Blood–Brain-Barrier-Penetrating Albumin Nanoparticles for

Nov 8, 2016 - Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medica...
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Blood−Brain-Barrier-Penetrating Albumin Nanoparticles for Biomimetic Drug Delivery via Albumin-Binding Protein Pathways for Antiglioma Therapy Tingting Lin,†,‡,§ Pengfei Zhao,†,∥ Yifan Jiang,† Yisi Tang,† Hongyue Jin,† Zhenzhen Pan,† Huining He,‡ Victor C. Yang,‡,⊥ and Yongzhuo Huang*,† †

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Shanghai 201203, China Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin 300070, China § Department of Pharmacy, Binzhou Medical University Hospital, 661 Huanghe Road, Binzhou 256603, China ∥ Nanchang University College of Pharmacy, 461 Bayi Road, Nanchang 330006, China ⊥ University of Michigan College of Pharmacy, 428 Church Street, Ann Arbor, Michigan 48108, United States ‡

S Supporting Information *

ABSTRACT: Nutrient transporters have been explored for biomimetic delivery targeting the brain. The albumin-binding proteins (e.g., SPARC and gp60) are overexpressed in many tumors for transport of albumin as an amino acid and an energy source for fast-growing cancer cells. However, their application in brain delivery has rarely been investigated. In this work, SPARC and gp60 overexpression was found on glioma and tumor vessel endothelium; therefore, such pathways were explored for use in brain-targeting biomimetic delivery. We developed a green method for blood−brain barrier (BBB)-penetrating albumin nanoparticle synthesis, with the capacity to coencapsulate different drugs and no need for crosslinkers. The hydrophobic drugs (i.e., paclitaxel and fenretinide) yield synergistic effects to induce albumin self-assembly, forming dual drugloaded nanoparticles. The albumin nanoparticles can penetrate the BBB and target glioma cells via the mechanisms of SPARC- and gp60-mediated biomimetic transport. Importantly, by modification with the cell-penetrating peptide LMWP, the albumin nanoparticles display enhanced BBB penetration, intratumoral infiltration, and cellular uptake. The LMWP-modified nanoparticles exhibited improved treatment outcomes in both subcutaneous and intracranial glioma models, with reduced toxic side effects. The therapeutic mechanisms were associated with induction of apoptosis, antiangiogenesis, and tumor immune microenvironment regulation. It provides a facile method for dual drug-loaded albumin nanoparticle preparation and a promising avenue for biomimetic delivery targeting the brain tumor based on combination therapy. KEYWORDS: albumin nanoparticle, albumin-binding protein, brain tumor targeting, SPARC, gp60, cell-penetrating peptide, biomimetic delivery

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transendothelial electrical resistance of brain microvessels is 100−500-fold higher than that with noncerebral capillaries.2 However, the BBB is not a static barrier, and actually, there are massive exchanges of substances on the BBB through nutrient transporters.

he global incidence rate of primary malignant brain tumors in 2012 was 3.4 per 100,000, representing an estimated diagnosed population of 139,608 males and 116,605 females (www.cbtrus.org). Specifically, for children, brain cancer becomes the leading cause of solid-tumor-related death in the world. To achieve effective brain cancer delivery, first of all, the brain−blood barrier (BBB) is the primary challenge against chemotherapy, where the cerebral microvessel endothelial cells are characterized by high expression of tight junction proteins and poor transcellular endocytosis.1 The © 2016 American Chemical Society

Received: June 28, 2016 Accepted: November 3, 2016 Published: November 8, 2016 9999

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ACS Nano Scheme 1. Schematic Illustration of LMWP-Modified Albumin NPs via a Green Synthesis Methoda

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The co-loaded hydrophobic drugs work synergistically to induce self-assembly of the NPs under a reduced and high salt condition. The BBBpenetrating NPs are delivered into glioma via the mechanisms by targeting the albumin-binding proteins (e.g., gp60 and SPARC), and the efficiency is further enhanced by CPP-assisted penetration.

Nutrient transporters have attracted great attention for their promising application in biomimetic delivery.3 Nutrient transporters on the BBB play an important role in maintaining the normal functions of the brain, the most energy-consuming organ, for actively fetching amino acids/peptides, sugars, proteins, and so on. Therefore, these transporters can potentially serve as portals for brain drug delivery, and indeed, they have been widely explored in recent decades, including LAT-1, GLUT-1, LDL, and transferrin receptors.4 Albumin is an important nutrient source for the body. However, albumin is normally excluded from entering the brain. Due to rapid growth, the metabolically active tumors are hungry for nutrients. In such cases, albumin intake into tumor tissues is greatly increased for use as a source of amino acids and energy for cancer cells.5,6 Albumin-binding proteins, such as SPARC (secreted protein acidic and rich in cysteine) and glycoprotein 60 (gp60), are the major mechanism responsible for albumin uptake by tumors, a process including transcytosis across endothelium and endocytosis in tumor cells.7 It has been reported that SPARC is overexpressed in brain tumors, serving as a promoter to glioma progression and invasion, suggesting its potential therapeutic value.8,9 To our interest, we conceived an idea of using the glioma-overexpressed albumin-binding proteins as transporters for drugs. However, little is known about the effects of albumin-binding proteins as targeting receptors on brain drug delivery, and indeed, their application for brain delivery has been scarcely investigated. Motivated by this idea, we herein presented an enhancedpenetrating albumin nanoparticulate system with coencapsulation of paclitaxel (PTX) and fenretinide (4-HPR). We expected that albumin serving as the drug carrier can not only solve the solubility problems of hydrophobic drugs but also enhance tumor-targeting delivery efficiency. Furthermore, poor intra-

tissue penetration imposes the second major barrier against drugs reaching the targeting cancer cells. For instance, the diffusion coefficient of macromolecules in the brain is estimated to be 10−6 cm2/s, and roughly it would take 3 days for a 1 mm diffusion.2 To address this problem, a cell-penetrating peptide LMWP (low molecular weight protamine) was employed to modify the albumin nanoparticles. The LMWP-modified albumin nanoparticulate system was developed for co-delivery of PTX and 4-HPR for brain cancer therapy (Scheme 1). An advantage of coencapsulation of PTX and 4-HPR was that their hydrophobic nature synergistically induced the self-assembly of albumin nanoparticles. Of note, the retinoid 4-HPR, a vitamin A analogue, has been demonstrated with potent pro-apoptotic effects against tumor growth and evaluated in clinical trials for combination therapy with cytotoxic drugs.10 However, both PTX and 4-HPR are poorly water-soluble, and a co-delivery system for them has not been reported yet. The presented delivery system bears several features as follows. First, enhanced brain delivery of the albumin nanoparticles can be achieved via the multiple mechanisms, such as albumin-binding protein-mediated active targeting to brain cancer, CPP-promoting BBB penetration and intratumoral infiltration, as well as intracellular delivery. Second, coencapsulation of two different drugs into the albumin nanoparticles can be achieved for combination therapy. Third, a green method was developed for albumin nanoparticle preparation based on urea/NaBH4-mediated denaturation.

RESULTS Chemical cross-linking is a critical method applied for stabilization of albumin nanoparticles, which, however, usually involves the use of toxic cross-linking agents (e.g., glutaralde10000

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Figure 1. Characterization of the albumin NPs. (A) Size and TEM of BSA-NP. (B) Size and TEM of L-BSA-NP. (C) Stability of NPs in PBS containing 10% FBS. (D) In vitro release of PTX.

Figure 2. Cellular uptake of the NPs in U87 cells. (A) Fluorescence image of U87 cells treated with albumin NPs. (B) FACS analysis of uptake efficiency on U87. (C) Schematic illustration of an in vitro BBB model. (D) FACS analysis of uptake by U87 in the lower chamber. (E) Western blotting analysis of SPARC expression. (F) FACS analysis of uptake in bEnd.3 and U87 cells.

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ACS Nano hyde).11 In this work, we developed a green method based on urea/NaBH4 denaturation and drug-induced self-assembly for albumin nanoparticle preparation. In a high-concentration urea solution and reducing condition (NaBH4), the noncovalent interactions (e.g., hydrogen bonding, hydrophobic effect) diminish and the disulfide bonds of albumin are cleaved, leading to protein unfolding into a linear structure. Following addition of drugs, water was given to decrease the salt concentration. The lipophilic drugs interacted with the hydrophobic domains and induced self-assembly into nanoparticulate forms. The subsequent formation of disulfide bridges further stabilized the nanoparticles. This method eliminated the need for cross-linking agents and energy consumption (e.g., emulsification process by high-pressure homogenization). LMWP is a naturally sourced CPP, derived from protamine (an FDA-approved drug) via enzymatic digestion. In our previous studies, we applied LMWP-assisted delivery strategies to overcome various biobarriers, such skin,12,13 intestinal mucosa,14 intratumoral heterogeneity, and transporter-mediated drug efflux.15 We previously found that the drug−albumin conjugates modified with LMWP were able to overcome the drug-resistant efflux and displayed enhanced cancer treatment efficacy.16 Recently, it has been reported that there is synergistic effect between cell-surface-binding domains and CPP in achieving exceptional transduction in the targeted tumor cells.17 Therefore, we expected brain delivery could be enhanced by the strategy of albumin-mediated targeting and LMWP-mediated penetration. LMWP−BSA synthesis is illustrated in Figure S1 (Supporting Information). The BSA nanoparticles (BSA-NPs) and LMWP-modified BSA nanoparticles (L-BSA-NPs) were fabricated through a self-assembly method developed by our lab. Figure 1A,B presents the size and transmission electron microscopy (TEM) photographs of the BSA-NPs and L-BSANPs, which are spherical in shape and uniform in size. Both NPs were less than 150 nm, with narrow size distribution and negative ζ-potential (Table S1, Supporting Information). The NPs exhibited good stability with very minor size changes after incubation in PBS containing 10% FBS at 37 °C for 72 h (Figure 1C). Both NPs showed similar drug release patterns and achieved comparable cumulative PTX release rates of 70 and 73% at 96 h, respectively (Figure 1D). The cellular uptake of the albumin NPs labeled with FITC was investigated in U87 cells. Due to the potent cell penetration ability, the L-BSA-NPs displayed a cellular uptake efficiency higher than that of the nonmodified BSA-NPs, as evaluated by microscopic observation and further confirmed by the quantitative results of fluorescence-activated cell sorting (FACS) (Figure 2A,B). The monolayer culture in Transwell using the immortalized mouse brain endothelial cells (bEnd.3) is a commonly used in vitro BBB model to study the brain delivery of NPs.18,19 The cellular uptake of the BSA-NP and L-BSA-NP in U87 cells on the lower compartment was measured. Flow cytometric analysis revealed that the uptake of L-BSA-NP by U87 cells across the bEnd.3 monolayer cells was 2.5-fold higher than with BSA-NP, demonstrating the ability of LMWP for enhancing permeation of the NPs through the bEnd.3 monolayer and uptake by the U87 cells (Figure 2C,D). The BSA-NP actually also showed substantial penetration ability in the cell culture model. It may be attributed to the high-level expression of the albuminbinding protein SPARC on U87 cells; moreover, the bEnd.3

cells also displayed minor SPARC expression (Figure 2E). The uptake efficiency in the U87 and bEnd.3 cells was in accordance with the SPARC level (Figure 2F). Poor drug intratumoral infiltration is a daunting obstacle against effective cancer therapy.20 By using a U87 spheroid culture model, we found that the L-BSA-NP displayed extensive penetration inside the spheroids, and confocal microscopic measurement showed the L-BSA-NP penetrated significantly deeper than the BSA-NP (176.7 vs 136.7 μm, p < 0.01) (Figure 3 and Figure S2). From the continual Y-axis scan layers (18 μm

Figure 3. Assessment of intratumoral penetration ability using tumor spheroids treated with L-BSA-NPs (A,B) and BSA-NPs (C,D). Representative images.

each), it can be seen that the fluorescence signal in the L-BSANP group is more intense and deeper than that in the BSA-NP groups. These findings suggested that LMWP was able to improve intratumoral infiltration of the drug-loaded NPs, which was in accordance with our previous observation in LMWPmediated delivery of PLGA NPs deep inside the tumor.15 Therefore, LMWP served multiple functions in this delivery system, such as enhanced BBB penetration and intratumoral infiltration and increased uptake by U87 cells, as well. The BSA-NPs and L-BSA-NPs with encapsulation of PTX and 4-HPR were tested in U87 cells for evaluation of their antitumor activity. The U87 cell viability was inhibited in a dose-dependent manner (Figure 4A). Compared with the free combo drugs, both NPs showed enhanced cytotoxicity and the L-BSA-NP displayed the highest antitumor activity, while a minor inhibition effect was seen in the groups with a single use of PTX or 4-HPR. The percentages of apoptotic cells detected by FACS were 15.1 and 24.6% for the BSA-NP and L-BSA-NP groups, respectively, compared to 10.8% for the free combo drugs (Figure 4B). Next, we took a look at the mechanisms of the combination therapy. The transformation from procaspase 3 to caspase 3 is a key step for induction of apoptosis even in cancer cells with deficient apoptotic machinery.21 PTX was able to activate the apoptotic caspase 3, and the combination of PTX/4-HPR showed enhanced transformation of procaspase 3 to the activated caspase 3; the NPs with coencapsulation of PTX and 4-HPR further enhanced caspase 3 activation, and the LBSA-NP displayed the highest capacity among the tested 10002

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Figure 4. Effect of albumin NPs on U87 cells. (A) MTT of the NPs in U87 cells for 48 h. (B) Cell apoptosis assay at a combo dose (2 μg/mL for each drug). (C) Western blotting analysis of caspase 3.

Figure 5. In vitro antiangiogenesis study. The endothelial cell tube formation on the Matrigel matrix was inhibited by 4-HPR, displaying potent effect similar to that with the antiangiogenic agents (regorafenib and sorafenib).

groups (Figure 4C). The results of MTT and caspase 3 assays were consistent. The antiglioma effect was also examined in other glioma cell lines (C6, GL216, and U251) (Figure S3), and the IC50 values are listed in Table S2. The L-BSA-NPs displayed the highest efficacy among all of the groups, demonstrating the potential of the L-BSA-NP-based combination therapy in antiglioma application. Notably, 4-HPR has been used for angioprevention.22 Our results also demonstrated that 4-HPR treatment significiantly inhibited the HUVEC tube formation on the Matrigel matrix, with a similar ability as the positive control (the antiangiogenic drugs of regorafenib and sorafenib), whereas the control group (PBS) clearly showed the appearance of capillary-like tubes (Figure 5). The in vitro angiogenesis study revealed that 4-HPR

was a potent antiangiogenic agent that may boost the antitumor effect of PTX. The mean residence times (MRT) of the BSA-NPs and LBSA-NPs were 8.36 and 7.42 h, respectively (Figure S4). The tumor-targeting efficiency was examined in two kinds of animal modelsthe mice harboring subcutaneous glioma and orthotopic glioma. In the subcutaneous glioma model, both the NPs achieved efficient tumor targeting. The distribution of the fluorescent dye-labeled NPs was observed at the tumor site within 2 h, and subsequently, the fluorescent intensity was increased gradually at the monitoring course from 2 to 8 h postdose (Figure 6A,B). The L-BSA-NPs exhibited tumor accumulation significantly higher than that of the BSA-NPs. The tumor displayed the highest fluorescence intensity among the dissected organs (Figure 6C,D). 10003

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Figure 6. In vivo imaging of drug distribution in mice bearing U87 xenograft tumors. (A) Whole body imaging. (B) In vivo radiant efficiency of the tumor site. (C) Ex vivo imaging of major organs dissected from mice. (D) Ex vivo radiant efficiency of tumors.

Figure 7. In vivo imaging of drug distribution in mice bearing U87 orthotopic glioma. (A) Whole body imaging of mice from 1 to 24 h. (B) In vivo radiant efficiency of the brain tumor site. (C) Ex vivo imaging of the major organs dissected from mice. (D) Ex vivo radiant efficiency of brain tumors.

considerable level of fluorescence intensity. It should be mentioned the wounded assessment of intracranial inoculation procedures showed the BBB integrity was fully recovered in a week (Figure S5), indicating that the surgery-induced BBB disruption was transient and recuperable. We further conducted cryosection to observe the intratumoral infiltration. Both of the albumin NPs displayed a comprehensive distribution in the intracranial glioma tissue. As expected, the L-BSA-NPs enhanced tumor penetration and were widespread across the tumor region (Figure 8A),

More importantly, in the orthotopic glioma model, the intracranial tumor-targeting effect was also found in both NPs (Figure 7). The L-BSA-NPs showed substantial brain accumulation within 2 h, and the fluorescence signal peaked at 10 h (Figure 7B). Meanwhile, the BSA-NPs also had considerable accumulation in the brain tumor but significantly less than that of the L-BSA-NPs (Figure 7C,D), as shown in the ex vivo imaging. In addition, since NPs are apt to be captured by the reticuloendothelial system (RES), our results showed that the RES-associated organs, such as lung and liver, had a 10004

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Figure 8. Intraglioma penetration and SPARC expression. (A) Confocal images of brain sections from orthotopic glioma of Cy5-labeled NPs (red). The NPs display preferential accumulation in the tumor. (B) Western blotting analysis of albumin-binding proteins (gp60 and SPARC) in the tumor and normal brain tissues.

Figure 9. Immunofluorescence studies. (A) Confocal images of brain sections from orthotopic glioma. Immunofluorescence of SPARC and its colocalization with Cy5-labeled NPs. (B) Immunofluorescence of SPARC and CD31 (tumor blood vessel). Colocalization indicates expression of SPARC on tumor blood vessel, too.

proteins (e.g., SPARC and gp60) in the glioma tissues by using Western blotting and found they were up-regulated, with overexpression in both the subcutaneous and orthotopic gliomas (Figure 8B). However, little expression was found in the normal brain issues, which accordingly exhibited insignificant drug distribution, as shown in Figure 8A. This indicated that SPARC and gp60 played an important role in brain delivery. As further evidence, the immunofluorescence observation was in accordance with the Western blotting and immunohistochemistry results (Figure S7), showing high SPARC expression. SPARC overlapped with the distribution of the albumin NPs, suggesting the effect of SPARC on

demonstrating the ability to overcome the intratumoral heterogeneity barrier. Notably, drug distribution was found predominantly in the tumor region, suggesting the site-specific action. It should be noted that the fluorescence imaging methods used our studies were verified, and under the selected experimental conditions, no autofluorescence at 488 nm in cells and tissue (also at 560 nm for Cy5) was observed (Figure S6). Apart from the EPR effect that is a major mechanism responsible for glioma preferential accumulation, the active targeting mechanisms for albumin-based brain tumor delivery was also involved, as demonstrated by the Transwell study above. We further investigated the levels of albumin-binding 10005

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Figure 10. Antiglioma efficacy on the subcutaneous glioma mouse model. (A) Tumor growth curve. (B) Tumor weight and tumor inhibition rate. (C) Representative tumor tissues. (D) Body weight variations in the treatment course. (E) Organ coefficients (*P < 0.05, **P < 0.01).

mediating albumin-based tumor-targeting delivery (Figure 9A). In the L-BSA-NP group, the fluorescence displayed a more comprehensive intratumoral distribution compared to that of the BSA-NPs. It corresponded to the results of penetration of the U87 tumor spheroid. In addition, the tumor blood vessels stained by anti-CD31 also highly expressed SPARC, and the immunofluorescence double staining showed clear colocalization of CD31 and SPARC (Figure 9B). The in vivo imaging and immunofluorescence results clearly demonstrated the significant enhancement of brain tumor delivery by the LMWP-based technology, by which the L-BSA-NP displayed superiority over the BSA-NPs. Moreover, the Cy5 fluorescence from the L-BSANP partially colocalized with the DAPI-stained nuclei, which accounted for the nucleus-targeting function of the argininerich CPP.15,23 The antiglioma activity of the albumin NPs was evaluated in the subcutaneous glioma mice at first. Both kinds of the NPs at a dose of PTX/4-HPR (2 + 2 mg/kg) could efficiently arrest the tumor growth, with an inhibition rate of 82% (L-BSA-NP) and 66% (BSA-NP), respectively (Figure 10A−C). Their efficacy was much higher than that of the combo free drugs (i.e., 40%) and single use of PTX (4 mg/kg) (i.e., 51%). The LBSA-NPs had the best antitumor activity among all groups, with statistical significance, confirming the enhanced brain delivery mediated by LMWP. The free PTX (4 mg/kg) showed

efficacy higher than that with the combo free drugs (2 mg/kg each), which could account for their different pharmacokinetics profiles, thereby leading to the reduced synergistic effect. In addition, it should be pointed out that the selected dose of 4HPR showed little impact on tumor growth in the preliminary studies, and thus it was not included in further studies. We further investigated the therapeutic effect on the orthotopic glioma (Luc-U87) model. The size of the intracranial glioma was monitored using the IVIS imaging system by giving the luciferase substrate luciferin. The L-BSANP group displayed clearly weaker bioluminescence intensity than other groups, with a smaller bioluminescence area, too (Figure 11A), indicating the shrinking tumors. The survival rate also demonstrated the improved treatment outcomes by applying the L-BSA-NPs; the median survival time was 24, 29, 31, 33, and 37 days for the groups of PBS, PTX, 4-HPR/ PTX, BSA-NPs, and L-BSA-NPs, respectively (Figure 11B). The group given L-BSA-NPs displayed the longest survival time among all of the groups, with very statistical significance compared to the free combo drug group. Although there was no statistical difference in significance between the groups treated with BSA and L-BSA-NP (33 vs 37 days), the animals given L-BSA-NP sustained less body weight loss than those with BSA-NP. 10006

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Figure 11. Treatment efficacy on the mice bearing orthotopic glioma. (A) Glioma growth inhibition monitored by using in vivo bioluminescence images. (B) Survival curve. (C) Body weight variations in the treatment course. (D) Organ coefficients.

It has been reported that retinoids (e.g., 4-HPR and retinoic acid) can act on immune cells (macrophages and dendritic cells) and serve as an immunostimulator for cancer therapy.24,25 Of interest, we found that the L-BSA-NPs encapsulated with 4HPR/PTX significantly suppressed the pro-tumor M2 phenotype of macrophages, as demonstrated by both the immunofluorescence and Western blotting assay (Figure 13). This preliminary result suggested that the effect of 4-HPR on tumor immune microenvironments, and the inhibition of tumor-associated macrophages contributed to the improved treatment outcomes. The safety of the treatments was evaluated. In the subcutaneous glioma model, body weight loss was observed

The TUNEL results showed that apoptosis in glioma was efficiently induced by the NPs, and the combination treatment displayed superiority to the monotherapy of PTX (Figure 12A). We further investigated the antiangiogenesis effect by immunohistochemistry. The tumor blood vessels in the giloma tissue slices were stained using anti-CD31, and it was shown that the vessel size and number in the groups treated with the combination of PTX/4-HPR were dramatically decreased compared to the single use of PTX (Figure 12B). Antiangiogenesis was also confirmed by Western blotting assay (Figure 12C), showing the reduced CD31 level. It indicated that angiogenesis inhibition was a main antitumor mechanism for this combination therapy. 10007

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Figure 12. TUNEL assay of apoptosis in orthotopic glioma (A) and detection of tumor-associated blood vessels with CD31 immunohistological staining (B) and Western blotting (C). Scale bar, 50 μm.

intake of the camouflaged drug cargos. Albumin is an important nutrient source for supporting the rapid proliferation of tumor cells, and albumin-binding proteins are overexpressed on many types of solid tumors, functioning as albumin intake transporters.27 However, the pathway of albumin-binding proteins has rarely been studied for delivery of brain chemotherapeutics. Our investigations showed that high expression of the albuminbinding proteins SPARC and gp60 was found in U87 human glioma cells and the transplanted xenografts on nude mice. There is a very low level of albumin-binding proteins on normal BBB capillaries, thereby accounting for poor permeation of native albumin,28 but their expression is up-regulated in tumor blood vessels. Our results also confirmed it by colocalization staining (anti-CD31 and anti-SPARC) in the glioma tissues. Therefore, albumin on its own can serve as both drug carrier and targeting ligand, making use of these pathways for glioma delivery. In addition, it is interesting that clusters of gp60 induced by anti-gp60 was reported to be able to activate enhanced albumin transport through microvessels via a transcellular pathway.29 Albumin NPs might act as an inducer for clustering the albumin-binding proteins because the NPs could bind with and cross-link multiple receptors. Considering the great potential of albumin NPs in brain drug delivery, further investigation in the BBB penetration mechanisms should be conducted. The efficiency of brain tumor delivery relies on two key steps that can be defined as targeting and penetration.30 Albumin not only functions as a targeting ligand to albumin-binding proteins but also triggers the transcytosis across the BBB and endocytosis into tumor cells. However, arrival at the tumor site is not a guarantee for effective intratumoral penetration, which is another formidable obstacle.20 Poor penetration leads to insufficient drug distribution and intracellular concentration in the tumor hypoxic area, accounting for drug resistance and cancer treatment failure.31,32 Recently, CPP has been explored for assisting intratumoral penetration, combined with peptide-

only in the PTX-treated group, while other groups gained weight (Figure 10D). The organ coefficients of spleen decreased in the mice receiving chemotherapy, compared with the PBS control group, while no significant difference was observed in the coefficients in other organs (Figure 10E). Moreover, the histopathological examination showed that the free drug groups caused substantial adverse effects, including focal hepatic necrosis, splenic nodule atrophy, pulmonary hemorrhage, and renal capillary congestion (Figure 14A). No pathological changes were seen in the BSA-NP and L-BSA-NP groups, which were similar to the PBS control group. In the orthotopic glioma model, the untreated group (PBS) sustained the most serious drop in body weight (Figure 11C), showing a tendency that was different from the subcutaneous model. It could be due to how the intracranial tumor affected the pituitary gland functions that are crucial for maintaining body growth and appetite. Therefore, in this case, body weight changes might not be able to reflect the biocompatibility. The organ coefficients of the spleen in the mice receiving PTX treatment and the NPs were lower than those with the PBS group, but no significant difference was seen in other organs (Figure 11D). The histological results clearly demonstrated that both NPs had improved safety as well as reduced toxicity in the treatment of orthotopic glioma (Figure 14B).

DISCUSSION Biomimetic design is a useful strategy that is often employed in drug delivery,26 in which drugs are cloaked by endogenous materials (e.g., cell membrane or ligands) for circumventing capture by RES or binding to the targeted receptors, thus improving the drug delivery efficiency. The brain is the most energy-consuming organ in the human body, and nutrient uptake by the transporters through the BBB is very active. The substrates of those transporters (e.g., glucose, transferrin, apolipoprotein E) are the ideal ligands that carry the drugs across the BBB, which identifies the substrates and induces the 10008

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conception has been gradually changing with the recent important findings that some CPP-binding receptors have been identified on cancer cells, such as neuropilin-1 and syndecan-4.34,35 Moreover, cancer cells overexpress certain glycosaminoglycans and present high levels of anionic lipids in the outer leaflet, conferring increased negatively charged character. Thereby, CPP displays stronger electrostatic interactions with cancer cells than the normal ones and improves the tumor target specificity and selective action.36 Our results demonstrated that the cell-penetrating LMWP could significantly improve albumin-based targeting delivery with enhanced tumor accumulation and intratumoral infiltration. The feature of the presented synthesis method is that a quick denaturation process occurs with exposure to the reducing agent NaBH4 and high-concentration urea solution. With addition of hydrophobic drugs (PTX and 4-HPR) and plenty of water, the drug-induced hydrophobic interactions facilitate the self-assembly of NPs. Both are water-insoluble and possess affinity to albumin. Therefore, these drugs tend to be entrapped into the hydrophobic domains of albumin molecules when they assemble into a nanoparticulate form. More interestingly, the two drugs can work synergistically together for induction of nanoparticle formation. The dual drug-loaded albumin nanoparticles showed better stability and higher loading efficiency compared to that of the single drug-loaded albumin nanoparticles (data not shown). It indicated that combination of the two drugs enhanced the intramolecular and intermolecular interaction of albumin and facilitated formation of hydrophobic cores, thus driving the self-assembly of albumin.

CONCLUSION In the present study, we developed a green synthesis method for preparation of albumin NPs, in which the hydrophobic drugs PTX and 4-HPR synergistically induced the formation of hydrophobic cores and self-assembly of albumin, readily achieving coencapsulation of drugs. The albumin-binding proteins (e.g., SPARC and gp60) were found with overexpression on tumor vessel epithelium and glioma cells, and they were mainly responsible for biomimetic delivery of albumin NPs to brain tumors. The modification with the cellpenetrating LMWP further enhanced the BBB penetration, intratumoral infiltration, and cellular uptake. The LMWPmodified albumin NPs with coencapsulation of PTX/4-HPR were demonstrated to be potent in arresting tumor growth in

Figure 13. Protumor phenotype M2 macrophages were suppressed by L-BSA-NPs. (A) Immunofluorescent detection of M2 macrophages in orthotopic glioma. (B) Western blotting assay of the orthotopic glioma tissues. CD206 is a surface maker of M2 macrophages. Scale bar, 50 μm.

targeting ligands (e.g., T7 and RGD), in order to simultaneously address both the issues of tumor targeting and penetration.30,33 Traditionally, CPP has been considered as a nonselective carrier, thereby compromising the targeting efficiency, but this

Figure 14. Histopathological examination of major organs collected after treatment on the animals with (A) subcutaneous and (B) orthotopic U87 tumors. Adverse toxicity was seen in free drug groups, including focal hepatic necrosis, splenic nodule atrophy, pulmonary hemorrhage, and renal capillary congestion, but not in both NP groups. Scale bar, 50 μm. 10009

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In Vitro Drug Release. The release profile of PTX from the NPs was studied using a dialysis method. Briefly, 1 mL of the bulk solution was placed into a dialysis bag (MWCO 10−12 kDa) with 20 mL of release medium (PBS, pH 7.4, containing 0.5% w/v SDS), and the experiment was conducted at 37 °C with continuous stirring at 140 rpm. At predetermined time intervals, 0.5 mL of the release medium was withdrawn for measurement and replenished with an equal volume of fresh medium. The released amount of PTX was determined by high-performance liquid chromatography (Agilent1200, USA). Cellular Studies. Cell Lines: Human glioma U87, U251 cells, mouse glioma C6, GL261 cells, and mouse brain endothelial bEnd.3 cells were incubated in Dulbecco’s modified Eagle’s medium containing 10% FBS and 1% antibiotics at 37 °C in humidified atmosphere and 5% CO2. In Vitro Uptake Studies: The U87 cells were seeded in 12-well plates at a density of 1 × 105 cells/well and cultured for 24 h. The cells were then incubated with the FITC-labeled BSA-NPs and L-BSA-NPs for 1 h. At the experimental end point, the cells were washed with PBS three times, fixed with 4% paraformaldehyde, and stained with DAPI. Fluorescent images were observed with a fluorescence microscope (Olympus, Japan). In addition, the cellular uptake efficiency was determined by using FACS. Penetration Studies Using an In Vitro BBB Model: The mouse brain endothelial bEnd.3 cells were seeded in the Transwell upper chamber (Corning, USA) for monolayer cell culture, which was ready for use with a transendothelial electrical resistance greater than 300 Ω. The U87 cells were seeded in the lower chamber. The FITC-labeled NPs were added to the upper chamber and incubated for 4 h, and then the U87 cells on the lower chamber were collected and washed with PBS three times for FACS assay. Penetration of the U87 Tumor Spheroid: The U87 cells were seeded at a density of 2 × 103 cells per well in 96-well plates coated with 50 μL of 1% (w/v) agarose gel to prevent cell adhesion. The cells were cultured at 37 °C in the presence of 5% CO2 for 7 days to form the three-dimensional tumor spheroids. The tumor spheroids were cultured with the NPs loaded with coumarin-6 for 4 h and subjected to confocal microscopy (Fluoview FV100 Olympus, Japan) after being washed with PBS three times. In Vitro Cytotoxicity Studies: The cell viability was measured by a standard MTT assay. The U87, U251, C6, and GL261 cells were seeded in a 96-well plate at a density of 5000 cells per well. After 24 h, the medium was substituted with the albumin NPs with different concentrations of PTX or PTX/4-HPR, followed by incubation for 48 h. The cell viability was measured using a standard MTT method. Furthermore, the U87 cell apoptosis was quantitatively measured using FACS (BD, USA). U87 cells were seeded in a 6-well plate and allowed to grow for 24 h. The cultured medium was then substituted with the BSA-NPs and L-BSA-NPs (equal to 2 μg/mL of PTX and/or 4-HPR), with subsequent 48 h incubation. After being washed with PBS, the cells were collected and washed again with PBS three times and stained with a FITC Annexin V apoptosis detection kit (Beyotime, China) according to the manufacturer’s protocol. The cells were subjected to apoptosis analysis by using FACS. In addition, the cells treated with the drugs were also collected for caspase 3 detection using Western blotting. Endothelial Cell Tube Formation Assay: The HUVEC cells (human umbilical vein endothelial cells) (4 × 104 cells/well) were seeded onto 24-well plates that were coated with Matrigel matrix (Corning, USA) and treated with 4-HPR (3 μg/mL). Sorafenib or regorafenib was used as the positive control. The cells were cultured for 12 h, and then the endothelial cell tube formation was observed. Animal Studies. Glioma Models: Balb/c nude mice (3−4 weeks old) were housed under specific pathogen-free conditions. Animals possessed continuous access to sterilized food pellets and distilled water, and a 12 h light/dark cycle. The animal experimental procedures were approved by the Institutional Animal Care and Use Committee. The subcutaneous U87 xenograft tumor model was established by inoculating 7 × 105 cells/mL subcutaneously into the back of nude

both subcutaneous and orthotopic glioma models via multiple mechanisms, such as antiangiogenesis, apoptosis, and tumor immune microenvironment regulation. The results indicated the significance of this brain delivery strategy and its application in combination therapy in antiglioma.

EXPERIMENTAL SECTION Materials. PTX was purchased from Melone Pharmaceutical Co., Ltd., and 4-HPR was from Nanjing Shengsai Chemical Co., Ltd. Bovine serum albumin (BSA) was obtained from RBC Life Sciences (USA). The Micro BCA protein assay kit and APO were acquired from the Beyotime Institute of Biotechnology (Haimen, China). Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) was obtained from ProteoChem (Loves Park, USA). Peptides were provided by Bankpeptide, Co., Ltd., China. Other reagents were of analytical grade. Anti-SPARC (H-90) and anti-gp60 antibodies were purchased from Santa Cruze (USA). Anti-caspase 3 antibody was purchased from Cell Signaling Technology (USA). Evans blue was purchased from Sigma-Aldrich (USA). Anti-CD31/PECAM1 antibody and anti-CD206/mannose receptor antibody was purchased from R&D Systems (USA). Sorafenib and regorafenib were purchased from Selleck (USA). Preparation of the LMWP−BSA. BSA was activated by SMCC. In brief, SMCC (20 mg/mL in DMSO) was added dropwise to the BSA solution (20 mg/mL in PBS, pH 7.2) at a molar ratio of 2:1 and reacted under magnetic stirring for 1 h. The activated BSA was purified using FPLC (Ä KTApurifier 10, GE Healthcare, USA) equipped with desalting column (GE Healthcare, USA). The activated BSA was reacted with thiolated LMWP (sequence: CVSRRRRRRGGRRRR) in PBS (pH 7.2) for 5 h at 4 °C, and then the unconjugated LMWP was removed by using a desalting column. The LMWP−BSA (L-BSA) conjugates were separated from the free BSA by using heparin affinity chromatography based on the interaction of LMWP and heparin. The concentration of the purified L-BSA was determined by a standard BCA method. Denaturation of BSA. The urea solution was prepared by dissolving tris(hydroxymethyl)aminomethane (3 g) and urea (29 g) in H2O and adjusting the pH to 8.3 with HCl. Following addition of 1.05 mL of ethanol as a defoamer, 0.1 mL of NaBH4 solution (72 mg/mL in 1 M NaOH solution) was rapidly added to 5.8 mL of BSA urea solution (100 mg/mL). The mixture was incubated at room temperature and then to 50 °C for 30 min, sequentially, and cooled to room temperature. Preparation of Drug-Loaded Albumin NPs. Briefly, a 100 μL ethanol solution of PTX and 4-HPR was added to 500 μL of denatured BSA or L-BSA urea solution (10 mg/mL). Then, 0.5 mL of H2O was added dropwise into the mixture under vortexing. The thusformed NPs (denoted as BSA-NP and L-BSA-NP, respectively) were purified using a Sephadex G50 column (GE Healthcare, USA). Characterization of the BSA-NP. The albumin NPs were analyzed by ZetaSizer nano-ZS90 (Malvern, UK) to determine size/ polydispersity index and ζ-potential. The morphology of albumin nanoparticles was investigated by TEM after the samples were deposited on a copper grid and stained with 1% acetic acid glaze. Drug-loading capacity and encapsulation efficiency were calculated using the following formula:

drug‐loading capacity (%) = (weight of entrapped PTX/weight of NPs) × 100%

encapsulation efficiency (%) = (weight of entrapped PTX/weight of PTX added) × 100% Stability of Nanoparticles. The size changes of the BSA-NPs and L-BSA-NPs were monitored by the dynamic light scattering technique in PBS containing 10% FBS for 72 h at 37 °C to study the physical stability. Mean size was determined by a ZetaSizer nano-ZS90 (Malvern, UK). 10010

DOI: 10.1021/acsnano.6b04268 ACS Nano 2016, 10, 9999−10012

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ACS Nano mice. The orthotopic glioma tumor mouse models were developed by inoculating the Luc-U87 cells (stable transfection of firefly luciferase) into the left cerebral hemisphere using a stereotaxic apparatus. The growth of an orthotopic glioma was monitored using bioluminescence imaging performed in the IVIS imaging system (Caliper PerkinElmer, Hopkinton, MA, USA) by intraperitoneal injection of luciferin substrate (150 mg/kg). In Vivo Imaging: The mice bearing subcutaneous or orthotopic gliomas were given the Cy5-labeled albumin NPs via tail vein injection. After the treatment, the mice were subjected to imaging at various time points. At 24 h postinjection, the mice were sacrificed, and the tumor and major organs were collected for fluorescent imaging. In addition, for the orthotopic glioma group, the brains were further fixed in 4% paraformaldehyde for 48 h, dehydrated with 20 and 30% sucrose solution for another 48 h, and processed with cryosection. The tissue slides were incubated with rabbit polyclonal anti-SPARC at 4 °C overnight and then stained with Alexa-488-conjugated goat rabbit IgG secondary antibody for 1 h at room temperature, followed by DAPI staining. Furthermore, the tumor blood vessel was detected using immunofluorescence with anti-CD31. The slides were subjected to confocal microscopic observation. The expression of the albuminbinding proteins (gp60 and SPARC) in the glioma tissue was detected by using Western blotting. In Vivo Antiglioma Therapy: The mice bearing subcutaneous glioma were randomly divided into five groups (four mice per group), receiving PBS (control), PTX (4 mg/kg), PTX/4-HPR (2 mg/kg each), BSA-NP (PTX/4-HPR, 2 mg/kg each), and L-BSA-NP (PTX/ 4-HPR, 2 mg/kg each) via tail vein injection every 2 days when the tumor volume was 100−200 mm3. The tumor volume was calculated using the following formula:

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank the National Basic Research Program of China (973 Program 2013CB932503, 2014CB931900) and NSFC, China (81172996, 81373357, 81422048, 81673382, 81521005, 81361140344) for the support. We also thank National Center for Protein Science Shanghai, CAS, for the technical support at Electron Microscopy Facility. REFERENCES (1) Brown, R. C.; Morris, A. P.; O’Neil, R. G. Tight Junction Protein Expression and Barrier Properties of Immortalized Mouse Brain Microvessel Endothelial Cells. Brain Res. 2007, 1130, 17−30. (2) Lo, E. H.; Singhal, A. B.; Torchilin, V. P.; Abbott, N. J. Drug Delivery to Damaged Brain. Brain Res. Rev. 2001, 38, 140−148. (3) Geldenhuys, W. J.; Mohammad, A. S.; Adkins, C. E.; Lockman, P. R. Molecular Determinants of Blood-Brain Barrier Permeation. Ther. Delivery 2015, 6, 961−971. (4) Mittapalli, R. K.; Manda, V. K.; Adkins, C. E.; Geldenhuys, W. J.; Lockman, P. R. Exploiting Nutrient Transporters at the Blood-Brain Barrier to Improve Brain Distribution of Small Molecules. Ther. Delivery 2010, 1, 775−784. (5) Stehle, G.; Sinn, H.; Wunder, A.; Schrenk, H. H.; Stewart, J. C.; Hartung, G.; Maier-Borst, W.; Heene, D. L. Plasma Protein (Albumin) Catabolism by the Tumor ItselfImplications for Tumor Metabolism and the Genesis of Cachexia. Crit. Rev. Oncol. Hematol. 1997, 26, 77− 100. (6) Wunder, A.; Stehle, G.; Sinn, H.; Schrenk, H.; Hoffbiederbeck, D.; Bader, F.; Friedrich, E.; Peschke, P.; Maierborst, W.; Heene, D. Enhanced Albumin Uptake by Rat Tumors. Int. J. Oncol. 1997, 11, 497−507. (7) Merlot, A. M.; Kalinowski, D. S.; Richardson, D. R. Unraveling the Mysteries of Serum AlbuminMore Than Just a Serum Protein. Front. Physiol. 2014, 5, 299. (8) Rempel, S. A.; Golembieski, W. A.; Fisher, J. L.; Maile, M.; Nakeff, A. SPARC Modulates Cell Growth, Attachment and Migration of U87 Glioma Cells on Brain Extracellular Matrix Proteins. J. NeuroOncol. 2001, 53, 149−160. (9) Shi, Q.; Bao, S.; Song, L.; Wu, Q.; Bigner, D. D.; Hjelmeland, A. B.; Rich, J. N. Targeting SPARC Expression Decreases Glioma Cellular Survival and Invasion Associated with Reduced Activities of FAK and ILK Kinases. Oncogene 2007, 26, 4084−4094. (10) Otterson, G. A.; Lavelle, J.; Villalona-Calero, M. A.; Shah, M.; Wei, X.; Chan, K. K.; Fischer, B.; Grever, M. A Phase I Clinical and Pharmacokinetic Study of Fenretinide Combined with Paclitaxel and Cisplatin for Refractory Solid Tumors. Invest. New Drugs 2005, 23, 555−562. (11) Elzoghby, A. O.; Samy, W. M.; Elgindy, N. A. Albumin-Based Nanoparticles as Potential Controlled Release Drug Delivery Systems. J. Controlled Release 2012, 157, 168−182. (12) Huang, Y.; Park, Y. S.; Moon, C.; David, A. E.; Chung, H. S.; Yang, V. C. Synthetic Skin-Permeable Proteins Enabling Needleless Immunization. Angew. Chem., Int. Ed. 2010, 49, 2724−2727. (13) Yang, Y. X.; Jiang, Y. F.; Wang, Z.; Liu, J. H.; Yan, L.; Ye, J. X.; Huang, Y. Z. Skin-Permeable Quaternary Nanoparticles with Layer-byLayer Structure Enabling Improved Gene Delivery. J. Mater. Chem. 2012, 22, 10029−10034. (14) He, H.; Sheng, J.; David, A. E.; Kwon, Y. M.; Zhang, J.; Huang, Y.; Wang, J.; Yang, V. C. The Use of Low Molecular Weight Protamine Chemical Chimera to Enhance Monomeric Insulin Intestinal Absorption. Biomaterials 2013, 34, 7733−7743.

V = (W 2 × L)/2 The mice bearing intracranial glioma were randomly divided into four groups (10 mice per group). The growth of orthotopic glioma (Luc-U87) was monitored using luciferin for in vivo imaging. The treatment of the albumin NPs was evaluated with the same dosage regimen as mentioned above for 3 weeks. The survival rate and median survival time were calculated, and the statistical difference was assessed by the Kaplan−Meier method. Mechanism Studies: After treatment, the intracranial glioma was dissected for immunohistochemical and immunofluorescent examination for detection of tumor blood vessels, apoptosis (TUNEL), and M2 macrophages. Histopathological Examination: At the experimental end point, major organs were collected for histopathological examination to assess the adverse effects. Western Blotting: SPARC expression in the cells (e.g., U87 and bEnd.3) and tissues (e.g., normal brain, subcutaneous, and orthotopic glioma) was examined. CD31 and CD206 expression in the tissues (e.g., normal brain, subcutaneous, and orthotopic glioma) after drug treatment was also examined. Protein concentration in the cell lysis and tissue homogenate was measured with a standard BCA method. The samples were resolved by 10% SDS-PAGE and analyzed by Western blotting analysis using polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA in Tris-buffered saline Tween (TBST) buffer. The membranes were incubated overnight at 4 °C with anti-SPARC and anti-GAPDH and then with HPR-conjugated second antibodies. The membranes were washed and stained for gel imaging analysis (ChemiDoc MPTM Imaging System, BIO-RAD).

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b04268. Additional tables and figures (PDF) 10011

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