Schematic of Cy7-micelle and Cy7-CREKA-micelle targeting glioma via the EPR effect and active binding to fi brin. 

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... Fig. 7 shows the biodistribution of micelles throughout the liver, spleen, kidneys, bladder, large intestines, heart, and lung at 3 h, 24 h, and 7 days after injection using optical imaging (Fig. 7). At all time points, both Cy7- and Cy7-CREKA-micelles were found to be primarily localized in the liver. Micelles also showed greater accumulation in the kidney and bladder when compared to the rest of the organs. By 7 days, both types of micelles were mostly cleared from circulation and were found to reside only within the liver. As the liver and kidney are vital organs for metabolism and excretion, tissues were stained with H & E, examined using light microscopy, and representative images from mice treated with PBS, Cy7-micelles, or Cy7-CREKA-micelle at all time points are pre- sented. As seen in Fig. 8, no apparent tissue or cellular damage was observed in mice injected with either micelles and were comparable to images obtained from mice injected with PBS, and no pathological changes were observed. The treatment of glioblastoma is limited due to the aggressive nature of the tumor and the inability for complete surgical excision. As a result, GBM prognosis remains poor, with a 14.6 month median survival time despite interventions [1]. Chemotherapy against glioblastoma has not translated into meaningful improvements in patient outcome due to low permeability and poor penetration across the BBB [23,24]. Furthermore, systemically delivered chemotherapy poses challenges including high toxicity and numerous side effects [24]. In contrast, the rational design of targeted delivery vehicles has the potential to ef fi ciently unload drugs to the tumor site, while sparing the surrounding healthy tissue and the function of vital organs. In this study, fl uorescently-labeled micelle nanoparticles functionalized with the pentapeptide, CREKA, were intravenously injected in a mouse model of glioma. Since CREKA has been shown to target other types of tumors in vivo by binding to fi brin intra- vascularly and fi brin deposition is also present in primary and metastatic brain tumors, CREKA-micelles were applied as targeting agents for gliomas [18 e 20]. The CMC of micelles was close to 1 m M , which is comparable to micelle systems composed of DSPE- PEG(2000) and 100 m l of 1 m M micelle solution was chosen to be administered in mice because the concentration remains above the CMC even upon injecting into approximately 2 mL of circulating blood volume (Fig. 1A) [21,25]. The conjugation of CREKA to DSPE- PEG(2000)-maleimide slightly increased the micelle size from 7.6 Æ 1.2 to 8.2 Æ 1.2 nm (Table 1, Fig. 1B). As particles from 5 to 250 nm are reported to exhibit higher transport ef fi ciency to tumors, the nanoparticles obtained here are within the range of this requirement [8,26 e 28]. The addition of the peptide also changed the zeta potential of micelles from À 27.4 Æ 4.5 to À 12.6 Æ 1.6 mV. The surface charge of Cy7-micelles can be attributed to the phos- phate group of the DSPE tail, PEG molecules, and the maleimide, which is increased by the addition of the arginine and lysine- containing peptide. Cy7 was chosen as the fl uorophore to be incorporated into micelles because its excitation and emission wavelength in the near infrared range is optimal to achieve both deep tissue penetration and low auto- fl uorescence for in vivo imaging [29]. Fig. 4 con fi rmed Cy7-CREKA-micelles accumulate in the brain by 1 h and that the amount of accumulation can be monitored and quanti fi ed through in vivo imaging, supporting the possibility for quantitative diagnostics using this system. When brains were excised after 3 h, 24 h, and 7 days post-injection, fl uorescence was detectable in tumors for both Cy7-micelles and Cy7-CREKA-micelles, con fi rming that the micelles passively target gliomas by the EPR effect and that the CREKA-modi fi ed micelles can further increase retention by active targeting (Fig. 5). Accumulation for both Cy7-micelles and Cy7-CREKA-micelles between 3 h and 24 h post-injection was not statistically signi fi cant although slightly higher at the latter time point, suggesting that our micelle delivery system has the potential to deliver the maximum amount of payload within 3 h after administration and is maintained for at least 24 h. Moreover, it is possible that all of the fi brin-binding sites of the tumor are occu- pied by 3 h; since micelles were constructed with a molar ratio of 10:90 for Cy7- and CREKA-containing amphiphiles, future drug delivery studies will alter the peptide amphiphile ratio to exploit micelle binding and offer the most ef fi cient and highest amount of therapy to the tumor site. After 7 days, micelles were cleared from the brain. Notably, no fl uorescence signal was found when either Cy7-micelles or Cy7- CREKA-micelles were administered in a brain injury model. This con fi rms that the intracranial injection technique did not contribute to micelle accumulation to the brain, but instead, micelles target speci fi cally to tumors via penetration of the compromised BBB. Upon histological examination, the fl uorescence signal followed the optical imaging trends in which Cy7-CREKA-micelles accumulate in the tumor to a greater extent than Cy7-micelles (Fig. 6). Concentrated, fl uorescent dots may represent micelles entrapped within the tumor blood vessels or thrombi, whereas dimmer regions of fl uorescence may represent extravascular diffusion across the blood-tumor-barrier (BTB) of Cy7-CREKA-micelles into the glioma tissue due to increased accumulation (Fig. 1) [27]. In order to verify whether Cy7-CREKA-micelles bind to intravascular fi brin within gliomas, the presence of fi brin was determined via immunohistochemistry. However, upon processing tissue sections for fi brin detection, the Cy7 signal was quenched. In addition to the in fl uence of material properties of nanoparticles on the penetration and accumulation in tumor tissues, previous studies have reported particle size to affect the clearance mechanisms of nanoparticles [26,30 e 33]. Since both Cy7- and Cy7- CREKA-micelles are approximately 8 nm in diameter, they fall in the range of particles that are capable of both renal clearance via the kidneys and bladder and elimination by macrophages in the spleen and liver through the reticuloendothelial system (RES), which is consistent with the biodistribution data found in Fig. 7 [34,35]. By 7 days, the majority of the micelles were cleared and only remained within the liver con fi rming RES to play a larger role in clearance. H & E images con fi rmed that the liver and kidney, key organs involved in renal clearance and RES, showed no signs of tissue damage and had morphologies that were similar to that of PBS-treated controls, establishing the biocompatibility and safety of these micelles as a targeted drug delivery system for the treatment of glioblastoma. Cy7-labeled micelles were modi fi ed with the fi brin-binding peptide, CREKA, to enhance the targeting of micelles to glioblastoma. Cy7-micelles and Cy7-CREKA-micelles were spherical in shape and the addition of the pentapeptide increased the average particle size and zeta potential from 7.6 to 8.2 nm, and À 27.4 to À 12.6 mV, respectively. Upon intravenous injection into a mouse model of intracranial glioma, Cy7-micelles passively accumulated at the tumor site via the EPR effect, whereas Cy7-CREKA-micelles displayed enhanced tumor homing via active targeting, con fi rmed by in vivo and ex vivo imaging and immunohistochemistry. Importantly, neither Cy7-micelles nor Cy7-CREKA-micelles localized to the brain in an injury model, conferring micelle speci fi city to brain tumors via penetration across the compromised BBB. Biodistribution of micelles showed high accumulation in the liver and kidneys, which suggests both renal clearance and RES plays a role in micelle elimination and organs showed no signs of cytotoxicity or tissue damage determined via histological evaluation. Our study demonstrates a new avenue for targeting glioblastoma, and future studies will harness these capabilities to achieve highly ef fi cient, chemotherapeutic drug delivery for the treatment of malignant ...

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... are primary brain tumors that are derived from as- trocytes, oligodendrocytes, or ependymal cells. Glioblastoma multiforme (GBM), a grade IV astrocytoma, accounts for 70% of all malignant gliomas and carries a 5-year survival rate of less than 5% with a 14.6-month median survival time even with the treatment of aggressive radiotherapy and concomitant or adjuvant temozolomide [1,2]. Treatment of glioblastoma represents one of the most formidable challenges in oncology due to the inability to completely surgically resect the tumor owing to a diffuse invasion of the surrounding normal brain tissue. The amount of tumor removed is also limited by a proximity to critical regions for brain function, unavoidably resulting in tumor recurrence [3]. Consequently, post-surgical chemotherapy regimens are considered to be essential for the treatment of gliomas, but therapeutic advancement for GBM has remained disappointing due to poor penetration of chemotherapy agents successfully crossing the blood e brain-barrier (BBB) [4]. Although this interface acts to protect the brain from exposure to harmful substances circulating in the bloodstream under normal physiological conditions, the BBB simultaneously limits penetration of drugs to cancerous tissue, even when partially compromised [5,6]. Recent developments in nanoparticle technology have yielded promising results in the delivery of therapeutic agents across the BBB [7 e 9]. Micelles are of particular interest due to their small size, biocompatibility, and hydrophobic core, which is especially ad- vantageous for loading poorly soluble drugs [10]. When properly designed, micelles can prolong drug bioavailability in circulation and deliver drugs in the optimum dosage range, which results in improved therapeutic ef fi cacy and reduced toxic side effects [11]. Importantly, micelles have been shown to chaperone chemotherapy agents across the BBB as active targeting drug delivery systems in addition to passively targeting tumor tissue via the enhanced permeability and retention (EPR) effect [12,13]. Through surface-modi fi cations with ligands such as transferrin, epidermal growth factor, folate, and a 2-glycoprotein, micelles can bind to receptors that are selectively expressed on cancer cells or the tumor vasculature [10,13 e 15]. Previous reports demonstrate GBM is accompanied by leaky, hemorrhagic vasculature, which contributes to thrombosis and fi brin deposition [16]. The presence of fi brin and proteins that become cross-linked to fi brin in tumors, but not in normal tissues, are thought to be a result of the leakiness of tumor vessels, which allows plasma proteins to enter from the blood into tumor tissue where fi brinogen is converted to fi brin via tissue pro- coagulant factors [17]. Since fi brin deposition within and around the tumor area is characteristic to primary and metastatic brain tumors, we examined the targeting capabilities of micelle nanoparticles functionalized with the clot-binding peptide, cysteine e arginine e glutamic acid e lysine e alanine (CREKA) [18 e 20]. In our earlier studies, fl uorescently-labeled micelles functionalized with the CREKA pentapeptide were reported to home to sites of fi brin found on thrombotic, atherosclerotic plaques [21]. Furthermore, CREKA has also been shown to target breast and prostate cancer by binding to fi brin and fi brin-associated clotted plasma proteins within the tumor vasculature [18,20]. For the fi rst time, the potential of CREKA-micelles as a targeting nanoparticle system for gliomas via active binding to fi brin within the tumor blood vessels was investigated in this study. To this end, peptide amphiphiles consisting of the CREKA peptide were used to self-assemble targeting micelles. We intravenously injected Cy7 labeled micelles with or without the CREKA peptide to mice bearing intracranial glioma tumors and hypothesized that the micelles would be capable of targeting brain tumor tissue through the EPR effect and that the CREKA modi fi cation could further enhance tumor homing of the micelles by binding to intravascular clots as well as interstitial areas of fi brin deposition (Fig. 1). In addition to targeting, the clearance properties of these micelles were evaluated for up to 7 days. The fi brin-binding pentapeptide, CREKA, was conjugated to DSPE-PEG(2000)-maleimide via a thioether linkage to cysteine, and the CMC of resulting PAs was determined to be 9.3 Â 10 À 7 m M (Fig. 2A). Fluorescently-labeled non-targeting (Cy7-micelle) and CREKA micelles (Cy7-CREKA-micelle) were constructed by mixing DSPE-PEG(2000)-Cy7 with either DSPE-PEG(2000)-CREKA or DSPE-PEG(2000)-maleimide in a 10:90 molar ratio, and the presence of spherical micelles with an average diameter of 7.6 Æ 1.2 and 8.2 Æ 1.2 nm was con fi rmed via TEM and DLS (Fig. 2B and Table 1). Zeta potentials of Cy7- and Cy7-CREKA-micelles were determined to be À 27.4 Æ 4.5 and À 12.6 Æ 1.6 mV, respectively (Table 1). In order to assess passive targeting of Cy7-micelles via the EPR effect and enhanced accumulation of Cy7-CREKA-micelles, micelles were intravenously administered in an intracranial GL261 glioma mouse model and assessed via optical imaging (Fig. 3). After 1 h in blood circulation, the localization of Cy7-CREKA-micelles at the tumor site was visible via in vivo imaging and the accumulation increased with time (Fig. 4). When brains were excised and imaged, both Cy7-micelles and Cy7-CREKA-micelles were localized at the tumor site within 3 h post-injection and cleared by 7 days (Fig. 5A). In contrast, no fl uorescence signal was found when micelles were administered in a brain injury model (Fig. 5A). Both Cy7- and Cy7- CREKA-micelles demonstrated increased accumulation from the 3 h e 24 h time points, but Cy7-CREKA-micelles accumulated to a signi fi cantly greater extent than their non-targeting counterparts at each time point (3 h: 8.7 Â 10 8 vs. 2.3 Â 10 8 p/s/cm 2 /sr; 24 h: 1.6 Â 10 9 vs. 1.7 Â 10 8 p/s/cm 2 /sr; Fig. 5B). When samples were sectioned and evaluated histologically, both Cy7- and Cy7-CREKA- micelles showed bright, individual, punctate patterns of Cy7 fl uorescence within the tumor region of the brain, and no Cy7 signal was found in the non-tumor tissue (Fig. 6A). Notably, Cy7-CREKA samples also showed a dimmer fl uorescence signal present throughout the sample. Immunohistochemistry also con fi rmed ...