D-T7 Peptide Modified PEGylated Bilirubin Nanoparticles Loaded with

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Biological and Medical Applications of Materials and Interfaces

D-T7 Peptide Modified PEGylated Bilirubin Nanoparticles Loaded with Cediranib and Paclitaxel for Antiangiogenesis and Chemotherapy of Glioma Meinan Yu, Dunyan Su, Yuanyuan Yang, Lin Qin, Chuan Hu, Rui Liu, Yang Zhou, Chuanyao Yang, Xiaotong Yang, Guanlin Wang, and Huile Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b16219 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 16, 2018

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ACS Applied Materials & Interfaces

D-T7 Peptide Modified PEGylated Bilirubin Nanoparticles Loaded with Cediranib and Paclitaxel for Antiangiogenesis and Chemotherapy of Glioma Meinan Yu1, 2, Dunyan Su2, Yuanyuan Yang2, Lin Qin2, Chuan Hu2, Rui Liu2, Yang Zhou2, Chuanyao Yang2, Xiaotong Yang2, Guanlin Wang1, *, Huile Gao2,* 1.

Faculty of Life Science and Technology, Kunming University of Science and

Technology, 727 South Jing Ming Road, Chenggong County, Kunming, 650500, P. R. China. 2.

Key Laboratory of Drug Targeting and Drug Delivery System of the Education

Ministry, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, P. R. China. *Corresponding

author:

Wang

G.:

[email protected];

Gao

H.:

[email protected]; [email protected] ABSTRACT: The blood brain tumor barrier (BTB) and blood-brain barrier (BBB) have always been the major barriers in glioma therapy. In this report, we proposed D-T7 peptide modified nanoparticles actively targeted glioma by overcoming the BBB and BTB to improve the anti-glioma efficacy. Glioma targeting experiment showed that the penetration effect of D-T7 peptide modified nanoparticles was 7.89-fold higher than that of unmodified nanoparticles. Furthermore, cediranib (CD) and paclitaxel (PTX) were used for the combination of the antiangiogenesis and chemotherapy for glioma. PEGylated bilirubin nanoparticles (BRNPs) were selected as a suitable drug delivery system (CD&PTX@TBRBPs) owing to the antioxidant, anti-inflammatory and reactive oxygen species (ROS)-responsive ability. MTT and apoptosis assays showed that CD&PTX@TBRBPs had the highest cytotoxicity and

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the median survival time of CD&PTX@TBRNPs group was 3.31-fold and 1.23-fold longer than that of saline and CD&PTX@BRNPs groups, respectively. All the results showed that we constructed a novel and accessible peptide-modified dual drug carrier with enhanced anti-glioma effect. KEYWORDS: blood-brain barrier, cediranib, paclitaxel, nanoparticles, bilirubin, glioma 1 INTRODUCTION For decades, cerebral tumors have been proved to be a great menace to the physical and mental health of human beings 1. Glioma is the most common and deadliest form with its invasive nature in all pernicious cerebral tumors 2. In 2005, radiotherapy with temozolomide chemotherapy was first introduced into glioma treatment, and then electromagnetic therapy combined with chemoradiotherapy was employed in 2015 3-4. Despite some progress made on glioma treatment and imaging 5, the poor drug distribution in glioma, to a great extent, contributes to the short survival of most patients 6, and the median survival period is only 14.6 months 7. With the rapid development of nanotechnology, it is expected that the newly developed nanoparticles yield progress in improving glioma targeting drug delivery and treatment 8. In glioma targeting delivery, the blood brain tumor barrier (BTB) and blood brain barrier (BBB) enormously limit the distribution of nanoparticles in glioma

9-12.

It is

promising to overcome the BBB and BTB by single ligand modified nanoparticles that target the receptors overexpressed on both glioma cells and BBB 11, 13. The brain capillary glioma cells and endothelial cells overexpress transferrin receptor (TfR), which makes it an attractive target for glioma

14-16.

Compared with proteins, small

peptides have gained much attention as targeting ligands owing to their facile synthesis, low cost, and stability. T7 peptide is a heptapeptide which showed high binding affinity with TfR 17-18. Therefore, several studies utilized T7 peptide to target glioma, which showed improved glioma treatment

18-20.

However, the proteolysis

property of L-form peptides attenuates their capacity in active targeting delivery. All D-form peptides with retro-inverso sequence showed higher stability and similarity, or even better receptor affinity than the L-form peptides 21-22. Our previous study also


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demonstrated the D-form T7 (D-T7) peptide could effectively bind with TfR and mediate brain targeting delivery

23.

Therefore, D-T7 peptide was modified on the

nanoparticles for glioma targeting delivery. Bilirubin (BR), which is an endogenous gall pigment with anti-inflammatory and antioxidant effectiveness, plays a crucial role in preventing tissues and cells from reactive oxygen species (ROS)-stimulus harm

24-26.

Studies showed that PEGylated

bilirubin nanoparticles (BRNPs) had been used as drug carriers to treat a wide range of inflammatory diseases

27-29.

Other researchers had also found that BRNPs were

fairly water-dispersible and circulated much longer in blood circulation and could be selected as a dual-responsive (light and ROS) drug-delivery system

30.

Based on the

above characteristics, BRNPs was selected as the drug-delivery system and D-T7 peptide was modified on the surface of BRNPs (TBRNPs) in this paper. It was expected to target glioma selectively and release drugs quickly with response to tumor microenvironment to improve the anti-glioma effect. Another reason for the poor survival of the patients with tumor is high tumor angiogenesis and VEGF is a key factor in angiogenesis 31. Cediranib (CD) can disrupt VEGF signaling pathways that cause the acute angiogenesis by inhibiting the tyrosine kinase activity of c-KIT and VEGFR1, 2, and 3. So, it could be incorporated into conventional chemotherapy for anti-angiogenesis

32.

Studies had shown that CD

combined with carboplatin and paclitaxel (PTX) were used for the treatment of patients with recurrent or metastatic cervical cancer, showing that the combinational drugs prolonged the patient’s life span 33. PTX plays a prominent therapeutic role in cancers by interacting with microtubules

34-35.

In this paper, PTX and CD were

encapsulated in TBRNPs to co-treat glioma and it was expected that the best anti-glioma effect was achieved, along with inhibiting angiogenesis (Scheme 1). In this study, we screened the appropriate drug proportion by MTT assay and drugs were encapsulated in BRNPs. Then the D-T7 peptide was modified onto PEGylated BR, and proved that TBRNPs obtained a better targeting ability. Anti-tumor effect of CD&PTX@TBRNPs was researched in this paper, and the result showed that CD&PTX@TBRNPs lengthened mice’s survival time, showing the potential



anti-glioma effects (Scheme 1).

Scheme 1. Schematic of transport mechanism and bio-responsive drug release of CD&PTX@TBRNPs. 2. EXPERRIMENTAL PART 2.1 Materials. PTX and CD were obtained from Dalian Meilun Biotech Co., Ltd. (Dalian, China) and Chemlin Chemical Industry Co., Ltd. (Nanjing, China), respectively. D-T7 peptide with a cysteine on C-terminal (D-T7-cys) (sequence: hrpyiahc, all D -form amino acids) was synthesized by Phtdpeptides Co., Ltd. (Zhengzhou, China). BR and Mal-PEG2000-NH2.TFA were obtained from Tokyo Chemical Industry (Tokyo, Japan) and Jenkem Technology (Beijing, China), respectively. Annexin V-FITC apoptosis detection kit was obtained from BD Pharmingen (USA). All cell lines were obtained from Chinese Academy of Sciences Cell Bank (Shanghai, China). Rat anti-mouse CD34 antibody was achieved from eBioscience, Inc., (San Diego, USA) and Rabbit anti-mouse TfR antibody was achieved from Novus Biologicals, Inc., (USA). Alexa Fluor® 488-labeled donkey anti-Rat secondary antibody was achieved from Invitrogen (USA) and Cy3-labeled Goat anti-Rabbit secondary antibody was achieved from Proteintech Group, Inc., (Chicago, USA). Male Kunming mice (20 ± 2g) were obtained from Chengdu Dashuo Biotechnology Co., Ltd. (Chengdu, China). All animal experiments were carried out under the approval of the ethics committee of Sichuan University. 2.2 Optimization the ratio of CD to PTX. MTT assay was selected to optimize the ratio of CD to PTX. CD and PTX (Cremophor EL/alcohol = 1/1) were prepared


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into a series of concentrations with serum-free medium and put into 96-well plate with HUVE cells. The concentrations of PTX and CD were prepared between 5 nM and 32.8 μM. After 24 h, MTT was added into each well and incubated. Then, formazan crystal was dissolved with dimethyl sulfoxide and the absorbance at 570 nm was detected by microplate reader (TECAN, Switzerland)

36.

The combined effect

between CD and PTX was evaluated by using combination index (CI) way by the median-effect analysis of Chou and Talalay and CI < 0.9 represents synergism 37. 2.3 Synthesis of T7-PEGylated BR. Mal-PEG2000-NH2.TFA (0.14 mM) and trimethyl-amine were firstly reacted with T7 (23.8 mg) in dimethyl sulfoxide at 25 oC for 24 h with a pH of 7.0 16. Then, BR (0.14 mM), NHS (0.14 mM) and EDC (0.14 mM) were reacted in dimethyl sulfoxide for 30 min at 25 oC

27, 38.

T7-PEG2000-NH2

(0.14 mM) was reacted with BR (0.14 mM) at 25 oC for 4 h. The mixture and chloroform were added into separate funnel and 0.1 N HCl was used to wash the mixture. The unreacted BR was then removed by centrifugation when the production was dissolved in methanol. The solution was rotated and evaporated to obtain T7-PEGylated BR (BR-PEG-T7). 2.4 Synthesis of CD or PTX @TBRNPs. 0.344 nM BR-PEG-T7 were dissolved in 200 μL chloroform and 2 mL deionized water, and 12 μL CD or PTX (10 mg/mL in DMSO) were added into the system for sonication (5 min, 5 s/5 s, 65 w). CD or PTX @TBRNPs were obtained by removing chloroform in vacuum and homogenizing again by sonication (10 min, 5 s/5 s, 65 w). Zeta potential and particle size were analyzed by particle size meter (Malvern, UK) through dynamic light scattering (DLS) principle. Nanoparticles’ morphology was observed by the transmission electron microscopy (TEM; Hitachi, H-600, Japan). 2.5 Quantification of CD and PTX. High-performance liquid chromatography (HPLC, Shimadzu, Japan) (C18 column-4.6 mm, 250 mm, Dikma) was used to measure the encapsulation efficiency and drug loading capacity of CD&PTX in nanoparticles. The mobile phase’s flow rate of CD was 0.5 mL/min

39.

The mobile

phase of PTX was water/acetonitrile (4/6, v/v), flow rate was 1 mL/min and detection wavelength was 227 nm with column temperature of 30 oC. Then, the drug content in



Page 6 of 27

nanoparticles was analyzed. CD or PTX@TBRNPs was centrifugated (6000 g, 5 min), then, the deposition was dissoved in methanol and measured by HPLC. 2.6 In vitro CD&PTX@TBRNPs toxicity test. HUVE, C6 and bEnd.3 cells were cultured in 96-well plate (2 × 103 cells per well) for one day. CD or PTX@BRNPs, CD or PTX@TBRNPs, BRNPs and TBRNPs were prepared into a series of concentrations in serum-free medium and placed into 96-well plate. After 24 h, MTT was added in 96-well plate and incubated. Then, formazan crystal was dissolved with dimethyl sulfoxide and the absorbance at 570 nm was detected by microplate reader 36.

2.7 Cellular uptake. Different forms of nanoparticles loaded with coumarin-6 (Cou6) were put into the 12-well plate with bEnd.3 and C6 cells at a concentration of 200 ng/mL. The cells were processed and observed by flow cytometry (BD FACSCelesta, USA) after incubation at 37 oC for 1 h 36. For qualitative assay, C6 cells and bEnd.3 cells were seeded onto cover glass in 6-well plate (1 × 105 cells per well). 24 h post incubation, different nanoparticles were put into the wells for 1 h at a concentration of 200 ng/mL per well. Then, the cells were imaged by confocal microscope (Zeiss, Germany) 36. 2.8 In vitro BBB model migration assays. BBB model were prepared by Millicell Cell Culture Inserts

40.

Briefly, bEnd.3 cells (1 × 105 cells per well) were plated in

6-well culture-insert for about one week. BBB model’s transendothelial electric resistance (TEER) was detected by Millicell ERS (Millipore, USA). When electric resistance was above 200 Ω, the system was selected for further application. Then, C6 cells were planted on cover glass in another 6-well plate. Then C6 cells and bEnd.3 monolayers were co-cultured for one day. Cou6@BRNPs and Cou@TBRNPs were putted into donor chamber of bEnd.3 monolayer. After 4 h and 12 h treatment, the bEnd.3 monolayers and C6 cells on the plates were processed and imaged by confocal microscope 40. 2.9 Cellular apoptosis. HUVE cells were treated with PTX@BRNPs, CD@BRNPs,

CD&PTX@BRNPs

and

CD&PTX@TBRNPs

(PTX

concentration-0.0854 μg/mL, CD concentration-0.18 μg/mL) at 37 oC with 24 h.


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While

C6

cells

were

incubated

with

PTX@BRNPs,

CD@BRNPs,

CD&PTX@BRNPs and CD&PTX@TBRNPs (PTX concentration-0.277 μg/mL, CD concentration-0.585 μg/mL) under 37 oC for 24 h. HUVE and C6 cells were stained with Annexin V-FITC and PI, and analyzed by flow cytometry

41.

Results were

analyzed by Flowjo 7.6 software. 2.10 Pharmacokinetic test. 12 male Kunming mice were divided into two groups, and were injected with DiD@BRNPs and DiD@TBRNPs (DiD 0.24 mg/kg), respectively. At 0.083 h, 0.167 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 10 h, 24 h and 36 h, 40 μL orbital bloods were collected and anticoagulated with heparin sodium. Then, 40 μL orbital bloods were diluted with 40 μL PBS (pH 7.4), and the fluorescent intensity was determined by Lumina III Imaging System (600-excitation, 710-emission). 2.11 In vivo imaging. C6 glioma bearing mice were constructed according to previous paper 42. 10 days later, DiR@BRNPs and DiR@TBRNPs (0.24 mg DiR/kg) were injected into the C6-bearing mice by tail vein. At 2 h, 4 h, 12 h, 24 h and 48 h after injection, mice were imaged using the Lumina III Imaging System (PerkinElmer, USA). Similarly, after injecting DiD@BRNPs and DiD@TBRNPs (0.25 mg DiD/kg) for 24 and 48 h, the mice were dissected after perfusion with normal saline and 4% paraformaldehyde, respectively. The organs were imaged using the Lumina III Imaging System. Then all organs were dehydrated and sectioned at 10 μm with the freezing microtome (Leica CM1950, Germany)

41.

At last, the brains slides were

stained with DAPI, antibodies of anti-CD34 and anti-TfR, and other organs were only stained with DAPI. The fluorescence distribution was observed using confocal microscope. 2.12 Biocompatibility analysis. 36 male Kunming mice were divided into three groups, and were injected with saline, free CD&PTX and CD&PTX@TBRNPs (PTX-1.7 mg/kg, CD-3.6 mg/kg), respectively, every other day for 6 times. 24 h after the last administration, 500 μL whole bloods were collected and anticoagulated with heparin sodium for complete blood count analysis. And another 1mL bloods were centrifuged with 3000 rpm for 5 minutes. The supernatants were taken for



biochemical analysis. 2.13 Anti-glioma effectiveness. Glioma bearing mice were divided into 6 groups (10 mice for each group). The mice were intravenous injected with saline, free CD & PTX, PTX@BRNPs, CD@BRNPs, CD&PTX@BRNPs and CD&PTX@TBRNPs (PTX-1.7 mg/kg, CD-3.6 mg/kg) at 10, 12, 14, 16, 18 and 20 days, respectively. The body weight and survival time were recorded. Two mice from each group were sacrificed at 24 h after the last treatment. 4% paraformaldehyde was used to fixed organs and paraffin was used to embed organs for section preparation and H&E staining. 2.14 Statistical analysis. Student’s t-test was used for statistical analysis. All data were analyzed by mean ± standard deviation (SD). Kaplan-Meier survival plot (SPSS 16.0) was selected to analyze survival time and log-rank test was selected to drawing survival curves, p < 0.05 (*), 0.01 (**) and 0.001 (***) represent significance. 3. RESULTS AND DISCUSSION 3.1 Optimization ratio of CD to PTX. The dose-response experiments of CD and PTX were used for assessing the inhibitory effect of two drugs on HUVE cells. The 50 % cell inhibiting concentration (IC50) of CD and PTX were 5.58 μM and 0.256 μM, respectively (Figure 1A&D). Fixed ratios of two drugs were determined by IC50 concentrations and used for subsequent CI calculation. To study whether CD strengthened the cytotoxicity effect of PTX, HUVE cells were incubated with gradually increased concentrations of PTX and fixed dose of CD, and cell viability was measured. Figure 1B showed CD strengthened tumor cell growth inhibition of PTX, and the CI value was less than 0.3 when PTX and CD were 25 nM and 100 nM, respectively. Figure 1E showed CI values were apparently lower than 1 at various concentrations, illustrating that the combinational drugs inhibited more cell growth. Then, A range of doses where CD was four times more than PTX was set to incubate HUVE cells and CI values were mostly less than 1 (Figure 1C&F). Thus, this ratio could be used for further research.


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Figure 1. Synergistic study of CD and PTX for HUVE cells and C6 cells. (A), (D) The IC50 of the CD and PTX for HUVE cells. (B), (C) Dose-response curve of two drugs for HUVE cells. (E), (F) CI values of B and C graphs, respectively. (G) Toxicity of TBRNPs. (H), (I) Analysis of synergy CD&PTX@TBRNPs for HUVE cells and C6 cells, respectively. a, b, c and d represent CD@BRNPs, PTX@BRNPs, CD&PTX@ BRNPs, CD&PTX@TBRNPs in H and I graphs, respectively. 3.2 Characterizations of nanoparticles. The mean size of CD@BRNPs and PTX@BRNPs was 71.5 and 77.2 nm, and the PDI of CD@BRNPs and PTX@BRNPs was 0.32 and 0.27, respectively (Table 1&Figure 2), showing a uniform distribution. The mean size of CD@TBRNPs and PTX@TBRNPs slightly increased, owing to D-T7 peptide modification. The zeta potential of nanoparticles was around 10 mV. The encapsulation efficiency of CD or PTX loaded nanoparticles was above 90%, and the drug loading capacity was between 8% and 9%, suggesting the peptide no influence on the encapsulation efficiency of nanoparticles. TEM images demonstrated that the size of nanoparticles

was consistent with the results of the DLS



Page 10 of 27

measurements. Table 1. Characterizations of nanoparticles (n = 3, mean ± SD). Nanoparticles

Size(nm)

Potential

PDI

(mV)

CD@BRNPs

71.9 ± 1.3

9.51 ±

0.325 ± 0.017

0.55 CD@TBRNPs

PTX@BRNPs

112.3 ±

11.33 ±

9.1

0.21

77.2 ± 9.1

6.81 ±

0.346 ± 0.037

0.273 ± 0.119

0.20 PTX@TBRNPs

117.8 ±

12.20 ±

3.5

0.26

0.336 ± 0.003

Drug

Encapsula

loading

tion

capacity

efficiency

8.35 ±

97.94 ±

0.01

0.15

8.80 ±

98.94 ±

0.01

0.13

8.11 ±

95.00 ±

0.04

0.42

8.51 ±

94.56 ±

0.03

0.39

Figure 2. Particle size characteristics of nanoparticles. Bars represent 100 nm. 3.3 In vitro TBRNPs and CD&PTX@TBRNPs toxicity test. When the TBRNPs concentration was up to 10 μM, the cell viability was around 80% and there was no obvious difference from the BRNPs group, indicating that the carrier was almost nontoxic in the normal range of administration dose (Figure 1G). According to above


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mentioned optimization, the ratio of CD/PTX (4/1) was chose for subsequent research. The IC50 of CD@BRNPs and PTX@BRNPs was 4.14 and 0.40 μM on HUVE cells, respectively (Figure 1H&I). However the IC50 of CD&PTX@BRNPs on HUVE cells significantly decreased to 0.183 μM (according to the concentration of CD). Similarly, the IC50 of CD@BRNPs, PTX@BRNPs and CD&PTX@BRNPs on C6 cells showed consistent trend with that of HUVE cells, suggesting that the combinational CD and PTX obtained a better cell killing capacity. The IC50 of CD&PTX@TBRNPs on HUVE cells was similar to that of CD&PTX@BRNPs. However, The IC50 of CD&PTX@TBRNPs on C6 cells was only 1.39 μM (according to the concentration of CD), which was 8.78-fold lower than that of CD&PTX@BRNPs. These results indicated that D-T7 peptide modification significantly improved the antitumor effect on C6 cells rather than HUVE cells, which accorded with the overexpression of TfR on C6 cells. 3.4 Cellular uptake. The fluorescence intensity of the bEnd.3 and C6 cells treated with Cou6@TBRNPs was much higher than that of Cou6@BRNPs, suggesting that the D-T7 peptide actively targeted C6 and bEnd.3 cells, which coincided with previous report 11. Quantitative analysis showed the fluorescence intensity of C6 and bEnd.3 cells incubated with Cou6@TBRNPs was 2.73-times and 1.15- times higher than that of Cou6@BRNPs, respectively, which was in accordance with the qualitative data (Figure 3A&B). To further verify that D-T7 peptide could penetrate through the BBB, the bEnd.3 cell monolayer was used as BBB model in vitro. As shown in Figure 3C, the fluorescence intensity on bEnd.3 monolayer treated with Cou6@TBRNPs was higher than that of Cou6@BRNPs at 4 h, and the fluorescence intensity was enhancive with the time extending to 12 h. After penetrating bEnd.3 cells monolayers, the uptake of nanoparticles on C6 cells was consistent with the uptake result on bEnd.3 cells monolayer in Figure 3D, indicating that D-T7 peptide penetrated through the BBB model and was taken up by C6 cells, and the amount of penetration and fluorescence intensity increased over the time.



Figure 3. Uptake in vitro. A: The uptake results of cell incubated with different nanoparticles for 1 h. Blue represents nucleus, green represents Cou6. Bar indicates 50 μm. B: Cou6@BRNPs and Cou6@TBRNPs uptake results by bEnd.3 and C6 cells for 1 h by the flow cytometry. C, D: Uptake results of bEnd.3 monolayer (C)and C6 cell (D) co-culture system, blue represents nucleus and green represents Cou6. Bars represent 50 μm and 100 μm for bEnd.3 and C6 cells, respectively. p < 0.05 (*) and 0.01 (**) represent significance. 3.5 Cellular apoptosis. In vitro C6 and HUVE cells apoptosis assay was evaluated by the Annexin-V/PI Kit. The total percentage of apoptosis and necrotic cells was 19.62% and 22.8% on HUVE cells incubated with CD@BRNPs and PTX@BRNPs, respectively (Figure 4A). The total percentage of apoptosis and necrotic cells was 24.59% on HUVE cells incubated with CD&PTX@BRNPs and was significantly higher than that of CD@BRNPs and PTX@BRNPs groups (Figure 4B). The total amount of apoptosis and necrotic cells of C6 cells was similar to that of HUVE cells incubated with CD@BRNPs, PTX@BRNPs and CD&PTX@BRNPs, indicating that the combinational drugs showed stronger toxicity. The total percentage of apoptosis and necrotic cells was 74.1% on C6 cells incubated with CD&PTX@TBRNPs, which


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was apparently higher than that of CD&PTX@BRNPs group (Figure 4C). However, the total percentage of apoptosis and necrotic cells was 24.9% on HUVE cells incubated with CD&PTX@TBRNPs and was not significantly different from CD&PTX@BRNPs group, which further proved that D-T7 peptide modification significantly improved the antitumor effect on C6 cells rather than HUVE cells. The result coincided with overexpression of TfR on C6 cells 14. Additionally, the data of apoptosis experiment was consistent with that of MTT assay.

Figure 4. Apoptosis in vitro. (A) The apoptosis assay of C6 and HUVE cells. (B), (C) The percentage of necrotic and apoptosis cells on HUVE and C6 cells, respectively, after different nanoparticles incubation. a, b, c and d represent CD@BRNPs, PTX@BRNPs, CD&PTX@BRNPs and CD&PTX@TBRNPs, respectively. p < 0.05 (*) and 0.001 (***) were considered as significance. NS represents no significance. 3.6 Tumor targeting efficiency study. Pharmacokinetic experiments were analyzed before tumor targeting efficiency analysis. The plasma half-life of DiD@TBRNPs and DiD@BRNPs were 15.638 and 16.123 h, respectively (Table S1),



Page 14 of 27

suggesting no significant difference in the plasma concentration of nanoparticles between the two groups. Next, brain tumor targeting experiments were studied. 2 h after nanoparticles injection, compared to DiR@BRNPs group, DiR@TBRNPs groups obtained higher fluorescence intensity (Figure 5A). Moreover, the fluorescence signal of DiR@TBRNPs group increased in

glioma site with the time

extending to 24 h, which was stronger than DiR@BRNPs, indicating that DiR@TBRNPs actively targeted glioma selectively, instead of passive penetration into the glioma through simplely enhanced permeability and retention (EPR) effectiveness

41,

owing to the overexpression of TfR on both glioma and brain

capillary endothelial cells. Due to the actively targeting capacity of DiR@TBRNPs, which showed better glioma targeting capacity than nanoparticles without D-T7 modification. On the other hand, the fluorescence signal of DiR@TBRNPs groups at 48 h was weaker than that at 24 h, suggesting the naonparticles were eliminated from brain. 3.7 Tissue distribution of DiD@TBRNPs. The fluorescence intensity of DiD@TBRNPs group in brain was 7.89 times higher than DiD@BRNPs group’s fluorescence intensity at 24 h. In above two groups, the fluorescence intensity in brain decayed at 48 h, consistent with the imaging in vivo (Figure 5B&C). To prove whether

TfR’s

overexpression

promoted

the

selective

accumulation

of

DiD@TBRNPs in glioma, TfR expression was detected by staining with anti-TfR antibody in frozen sections of glioma (Figure 5D). The red fluorescence signal at glioma was strong, while the intensity of red fluorescence signal in normal brain was weaker, suggesting that TfR’s overexpression was consistent with the progression and proliferation of C6 glioma cells. Evidently, the signal of DiD was the strongest in glioma region of DiD@TBRNPs group, which further validated the capacity of D-T7 peptide targeting TfR. Neovasculature was clearly found in glioma, after staining by anti-CD34 antibody (Figure 5E). This result confirmed that mass accumulation of DiD@TBRNPs was due to the D-T7 peptide binding to TfR, which promoted the accumulation of nanoparticles in the glioma. The nanoparticles primarily accumulated in the spleen and liver at 24 h, while the


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fluorescence signal decreased with the time extending to 48 h (Figure 5B&C), suggesting nanoparticles might be eliminated in the liver. In addition, the fluorescence intensity in other organs also decreased at 48 h. However, the fluorescence signal of DiD@BRNPs group increased in the heart at 48 h, indicating that this group might be of higher cardiotoxicity. DiD@TBRNPs group obtained a lower fluorescence signal than DiD@BRNPs group in heart, liver and lung both at 24 h and 48 h, suggesting that DiD@TBRNPs were less toxic to these normal organs. However, in spleen and kidney, DiD@TBRNPs group showed a stronger fluorescence signal than DiD@BRNPs both at 24 h and 48 h, suggesting that DiD@BRNPs were less toxic to these normal organs. The results of frozen sections were consistent with those results (Figure 6).



Figure 5. Fluorescence distribution in vivo. (A) Fluorescence signal distribution of different nanoparticles with the time extending in vivo. Bar represents radiant efficiency between 1935 and 6154. (B) Fluorescent imaging of organs ex vivo. Bar represents radiant efficiency between 8.26 × 107 and 3.11 × 109. (C) Fluorescent signal’s semi-quantification. (D), (E) Fluorescence distribution of DiD@BRNPs and DiD@TBRNPs in glioma sections after intravenous injection at 24 h and 48 h, green indicates DiD, blue indicates nucleus and red indicates the TfR and CD34, respectively. N represents normal brain, G represents glioma, and bars represent 50 μm.

Figure 6. Fluorescence distribution of DiD@BRNPs and DiD@TBRNPs in slices. Blue represents nucleus, green represents DiD, and bar represents 50 μm. 3.8 Anti-glioma efficacy and biocompatibility. Based on the above investigations, we further evaluated anti-glioma efficacy of CD&PTX@TBRNPs after systemic administration using the body weight and survival study (Figure 7A&B, Table 2). Mean body weights of saline and free CD&PTX groups were rapid loss and early


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mortality of those was high. C6 bearing mice in other groups showed no obvious body weight change within 20 days of treatments, which indicated drug loaded nanoparticles controlled the weight loss of mice. PTX@BRNPs and CD@BRNPs groups’ median survival time were 32 and 38 days, respectively, verifying the better treatment effectiveness than free CD&PTX group. CD&PTX@BRNPs group’s median survival time was 43 days, showing that the survival period of mice prolonged than mice treated with single drug loaded nanoparticles. This result showed that combinational drugs obtained better glioma treatment effect. The median survival time of CD&PTX@TBRNPs group was 53 days, which was prolonged by 23% in comparison with CD&PTX@BRNPs group, demonstrating D-T7 peptide contributed to the best anti-glioma effectiveness of CD&PTX@TBRNPs. To obtain the conceivable mechanism behind the prolonged survival time of treated mice, the brain slices of glioma-bearing mice were observed. H&E staining results showed that glioma size of saline and free CD&PTX groups was larger compared with other groups (Figure 7C), which was consistent with their shorter survival time. Glioma size from PTX@BRNPs and CD@BRNPs groups reduced, suggesting slight anti-glioma effect of the two drugs. Compared to PTX@BRNPs and CD@BRNPs groups, glioma size of CD&PTX@BRNPs group further reduced, demonstrating the strengthened anti-glioma effect. Furthermore, glioma size of CD&PTX@TBRNPs group was the lowest in all groups, demonstrating actively targeting nanoparticles with two drugs achieved the best anti-glioma effect. Compared to saline group, apparent typical tubular necrosis and atrophy were found in kidneys of PTX@BRNPs and free CD&PTX groups, demonstrating potential nephrotoxicity of PTX. Compared to saline group, the kidneys of CD&PTX@BRNPs and CD&PTX@TBRNPs groups achieved weaker tubular atrophy, indicating that the combinational drugs reduced the nephrotoxicity of PTX. Compared with the saline group, H&E staining of other organs in CD&PTX@TBRNPs group showed no significant toxicity (Figure 8), and suggesting a better biocompatibility of PEGylated BR nanoparticles in vivo. Several parameters, including mean platelet volume (MPV), mean corpuscular volume (MCV), red blood cell distribution width (RDW), mean corpuscular hemoglobin



(MCH), hematocrit (HCT), lymphocyte (Lymph), white blood cell (WBC), monocyte (Mon), neutrophile granulocyte (Gran), hemoglobin (HGB), platelet (PLT) and red blood cell (RBC) were evaluated after two weeks treatment by saline, free CD&PTX and CD&PTX@TBRNPs. Most parameters of CD&PTX@TBRNPs groups were within the normal range (Figure S1), suggesting that CD&PTX@TBRNPs displayed no obvious hematological toxicity in vivo. However, in the free CD&PTX group, the parameters of alanine transaminase (ALT), aspartate transaminase (AST) and albumin （ AL ） were beyond the normal range, and they were obviously higher than saline group, suggesting that free drugs displayed higher hepatotoxicity (Figure S2). In CD&PTX@TBRNPs group, although the ALT was beyond the normal range, other parameters were in the normal range, which indicated that the hepatotoxicity of the CD&PTX@TBRNPs group was lower than that of the free CD&PTX group. The creatinine (CRE), blood urea nitrogen (BUN), urea (UA), lactic dehydrogenase (LDH) and creatine kinase (CK) of the saline, free CD&PTX and CD&PTX@TBRNPs groups were all in normal range (Figure S2), indicating that the CD&PTX@TBRNPs displayed no obvious toxicity to kidneys and hearts of mice. These experiments proved that the CD&PTX@TBRNPs displayed a good biocompatibility.


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Figure 7. Anti-glioma effectiveness of different nanoparticles after treatment in vivo. (A) Body weight analysis of C6-bearing mice. (B) Survival curves of mice were calculated by Kaplan Meier survival assay. (C) H&E staining experiments of model mice in brains and bar indicates 100 μm. a, b, c, d, e and f represent CD&PTX@TBRNPs, CD&PTX@BRNPs, CD@BRNPs, PTX@BRNPs, CD&PTX, Saline in Figure A&B, respectively.



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Figure 8. H&E staining experiments. Bar represents 100 µm. Table 2. The survival period of C6-bearing mice after administration (n = 8). Groups

Median

SD

Significance

(day)

Incremental survival time

Saline

16

3.300

-

-

CD&PTX

19

2.828

-

19%

PTX@BRNPs

32

12.728

-

100%

CD@BRNPs

38

11.785

-

137%

PTX&CD@BRNPs

43

14.849

a, b, c, d

168%

PTX&CD@TBRNPs

53

13.093

a, b, c, d

231%

a, b, c and d represent p< 0.05 (*), compared with saline, CD&PTX, PTX@BRNPs, and CD@BRNPs groups, respectively. 4. CONCLUSIONS In summary, we established a CD and PTX co-loaded PEGylated BR nanocarrier with D-T7 peptide modification across the BBB and BTB for improving anti-glioma


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effectiveness. Uptake and glioma targeting efficiency trials proved that D-T7 peptide promoted nanoparticles accumulation and penetration into glioma. MTT and apoptosis trials verified that combinational CD&PTX obtained a better effect than single use. Glioma bearing mice treated with CD&PTX@TBRNPs obtained the longest survival period, indicating that D-T7 peptide promoted CD&PTX accumulation and penetration into glioma for improving anti-glioma effectiveness. ASSOCIATED CONTENT Supporting Information The pharmacokinetic parameters, complete blood count and biochemical parameter analysis of mice after treated with different nanoparticles. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] or [email protected]. ORCID Huile Gao: 0000-0002-5355-7238 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS The work was supported by National Natural Science Foundation of China (81872806, 31571016, 81360162 and 81260351), Sichuan Youth Science and Technology Innovation Research Team Funding (2016TD0001) and the Fundamental of Research Funds for the Central University (SCU2017A001). REFERENCES (1) Lapointe, S.; Perry, A.; Butowski, N. A., Primary Brain Tumours in Adults. Lancet 2018, 392, 432-446. (2) Harris, M.; Svensson, F.; Kopanitsa, L.; Ladds, G.; Bailey, D., Emerging Patents in the Therapeutic Areas of Glioma and Glioblastoma. Expert Opin. Ther. Pat. 2018, 28, 573-590. (3) Stupp, R.; Taillibert, S.; Kanner, A. A.; Kesari, S.; Steinberg, D. M.; Toms, S. A.; Taylor, L. P.; Lieberman, F.; Silvani, A.; Fink, K. L.; Barnett, G. H.; Zhu, J. J.;



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Table Of Contents Graphic 143x60mm (300 x 300 DPI)


D-T7 Peptide Modified PEGylated Bilirubin Nanoparticles Loaded with

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