaining PEG-T-PCL nanoparticles and PEG-M-PCL nanoparticles were 1,283 nm and 1,118 nm, respectively. The observed responsiveness is specific to the cleavage motifs in the corresponding nanoparticles, as we found that the treatment with MMP-9 did not change the size of PEG-T-PCL nanoparticles, and the treatment with thrombin did not change the size of PEG-M-PCL nanoparticles. As a control, PEG-PCL nanoparticles synthesized using the same polymer but without proteasecleavable peptides did not respond to thrombin or MMP-9 (Figure 2e). The responsiveness of nanoparticles to protease treatments was confirmed by transmission electron microscopy (TEM) (Figure 2c-e). We determined if the protease-responsive property of PEG-T-PCL and PEG-M-PCL nanoparticles was capable of enhancing drug delivery for stroke treatment. PEG-T-PCL nanoparticles and PEG-M-PCL nanoparticles, along with control PEG-PCL nanoparticles were synthesized encapsulating IR780, a near-infrared fluorescence dye that allows for non-invasive detection in live animals. The resulting IR780-encapsulated nanoparticles were administered to stroke-bearing mice. After 24 hours, mice were imaged using IVIS. Because the BBB is partially compromised by stroke insults,5,22 PEG-PCL nanoparticles were found to slightly accumulate in the ischemic region. However, compared to PEG-PCL nanoparticles, both PEG-T-PCL nanoparticles and PEG-M-PCL nanoparticles demonstrated significantly greater efficiencies (Figure 2f). Among all the three tested formulations, PEG-T-PCL nanoparticles exhibited the greatest efficiency. The average fluorescence intensity in the ischemic region in mice treated with PEG-T-PCL nanoparticles was 5.5- fold and 2.2- fold greater than those for mice treated


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with PEG-PCL nanoparticles and PEG-M-PCL nanoparticles, respectively (Figure 2g). The enhanced delivery of PEG-T-PCL nanoparticles is likely mediated by thrombin accumulated in the ischemic brain, which maintains the activity to cleave substrates (Figure S1a,d). Therefore, we selected the PEG-T-PCL nanoparticles for further optimization.

Figure 2. Synthesis and evaluation of expandable nanoparticles. (a) Scheme of chemical synthesis of PEG-T/M-PCL block copolymers. (b) Sequences of thrombin- and MMP-9- cleavable peptides. (c) DLS (left) and TEM (right) analyses of PEG-T-PCL nanoparticles in PBS with or without thrombin/MMP-9 (100 nM). (d) DLS (left) and TEM (right) analyses of PEG-M-PCL nanoparticles in PBS with or without MMP-9/thrombin (100 nM). (e) DLS (left) and TEM (right) analyses of PEG-PCL nanoparticles in PBS, and PBS containing either thrombin or MMP-9. (f) Representative images of brains isolated from mice


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that received the indicated treatments. (g) Semi-quantification of nanoparticles in the excised brains based on IR780 fluorescence intensity. Intensity was quantified using Living Image 3.0.

We set to compare expandable and shrinkable nanoparticles for drug delivery for stroke treatment. Shrinkable nanoparticles were synthesized using PEG-PCL-T-PEG, which was obtained a two-step reaction (Figure 3a). First, mPEG-OH was used to initiate the ring-opening polymerization of ε-caprolactone. The resulting PEG-PCL was then activated by CDI and reacted with NH2-norleucine-TPRSFL-C-PEG, which was prepared by coupling NH2norleucine-TPRSFL-C with mPEG-MAL. Analysis by 1H NMR showed that PEG-PCL-T-PEG has a molecular weight of 16,400 Da (Figure S3), which is comparable to that of PEG-T-PCL. PEG-PCL-T-PEG nanoparticles were synthesized using standard nanoprecipitation procedure. DLS analysis showed that PEG-PCL-T-PEG nanoparticles were stable in PBS and PBS containing MMP-9. However, upon treatment with thrombin, the average size of the nanoparticles decreased gradually from 218.2 nm to 79.3 nm within 24 h, which was further confirmed by TEM analysis (Figure 3b). We compared PEG-PCL-T-PEG shrinkable nanoparticles with PEG-T-PCL expandable nanoparticles for drug delivery to the ischemic brain. Non-responsive PEG-PCL nanoparticles were used as a control. All nanoparticles were synthesized with encapsulation of IR780 and intravenously administered to stroke-bearing mice using the same procedures as described above. PEG-PCL-T-PEG shrinkable nanoparticles were found to exhibit the greatest delivery efficiency (Figure 3c). Based on the fluorescence intensity, the accumulation of PEG-PCL-T-PEG nanoparticles in the ischemic region was 9- fold and 4.5fold greater than those of PEG-PCL nanoparticles and PEG-T-PCL nanoparticles, respectively (Figure 3d). Based on those results, we selected PEG-PCL-T-PEG shrinkable nanoparticles for the remainder of the study.


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Figure 3. Synthesis and evaluation of shrinkable nanoparticles. (a) Scheme of chemical synthesis of PEGPCL-T-PEG. (b) DLS (left) and TEM (right) analyses of PEG-PCL-T-PEG nanoparticles in PBS with or without thrombin/MMP-9 (100 nM). (c) Representative images of the brains isolated from mice received the indicated treatments. (d) Semi-quantification of nanoparticles in the excised brains based on IR780 fluorescence intensity.

Targeted delivery of nanoparticles for stroke treatment can be achieved using surface conjugation of ligands that interact with molecular targets enriched in the ischemic microenvironment. To identify such targets, we profiled proteins in both the ischemic brain and normal brain using RayBio L-90 Antibody Array (RayBiotech, USA), which is capable of simultaneously detecting 90 proteins. Most of the selected proteins detected by the L-90 Array were growth factors, angiogenic factors, proteases, soluble receptors, soluble adhesion molecules, cytokines, chemokines, and adipokine, most of which are potential receptors for targeted delivery. In order to identify proteins that are associated with the severity of ischemia,


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we dissected the right hemisphere containing the ischemic region and the contralateral left hemisphere without ischemia from two groups of animals, which received occlusion for 60 min and 90 min, respectively. Expression of candidate proteins was quantified based on their signal intensities detected in the array. For each protein, the ratio of its signal intensity in the ischemic hemisphere to that in the contralateral hemisphere without ischemia was calculated. We found that 19 out of the 90 candidate proteins had significantly different levels of expression in the ischemic hemisphere, compared to those in the contralateral control (Figure 4a,b and Table S1). Among them, Mac-1, TIE-2, CXCR4 and MMP-2 had been previously studied for their binding ligands. Based on previous studies, we selected ligands that are known to bind those candidate proteins, including P2 recognizing integrin αmβ2,23 TH1 recognizing TIE-2,24 and AMD3100 recognizing CXCR4 (Figure 4b).16 We previously showed that targeting MMP2 using CTX enhanced drug delivery to the ischemic brain.7 Therefore, we chose CTX as a benchmark to evaluate other candidate ligands. Among all candidate proteins, Mac-1, which is also called integrin αmβ2, was found to have the greatest increase in expression level in the 90 min occlusion cohorts, suggesting that other integrin family proteins may also be elevated in the ischemic brain. Therefore, we also included cRGD, which recognizes a variety of integrins,25 in the evaluation. To enable surface modification, we synthesized MAL-PEG-PCL-T-PEG using the same procedure that was used for synthesis of PEG-PCL-T-PEG, except that MAL-PEG-OH was used to initiate the ring-opening polymerization of ε-caprolactone (Figure S4a). After standard nanoprecipitation procedures, nanoparticles were obtained with surface display of maleimide groups. Peptide ligands, which were synthesized with a cysteine terminal (P2 and TH1) or engineered to display a SH group through thiolation (CTX and cRGD), were conjugated to the surface of nanoparticles through a thiol-maleimide reaction. AMD3100 was conjugated


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according to a previously reported method.26 We found that ligand conjugation did not alter the size and shape of nanoparticles (Figure S4b). Ligand-conjugated nanoparticles, along with control PEG-PCL-T-PEG nanoparticles without surface modifications, were evaluated in strokebearing mice using the same procedures as described above. Among all of the candidate ligands, AMD3100 demonstrated the greatest targeting effect (Figure 4c). Based on the fluorescence intensity, the accumulation of AMD3100-conjugated PEG-PCL-T-PEG nanoparticles in the ischemic region was 30- fold and 3.6- fold greater than those of control nanoparticles without ligands and CTX-conjugated nanoparticles, respectively (Figure 4d). Biodistribution analysis showed that the concentration of nanoparticles in the ischemic brain is comparable to that in the liver (Figure S4c). In addition to the high efficiency, AMD3100-conjugated nanoparticles also demonstrated great specificity to the ischemic region. There was high overlap between the location of ischemia (white, TTC staining) and the location of nanoparticles (red to yellow, IR780 signal) (Figure 4e). Western Blot confirmed that the level of CXCR4 was significantly elevated in the ischemic brain (Figure 4f), suggesting enhanced delivery of the nanoparticles is likely mediated through the interaction between AMD3100 and CXCR4. To simplify the nomenclature, we designated AMD3100-conjugated, size shrinkable nanoparticles as ASNPs. The upregulation of CXCR4 protein determined by Western Blot (6-fold at the 12 h time point) is greater than that determined by the Antibody Array. This is because that the array experiment was performed using the entire right hemisphere containing the ischemic region and the contralateral left hemisphere without ischemia. Considering the fact that the ischemia occurs in a small region of the right hemisphere, the upregulation determined by the array was underestimated that specifically in the ischemic tissue. To identify the cell types expressing CXCR4, we performed immunofluorescence staining on the ischemic brains isolated from


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MACO mice 24 h after surgery. Antibodies targeting GFAP, Tuj1, and Olig1 were used to identified astrocytes, neurons, and oligodendrocytes, respectively. The results shown in Figure S5 suggested that CXCR4 were mainly expressed in neurons and oligodendrocytes, which is consistent with previous reports.27,28 We further characterized the ASNPs. 1H NMR analysis by calculating the integral intensity ratios suggests that 20% of PEG-PCL-T-PEG was conjugated with AMD3100 molecules. The disappearance of the characteristic peaks of hydrophobic PCL block in D2O indicates that the AMD3100-PEG-PCL-T-PEG polymer was successfully self-assembled into a core/shell micellar structure in aqueous solution (Figure S6). DLS analysis showed that the ASNPs are in diameter of 226.3 nm with a polydispersity index of 0.121, and bear a surface charge of +5.85 mV (Figure S7). We determined the kinetics of ASNP accumulation in the ischemic brain. After MCAO surgery, mice were treated with IR780-loaded ASNPs through intravenous administration and imaged at 1, 3, 6, and 24 h by IVIS. Results in Figure S8 showed that the intensity of IR780 fluorescence in the ischemic region continuously increased over the first 24 hours. IR780 is a lipophilic cyanine dye. A recent study found that IR780 may not be a reliable indication of the extravasation/penetration of certain nanoparticle, as the observed distribution and kinetics of fluorescence may be originated from the dye payload released into the blood stream during circulation.29 To exclude the possibility that the observed accumulation of ASNPs in this study based on IR780 fluorescence was originated from free IR780 released from the nanoparticles, we intravenously administered free IR780 to MCAO mice and compared the accumulation of fluorescence in the ischemic region with that in mice received ASNPs encapsulated the same amount of IR780. The comparison study was further repeated using a non-lipophilic dye, rhodamine B (RhoB). Results in Figure S9a,b showed that delivery via


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ASNPs enhanced the accumulation of both dyes in the ischemic region by 11-fold, suggesting that the impact of released payload dyes is limited in this study. Nonetheless, this observation doesn’t exclude the possibility that the observed fluorescence is partially from the free dyes released from ASNPs, which does not directly indicate the localization of ASNPs. Therefore, determination of the accumulation of ASNPs in the brain using fluorescent dyes as surrogates may suggest the overall trend of ASNP accumulation but not the actual quantity. Last, we characterized the pharmacokinetics of ASNPs in mice. RhoB-loaded ASNPs were intravenously injected into mice. Afterwards, the blood was collected at various time points. The concentration of RhoB in the plasma was determined and plotted versus time (Figure S10). We found that the half-life of ASNPs in the circulatory system was 22.4 h. Other pharmacokinetic parameters were summarized in Table S2. Different from the observation in the brain in which the fluorescence intensity continuously increased over the first 24 hours (Figure S8), the fluorescence intensity in the blood reached a peak around 1 hour following injection (Figure S10). The observed difference in nanoparticle kinetics is likely because of two reasons. First, due to the existence of the BBB, nanoparticles after penetration into the brain may not be able to efficiently transport back to the circulatory system. Second, nanoparticles in the blood circulation may be efficiently eliminated by the mononuclear phagocyte system (MPS).14 By contrast, microglia, the resident immune cells in the brain, may not have the quantity and efficiency to allow eliminating nanoparticles in a rate comparable to that in the circulatory system.


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Figure 4. Identification and selection of ligands for targeted delivery of nanoparticles to stroke. (a) Heat diagram of proteins that are differentially enriched in the ischemic brain and in the control normal brain (*P < 0.05). (b) Candidate ligands that bind to the selected proteins. (c) Representative images and (d) semi-quantification of nanoparticles in the brains of MCAO mice received treatments with the indicated ligand-conjugated nanoparticles. (e) Representative images of brain slices with TTC staining (left) and fluorescence imaging (right). (f) Western Blot confirmed that the level of CXCR4 significantly elevated in the ischemic brain. Negative control: 293T cell lysate. Positive control: U87 flank tumor lysate. Control: normal brain tissue.

We assessed if ASNPs could improve stroke treatment. Glyburide, a promising stroke therapeutic drug whose efficacy has been significantly limited by the dose that can be administered,17 was used as a cargo drug. Glyburide was encapsulated into ASNPs with efficiency of 65% or loading of 0.33% by weight. Encapsulation of glyburide did not alter the


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morphology of nanoparticles (Figure 5a). In presence of thrombin, ASNPs released over 90% of glyburide within 48 h. By contrast, without thrombin, only 28% of glyburide was released (Figure 5b). We evaluated glyburide-loaded ASNPs in MCAO mice. ASNPs were administered at a dose equivalent to 5 µg/kg of glyburide through i.v. injection 0, 24, and 48 h after surgery. Mice were monitored for survival and behavior, and were euthanized at day 7. We found that treatment with glyburide-loaded ASNPs significantly improved mouse survival (Figure 5c, P = 0.02), reduced infarct volumes by 36% (Figure 5d and Figure S11), and improved neurological scores (Figure 5e). The biological activities of glyburide on stroke mainly depend on its antiswelling effect.17,30 We determined the impact of treatment with glyburide-loaded ASNPs on brain edema. After MCAO surgery, mice were randomly grouped and treated with PBS or glyburide-loaded ASNPs. Twenty-four hours later, the mice were euthanized and the brains were isolated and weighted. We found that the average water content in the ipsilateral hemispheres isolated from mice received glyburide-loaded ASNPs was 80.8 ± 2.2%, which is significantly lower than that in control mice, which was 84.0 ± 1.3% (Figure S12). These results suggest that the observed therapeutic benefit is likely resulted from reducing cerebral edema.

Figure 5. AMD3100-conjugated shrinkable nanoparticles for intravenous delivery of glyburide. (a) The morphology of nanoparticles was determined by TEM. (b) Release of glyburide in PBS with and without thrombin (100 nM). (c) Kaplan-Meier survival analysis (n=10), (d) infarct area (day 3 after surgery, n =


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5), and (e) neurological scores (day 3 after surgery, n = 5) of MCAO mice receiving the indicated treatments.

The targeting effect of ASNPs is largely mediated by AMD3100, which interacts with CXCR4 in the ischemic brain. AMD3100 is a potent antagonist of CXCR4. Accumulation of AMD3100 in the ischemic brain may antagonize CXCR4, one of the major chemotactic factors that determine the sequelae of inflammatory.31 However, this was unlikely happened in our animal studies, in which glyburide- loaded ASNPs were administered at a dose equivalent to 5 µg/kg of glyburide, or equivalent to 1.8 nM PCL-M-PEG, per injection. We determined that 20% of PEG-PCL-T-PEG was conjugated with AMD3100 molecules (Figure S6). Therefore, in the treatment studies, each mouse received intravenous administration of AMD3100 at 0.4 nM, which is significantly below 44 nM, the concentration required for inhibiting 50% of CXCR4 activity.32 Therefore, it is unlikely that treatment with ASNPs has a significant biological effect on CXCR4 activity. To confirm this, we performed an immunostaining study, in which the impact of ASNP treatment on macrophages was evaluated using an anti-CD68 antibody. Treatments with PBS, free AMD3100 at 1.2 µg, and free AMD3100 at 20 µg were included as controls. Results in Figure S13 showed that treatment with ASNPs or free AMD3100 at 1.2 µg did not alter the presence of macrophages, although the presence of macrophages was reduced by 62% in mice treated with free AMD3100 at 20 µg. Improved treatment of stroke requires developing new approaches to enhance the delivery of therapeutics to the ischemic brain, which can be achieved by surface conjugation of ligands targeting the BBB,10 or the ischemic microenvironment.7,

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However, ligands for targeted

delivery for stroke treatment has not been systemically explored. Previously, only a handful of ligands,

including

CTX,

anti-TfR

antibody,

and

anti-fibrin

antibody,

have

been


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characterized.7,10,11 In this study, by profiling proteins enriched in the ischemic brain, we identified several molecules that can be targeted for enhanced drug delivery for stroke treatment. After further evaluation, we found that AMD3100, a ligand that was previously explored by others for targeted cancer delivery,26 enhances delivery of nanoparticles to the ischemic brain with an efficiency 3.6 times greater than CTX, which we recently identified as a therapeutic for brain-targeted drug delivery.7 In addition to surface modification, we also showed that improved delivery of nanoparticles to the ischemic brain can be achieved by engineering nanoparticles to respond to biological cues in the ischemic microenvironment. Size-shrinkable nanoparticles have been recently explored by others for cancer drug delivery. A previous study demonstrated that gelatin nanoparticles, which are 100 nm in diameter, can respond to MMP-2 in the tumor microenvironment and release cargo nanoparticles 10 nm in diameter.33 Another study used polymeric clustered nanoparticles, which have an initial diameter of 100 nm. After reaching the tumor microenvironment, the nanoparticles were able to respond to extracellular acidity and release 5 nm nanoparticles.34 In both studies, the authors found that these “shrinkable” nanoparticles significantly enhanced drug delivery to tumors.33,34 However, size expandable nanoparticles had yet not been explored. In this study, we found that, both size shrinkable and expandable nanoparticles penetrate the ischemic brain with efficiency greater than non-responsive nanoparticles. Compared to the expandable nanoparticles, the size shrinkable nanoparticles demonstrated a greater efficiency. We found that the upregulation of thrombin was detected between 3-8 h after ischemic insult and the expression level of thrombin increased with time over at least 12 hours (Figure S1). The half-life of ASNPs was determined to be 22.4 h (Table S2). Therefore, the expression of thrombin is early enough to make the thrombin-depending strategy useful. Consistently, results


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in Figure S8 suggested that the accumulation of ASNPs in the ischemic region increases continuously with time over the first 24 h. For the treatment of acute diseases such as stroke using nanoparticles, fast response/drug release may be desired. We showed that ~30% and ~80% of ASNPs responded to thrombin cleavage and reduced their sizes at 1 h and 6 h, respectively (Figure 3b). Accordingly, in the presence of thrombin, 40.2%, 69.5%, and 83.7% of cargo molecules was released from ASNPs within 1, 6 and 24 h, respectively (Figure 5b). The degree of drug release rate appears to be sufficient for stroke treatment (Figure 5c-e). We showed that ASNPs, the formulation that demonstrated the greatest delivery efficiency, significantly enhanced the therapeutic benefit of glyburide for stroke treatment (Figure 5). Glyburide is an FDA-approved diabetes medication, which binds to SUR1 with subnanomolar affinity.30 Recently, the SUR1-regulated NCCa-ATP channel was found to be a major contributor to cerebral edema, which is responsible for high mortality and morbidity rates in stroke patients.30 To determine the therapeutic benefits of targeting SUR1, we tested glyburide in human stroke patients.17 Our results showed that there was a trend toward improved survival and reduced midline shift, a surrogate biomarker of brain edema, by ~40%.17 However, we found that further enhancing the clinical efficacy of glyburide could not be achieved due the limitations associated with the current glyburide formulation, which is free drug in solution. Free glyburide is known to have a limited ability to penetrate the brain.35 Additionally, as a diabetes medication, free glyburide can only be given at a low dose (3 mg/d) to avoid hypoglycemia. In this study, we were able to overcome these limitations associated with the free glyburide formulation and may fully utilize glyburide’s therapeutic potential. We showed that ASNPs were able to alter the biodistribution of glyburide by increasing its accumulation in the brain (Figure 4). Thus, a greater dose of glyburide can be administered without adverse side effects. In addition, ASNPs


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can provide controlled release of glyburide over time (Figure 5b), whereas current clinical applications of glyburide require continuous infusion.

CONCLUSIONS In conclusion, we have developed protease-responsive, brain-targeting nanoparticles for targeted delivery to the ischemic brain. Major components of ASNPs include PCL, PEG, and AMD3100, all of which have been approved by the FDA for clinical use. Due to its high efficiency in penetrating the ischemic brain and its construction from safe materials with minimal toxicity, glyburide-loaded ASNP have the potential to be translated into clinical applications for improved treatment of patients with stroke.

MATERIALS AND METHODS Materials. Matrix metalloprotease 9 (MMP-9)-cleavable peptide (NH2-LGRMGLPGK-CSH), thrombin-cleavable peptide (NH2-norleucine-TPRSFL-C-SH), P2, CTX, cRGD, and TH1 were purchased from AnaSpec. Methoxy poly(ethylene glycol) maleimide (mPEG-MAL, MW = 5000) and maleimide poly(ethylene glycol) (MAL-PEG-OH, MW = 5000) were purchased from Jenkem Technology. Polycaprolactone, ε-caprolactone, and Tin(II) 2-ethylhexanoate were purchased from Sigma-Aldrich. AMD3100 octahydrochloride was purchased from Santa Cruz Biotechnology. N,N′-Bis(acryloyl)cystamine was purchased from Alfa Aesar. Synthesis and Characterization of PEG-M/T-PCL and PEG-PCL-T-PEG. Detailed procedure of nanoparticle synthesis and characterization is described in the Supporting Information.


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Enzyme-Triggered Size Change. To test the response of nanoparticles to enzyme, 1 mg/mL nanoparticles were incubated with 100 nM MMP-9 or thrombin at 37 °C. As a control, PEG-PCL nanoparticles without enzyme-sensitive linkers were also incubated with 100 nM MMP-9 or thrombin. DLS and TEM were used to detect the time-dependent size distribution and morphological change of the nanoparticles. Thrombin-Triggered Drug Release. To investigate the thrombin-triggered drug release, the glyburide-loaded PEG-PCL-T-PEG nanoparticles were placed into a dialysis tube (MWCO 3000) against PBS buffer in the presence or absence of 100 nM thrombin. The tubes were kept at 37 °C in an incubator at a shaking speed of 100 cycles/min. 200 µL buffer was withdrawn at different time points for glyburide analysis by using a HPLC instrument consisting of a Shimadzu SIL-10A system (Kyoto, Japan) with an Ascentis C18 separation column. The mobile phase, consisting of 45% buffer (50 mM NH4H2PO4) and 55% acetonitrile, was pumped at a flow rate of 1 mL/min. Detection was performed using a UV detector (Shimadzu RF-10A, Kyoto, Japan) with an absorbance wavelength of 254 nm. The glyburide standards were prepared by dilution of a glyburide stock solution (1 mg/mL in methanol) in PBS at 20 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, and 1 µg/mL. Animals. Male C57BL/6 mice (Charles River Laboratories, Willimantic, 191 CT, USA), 1822 g each, were given free access to food and water before all experiments. All animal experiments were approved by the Yale University Institutional Animal Care and Utilization Committee. Middle Cerebral Artery Occlusion (MCAO). MCAO models were generated according to methods that we recently reported.7, 36 Briefly, the animals were anesthetized with 5% isoflurane (Aerrane, Baxter, Deerfield, IL) in 30%O2/70%N2O using a Tabletop Anesthesia system


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(Harvard Apparatus, USA). Isoflurane was then maintained at 1.5%. During the procedures, the body temperature of mice was maintained at 37.0 ± 0.5 °C. Regional cerebral blood flow (rCBF) was monitored using a laser Doppler flowmeter (AD Instruments Inc.) duration the course of surgery. Mice were placed in the supine position, and a middle neck incision was made under a dissecting microscope (Leica A60). The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully exposed and dissected from the surrounding tissue. Then, a small hole in the ECA was made using Vanes-style spring scissors. A 6-0 silicon-coated mono-filament suture (Ducal Corporation) was introduced into the ECA and gently advanced from the lumen of the ECA into the ICA at a distance of 8-10 mm beyond the bifurcation to occlude the origin of middle cerebral artery. Successful MCA occlusion was confirmed by a reduction of rCBF by over 80%. The occlusion lasted 90 min and the monofilament was withdrawn to allow for reperfusion. Protein Profiling Using RayBio® Antibody Array. Ischemic tissue and control normal tissue were dissected from the same animal, lysed in Cell Lysis Buffer with protease inhibitors, and subjected to quantification using RayBio L-90 Antibody Arrays (AAR-BLG-1, RayBiotech) in accordance with the manufacturer’s instructions. Signal intensity for each protein was obtained using GenePix 4000B Microarray Scanner. A positive control (IgG) and negative control (buffer) were used to normalize the results from different membranes. Fluorescent Imaging of Nanoparticles. Mice with successful MCAO surgery were prepared and randomly assigned into experimental groups (n = 3). Immediately after surgery, IR780- or RhoB- loaded nanoparticles were administered intravenously through the tail vein. Dose for each group was adjusted according to the fluorescence intensity to ensure that each mouse received the same amount of dye. Twenty-four hours later, mice were sacrificed to isolate the brain and


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imaged by IVIS imaging system (Xenogen) with excitation wavelength of 745 nm and emission wavelength of 820 nm for IR780 or IR780- loaded nanoparticles, and excitation wavelength of 535 nm and emission wavelength of 600 nm for RhoB or RhoB- loaded nanoparticles. Fluorescence intensity in each brain was quantified using Living Image 3.0 (Xenogen). Determination of the Therapeutic Benefits. For the study to determine the effect of treatments on survival, mice with successful MCAO surgeries were randomly divided into 4 groups (n = 10 for each group), which received treatment of PBS, blank ASNPs, glyburideloaded ASNPs at a dose equivalent to 5 µg/kg of glyburide, and the same amount of free glyburide, respectively. Different from the aforementioned characterization studies, in which mice received single injection of nanoparticles, mice in the treatment studies received three injections, which were given intravenously at 0, 24 and 48 h after surgery. Mice were monitored for survival for 7 days and were euthanized if one of the following criteria was met: (1) the mouse's body weight dropped below 15% of its initial weight, or (2) the mouse became lethargic or sick and unable to feed. For the study to determine the impact of treatments on infarct volume and neurological score, a cohort of mice were prepared (n = 5 for each group) and received the same treatments as described above. Three days later, the neurological score of each mouse was assessed by a standard behavioral test,7, 36 and were scored as follows: (1) normal motor function, (2) flexion of torso and contralateral forelimb when animal was lifted by the tail, (3) hemiparalysis resulting in circling to the contralateral side when held by tail on flat surface, but normal posture at rest, (4) leaning to the contralateral side at rest, and (5) no spontaneous motor activity. Therapeutic evaluations were carried out using an unbiased approach; the reviewer who scored mouse function was unaware of which treatment group each mouse belonged to. After the evaluation, the mice were sacrificed and the brains were excised, sectioned, and stained with


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TTC. The infarct area in each slice was quantified using ImageJ. The infarct volume was calculated by the formula described as: Corrected infarct volume (%) = (contralateral hemisphere volume – non-infarcted ipsilateral hemisphere) / contralateral hemisphere volume × 100. For the study to determine the effect of treatments on brain edema, MCAO mice were prepared and received a single injection of PBS, or glyburide-loaded ASNPs at a dose equivalent to 5 µg/kg of glyburide immediately after surgery (n = 5 for each group). After 24 hours, the mice were sacrificed and the brains were excised, and weighted to obtain the wet weight. Then, the brains were lyophilized for 24 h and weighted to obtain the dry weight. Tissue water content was calculated as: Tissue water (%) = (wet weight-dry weight)/wet weight × 100. Statistical Analysis. All data were collected in triplicate and reported as mean and standard deviation. Comparison between the groups were performed using a t-test. One-way ANOVA was used to analyze multiple comparisons by GraphPad Prism 7.0. *P < 0.05, **P < 0.01 and ***P < 0.001 were considered significant.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Experimental details including Supporting Materials and Methods, Characterization of protease expression and polymers (Figure S1), Schematic illustration showing the procedure of nanoprecipitation (Figure S2), 1H NMR spectrum of PEG-PCL-T-PEG polymer (Figure S3), Synthesis and characterization of polymers and nanoparticles (Figure S4), Immunostaining using anti-GFAP, anti-Tuj1 and anti-Olig1 antibodies (Figure S5), 1H NMR spectrum of ASNPs (Figure S6), DLS characterization of ASNPs


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(Figure S7), Semi-quantification of the accumulation of ASNPs in the ischemic brains (Figure S8), Imaging and semi-quantification of IR780 and RhoB (Figure S9), Plasma concentrations of RhoB verse time (Figure S10), Representative TTC staining images of brain slices (Figure S11), Effects the indicated treatment on brain edema (Figure S12), Representative images and quantification of macrophages (Figure S13), List of candidate proteins (Table S1), and Pharmacokinetic parameters of RhoB-loaded ASNPs (Table S2).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] *E-mail: [email protected] . Author Contributions #

X. G. and G. D. contributed equally to this work. J. Z. designed the experiments. X. G., G. D., J.

L., P. Z., F. L., Z. T. R.H., M.L., and G. X. performed the experiments. All the authors were involved in the analyses and interpretation of data. J. Z., X. G., and G. D. wrote the paper, with the help of the co-authors.

ACKNOWLEDGMENT This work was supported by NIH Grants NS095817 (JZ) and NS095147 (JZ), AHA grants 15GRNT25290018 and 18TPA34170180 (JZ), State of Connecticut (JZ). X. Guo was partially supported by NSFC grant 51603172, and X. Gou was partially supported by NSFC grant 81471415 and Projects of International Cooperation and Exchanges Natural Science Foundation of Shanxi Province of China (No. 2018KW-038).


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