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Peptide-22 and cyclic RGD functionalized liposomes for glioma targeting drug delivery overcoming BBB and BBTB Cuitian Chen, Ziqing Duan, Yan Yuan, Ruixiang Li, Liang Pang, Jianming Liang, Xinchun Xu, and Jianxin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15831 • Publication Date (Web): 27 Jan 2017 Downloaded from http://pubs.acs.org on February 1, 2017

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ACS Applied Materials & Interfaces

Peptide-22 and cyclic RGD functionalized liposomes for glioma targeting drug delivery overcoming BBB and BBTB Cuitian Chen,a Ziqing Duan,a Yan Yuan,a Ruixiang Li,a Liang Pang,a Jianming Liang,a Xinchun Xu, b,* and Jianxin Wang a,* a

Department of Pharmaceutics, School of Pharmacy, Fudan University & Key Laboratory of

Smart Drug Delivery, Ministry of Education, Shanghai 201203, PR China. b

Shanghai Xuhui Central Hospital, Shanghai 200031, PR China.

KEYWORDS: Glioma, Blood-brain barrier, Blood-brain tumor barrier, Liposome, c(RGDfK), Peptide-22

ABSTRACT: Chemotherapy outcomes for the treatment of glioma remain unsatisfied due to the inefficient drug transport across BBB/BBTB and poor drug accumulation in the tumor site. Nanocarriers functionalized with different targeting ligands are considered as one of the most promising alternatives. However, few studies were reported to compare the targeting efficiency of the ligands and develop nanoparticles to realize BBB/BBTB crossing and brain tumor

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targeting simultaneously. In this study, six peptide-based ligands (Angiopep-2, T7, Peptide-22, c(RGDfK), D-SP5 and Pep-1), widely used for brain delivery, were selected to decorate liposomes respectively so as to compare their targeting ability to BBB or BBTB. Based on the in vitro cellular uptake results on BCECs and HUVECs, Peptide-22 and c(RGDfK) were picked out to construct a BBB/BBTB dual-crossing, glioma-targeting liposomal drug delivery system c(RGDfK)/Pep-22-DOX-LP. In vitro cellular uptake demonstrated that the synergetic effect of c(RGDfK) and Peptide-22 could significantly increase the internalization of liposomes on U87 cells. In vivo imaging further verified that c(RGDfK)/Pep-22-LP exhibited higher brain tumor distribution than single ligand modified liposomes. The median survival time of glioma-bearing mice treated with c(RGDfK)/Pep-22-DOX-LP (39.5 days) was significantly prolonged than those treated with free doxorubicin or other controls. In conclusion, the c(RGDfK) and Peptide22 dual-modified liposome was constructed based on the targeting ability screening of various ligands. The system could effectively overcome BBB/BBTB barriers, target to tumor cells and inhibit the growth of glioma, which proved its potential for improving the efficacy of chemotherapeutics for glioma therapy.

1. INTRODUCTION Glioma is one of the most common and aggressive intracranial malignancies with high morbidity and mortality. The median overall survival of glioma patients is 12-18 months and the 5-year survival rate is less than 10%.1, 2 Because of the invasive growth and an undefined tumor edge, surgery can hardly remove the tumor completely and the recurrence rate is very high.3, 4 Once relapse, the average survival duration is only 1.5 years.1 Systemic chemotherapy has been widely


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used in the treatment of glioma.5-7 Unfortunately, clinical outcomes remain unsatisfied due to the unfavorable pharmacokinetics, severe side effects and poor drug accumulation in the brain parenchyma.7, 8 Recently, the exploitation of nanocarriers for drug delivery has emerged as a promising alternative to conquer these problems.9 However, the effective transport of nanocarriers to the glioma site is still limited.10, 11 It is essential to deliver anti-cancer drug loaded nanocarriers across blood-brain barrier (BBB) and blood–brain tumor barrier (BBTB) simultaneously in order to treat glioma. The BBB, an endothelial cell monolayer associated with pericytes and astrocytes, prevents approximately 98% small molecules and nearly 100% large molecules from transporting into the brain.12 Meanwhile, it behaves as the main obstacle for nanocarriers to enter the brain parenchyma in the early stage of glioma.13 With the gradual progression of glioma, the integrity of BBB is compromised to some extent while the BBTB emerged. The BBTB exists between the brain tumor tissues and capillary vessels, which also impedes the effective transport of nanocarriers into tumor.14,

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Many efforts have been made to overcome BBB or BBTB separately. However, in most cases, BBB and BBTB exist simultaneously when glioma is diagnosed.16 Therefore, it is significant to develop a novel drug delivery system possessing BBB and BBTB dual-targeting ability. As is reported previously, receptor-mediated transcytosis (RMT) is one of the most common strategies for nanocarriers to penetrate BBB/BBTB.17 Studies have found that brain capillary endothelial cells and endothelial cells of tumor angiogenic vessels express numerous different receptors.14, 18 Nanocarriers decorated with specific ligands can efficiently bind to corresponding receptors overexpressed on BBB/BBTB and then initiate the RMT process, resulting in the enhancement of drug penetration across the barriers and accumulation in the glioma site.19 Various ligands have been demonstrated to possess high binding affinities to specific receptors


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and exhibited obvious brain tumor targeting properties when conjugated to nanoparticles. There are thousands of reports about the modification with targeting ligands on nanocarriers. Almost all the research results are positive and prove that the application of ligands could significantly improve the targeting efficiency of the nanosystems. However, it is difficult to compare the results and determine which ligand is more effective for brain delivery, since the important factors influencing the targeting efficiency, such as particle type, formulation parameters and the ligand density, were quite different in various studies.20-22 Therefore, it is essential to evaluate the BBB/BBTB targeting ability of different ligands in the same nanocarrier, which will pick out the most efficient ligand for the establishment of brain targeting drug delivery system. Angiopep-2,23 T7,

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and Peptide-2225 are widely used as specific ligands for low-density

lipoprotein receptor-related protein-1 (LRP1), transferrin receptor (TfR) and Low-density lipoprotein receptor (LDLR), respectively, which are overexpressed on BBB as well as glioma cells. c(RGDfK)16 (a ligand of integrin αvβ3 /αvβ5), Pep-126 (a ligand of IL-13Rα2) and D-SP527 (a novel retro-inverso analogue of L-SP5) have been proved to display dual-targeting bioactivity for both tumor neovasculature (BBTB) and tumor cells. It was reported that nanocarriers functionalized with any of the above ligands could be served as an effective targeting delivery system to deliver chemotherapeutics across BBB or BBTB and accumulate in the tumor site. 26-31 However, few studies were reported to compare the targeting efficiency of these ligands and combine the specific ones to decorate nanocarriers so as to overcome multiple barriers. In this study, we try to develop a glioma-targeted delivery system modified with ligands facilitating across not only BBB, but also BBTB, and targeting tumor cells and neovasculature as well. Liposomes were chosen as the delivery vehicle because of the nontoxic and nonimmunogenic characteristics, the high drug loading ability and the possibility to be decorated


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with different ligands. Six peptide-based ligands with different density were screened and compared the targeting efficiency. We divided these functionalized liposomes into two groups, one for the evaluation of BBB targeting capacity (Angiopep-2-LP, T7-LP, Peptide-22-LP) and the other for the investigation of BBTB targeting ability (c(RGDfK)-LP, Pep-1-LP, D-SP5-LP). Based on the comparison results, we selected the optimal ligand from each group and combined them to develop a BBB/BBTB dual-crossing, glioma-targeting liposomal drug delivery system (c(RGDfK)/Pep-22-DOX-LP). To further verify the brain tumor targeting efficacy, in vitro cellular uptake and in vivo imaging study were performed. Eventually, therapeutic effects of different formulations were evaluated on intracranial glioma-bearing mice model, choosing doxorubicin (DOX) as the model drug.

2. MATERIALS AND METHODS 2.1. Materials. T7 with a cysteine on the N-terminal (Cys-T7) (CHAIYPRH), D-SP5-Cys (D(PRPSPKMGVSVSC)),

Pep-1(CGEMGWVRC),

Angiopep-2-Cys

(TFFYGGSRGKRNNFKTEEYC), c(RGDfK) (c(Arg-Gly-Asp-d-Phe-Lys)) and Peptide-22 (NH2-C6-[cMPRLRGC]c-NH2) were synthesized by Bankpeptide biological technology Co. LTD. (Hefei, China) with the purity above 95%. Soyabean phosphatidylcholine (SPC) was purchased from Lipoid GmbH (Ludwigshafen, Germany). Cholesterol was from Sinopharm Chemical Reagent Co. LTD. (Shanghai, China). mPEG2000-DSPE was obtained by NOF Co. (Tokyo, Japan). DSPE-PEG3400-NHS was provided by Nanocs Co. (NewYork, USA). DSPEPEG3400-Mal was obtained from Laysan Bio Co. (Arab, AL). 5-carboxyfluorescein (FAM), Hoechst 33342 and near infrared dye DiR were received from FanboBiochemicals (Beijing, China). 5-(dimethyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) was purchased from


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sigma (St. Louis, MO, USA). Doxorubicin hydrochloride (DOX·HCl) was obtained from Melonapharma (Dalian, China). Human umbilical vascular endothelial cells (HUVECs), Brain capillary endothelial cells (BCECs) and Human glioblastoma cells (U87s) were obtained from Cell Bank, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, 1% L-glutamine, 1% nonessential amino acids, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C under a humidified atmosphere containing 5% CO2. Balb/c nude mice (Female, 18–20 g) were purchased from Shanghai SLAC laboratory animal Co. LTD (Shanghai, China). All animal experiments were performed in accordance with the principles of care and use of laboratory animals. The protocol of animal experiments was approved by the Animal Experimentation Ethics Committee of Fudan University. 2.2. Synthesis and Characterization of Functionalized Phospholipid. DSPE-PEG3400-T7, DSPE-PEG3400-D-SP5, DSPE-PEG3400-Pep-1, DSPE-PEG3400-Angiopep-2 were synthesized by conjugating DSPE-PEG3400-Mal to the cysteine residue on cys-T7, cys-D-SP5, Pep-1 and Angiopep-2, respectively.27 DSPE-PEG3400-Peptide-22 and DSPE-PEG3400-c(RGDfK) were synthesized by the reaction of amino groups on Peptide-22 and c(RGDfK) with the active group NHS contained in DSPE-PEG3400-NHS, respectively.32 In brief, DSPE-PEG3400-Mal and peptide (molar ratio 1:1.5) were dissolved in warm PBS (pH 7.0) with sonication. DSPE-PEG3400-NHS and peptide (molar ratio 1:2) were dissolved in anhydrous DMF with DIEA (0.5 equiv). Mixtures were gently stirred for 24 h at room temperature. Excessive peptides were removed by dialysis against distilled water. Pure products were freeze dried and stored at -20 °C until use. These


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functionalized phospholipids were confirmed by MALDI-TOF mass spectrometry (MALDI TOF/TOF, SCIEX TOF/TOF™ 5800, AB SCIEX, USA). 2.3. Preparation and Characterization of Liposomes. Liposomes were prepared by thinfilm hydration and extrusion method. Eight kinds of liposomes were prepared with different lipid compositions. Blank liposomes (LP), which were not modified with any ligands, were taken as the control and made of SPC, cholesterol and mPEG2000-DSPE (60:33:7, molar ratio). The liposomes modified with different ligands, including c(RGDfK) modified liposomes (c(RGDfK)LP), D-SP5 modified liposomes (D-SP5-LP), Pep-1 modified liposomes (Pep-1-LP), Angiopep-2 modified liposomes (Angiopep-2-LP), T7 modified liposomes (T7-LP) and Peptide-22 modified liposomes (Pep-22-LP), were prepared with the same formulation as blank liposomes except that part of mPEG2000-DSPE was substituted by DSPE-PEG3400-c(RGDfK), DSPE-PEG3400-D-SP5, DSPE-PEG3400-Pep-1, DSPE-PEG3400-Angiopep-2, DSPE-PEG3400-T7 and DSPE-PEG3400Peptide-22. In order to search for the optimal amount of specific DSPE-PEG3400-ligand in the liposomes, the ratio of 1%, 3% and 5% were compared. Based on the screening results, a kind of dual-modified liposomes, c(RGDfK) and Peptide-22 (c(RGDfK)/Pep-22-LP), composed of SPC, cholesterol,

mPEG2000-DSPE,

DSPE-PEG3400-c(RGDfK)

and

DSPE-PEG3400-Peptide-22

(60:33:1:3:3, molar ratio) were developed to validate the synergetic effect of two ligands. All lipid materials were dissolved in CHCl3, and followed by rotary evaporation to form a lipid film using a ZX-98 rotary evaporator (LOOYE, China) at 35 °C. After maintained in vacuum overnight, the thin film was hydrated with saline at 37 °C and successively extruded through polycarbonate membranes with the pore size of 200 nm, 100 nm and 50 nm, using an Avanti Mini Extruder (Avanti Polar Lipids). DiR and FAM loaded liposomes were prepared by the same method described above except that DiR was added in the organic solution (CHCl3) to


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form the thin film, while FAM was added in the hydration medium (saline). DOX loaded liposomes were prepared using an ammonium sulfate gradient loading method according to the previous report.33 Particle size and zeta potential of various liposomes were measured by dynamic light scattering detector (Zetasizer, Nano-ZS, Malvern, UK). The morphology of liposomes was detected by transmission electron microscopy (TEM, Tecnai G2 F20 S-Twin, FEI, USA). The encapsulation efficiency and the loading efficiency of DOX were measured using a previously reported method.34 2.4. In Vitro DOX Release. The release of DOX was performed by a dialysis method. Briefly, 0.4 mL liposome suspension or free DOX solution (0.5 mg DOX/mL) was placed into a dialysis tube (MWCO = 3500) and tightly sealed. Then the dialysis tube was immersed into 40 mL PBS (pH 7.4) in a 37 °C shaker for 48 h at 100 rpm. At predetermined time points, 0.5 mL of release medium was sampled and immediately replaced with an equal volume of fresh release medium. The concentration of DOX was measured by a microplate reader (synergy TM 2, Bio Tek, USA) at Ex/Em 480 nm/525 nm. 2.5. In Vitro Cellular Uptake. To evaluate the BBB targeting ability of Angiopep-2, T7 and Peptide-22 modified liposomes in vitro, BCECs were seeded into a 12-well plate at a density of 5×105 cells/well. After 24 h incubation, the culture medium was replaced with 5 µM FAMloaded liposomes of different formulations in DMEM containing 10% FBS and allowed for further co-incubation for 4 h at 37 °C. Following that, cells were rinsed with cold PBS twice, then trypsinized, centrifuged and finally resuspended in 0.3 mL PBS. The fluorescence intensity of cells treated with different type and different ligand density (1%, 3% and 5%) of liposomes was analyzed by a flow cytometer (BD Biosciences, Bedford, MA, USA). Similarly, the BBTB


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targeting capacity of c(RGDfK), D-SP5 and Pep-1 modified liposomes was evaluated on HUVECs and the effect of ligand density (1%, 3% and 5%) on cellular uptake was also investigated by flow cytometry as described previously. To qualitatively investigated the tumor targeting ability of LP, c(RGDfK)-LP, Peptide-22-LP and c(RGDfK)/Peptide-22-LP, the cellular uptake of the liposomes on U87 cells was studied. U87 cells were seeded into a 12-well plate at a density of 5×104 cells/well. Twenty-four hours later, 5 µM FAM loaded liposomes in DMEM containing 10% FBS were added into each well. After 4 h incubation, the cells were stained with Hoechst 33342 and then were washed with cold PBS for three times. Finally, the samples were visualized by fluorescent microscopy (Leica, DMI4000D, Germany). For quantitative study, flow cytometry was conducted on U87 cells and the detailed process was in accordance with the above description. 2.6. In Vivo Animal Imaging. The intracranial glioma-bearing mice model was established as previously described. 35 Fifteen days after inoculation, glioma-bearing mice were intravenously injected via the tail with DiR loaded LP, c(RGDfK)-LP, Pep-22-LP and c(RGDfK)/Pep-22-LP respectively at a dose of 0.3 mg DiR/kg. The distribution of fluorescence was observed at predetermined time points (2 h, 6 h, 12 h and 24 h) using an in vivo imaging system (IVIS Spectrum, Caliper, USA). Twenty-four hours later, the mice were sacrificed after heart perfusion with saline. Brains and peripheric organs were harvested and imaged as well. 2.7. In Vitro Cytotoxicity. The cytotoxicity of free DOX, DOX-LP, c(RGDfK)-DOX-LP, Pep-22-DOX-LP and c(RGDfK)/Pep-22-DOX-LP against U87 cells was assessed by MTT assay. Briefly, U87 cells were seeded into 96-well plates at a density of 3× 103 cells/well and cultured for 24 h at 37°C. Different formulations of doxorubicin loaded liposomes were diluted to predetermined concentrations with complete medium and then were added into each well.


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Forty-eight hours later, the medium was removed and the cells were washed twice with PBS, then 100 µL MTT solution (0.5 mg/mL) was added. After 4 h co-incubation at 37 °C, MTT solution was replaced with 150 µL DMSO, followed by gently shaking to dissolve the formazan crystals. The absorbance was measured via a microplate reader (synergy TM 2, BioTek, USA) at 490 nm. The cells without treatment were served as control. 2.8. In Vivo Anti-glioma Efficacy. In vivo anti-tumor efficacy was performed in intracranial glioma-bearing mice. Seven days after tumor inoculation, the mice were randomly divided into six groups (n=6). The mice in blank control group were intravenously injected with saline and the other five treatment groups were administered with free DOX, DOX-LP, c(RGDfK)-DOXLP, Pep-22-DOX-LP and c(RGDfK)/Pep-22-DOX-LP via tail vein at day 7, 14 and 21 with 5 mg DOX/kg, respectively. Body weights were monitored every other day. Survival time was recorded and analyzed by GraphPad Prism 5 (GraphPad Software Inc., California, USA). 2.9. Cardiotoxicity Evaluation. Thirty-six intracranial glioma-bearing mice were divided into 6 groups randomly and given injections of saline, free DOX, DOX-LP, c(RGDfK)-DOX-LP, Pep-22-DOX-LP and c(RGDfK)/Pep-22-DOX-LP, respectively, at a dose of 5 mg DOX/kg at day 7, 14, and 21. Two days after the last administration, the mice were sacrificed. The sections of heart were stained with hematoxylin and eosin to evaluate the cardiotoxicity of DOX. 2.10. Statistical Analysis. All data were analyzed using Graphpad Prism 5 software. Statistical comparisons were performed by one-way ANOVA. Data were expressed as mean ± SD. Statistical significance was defined as *p < 0.05 and extreme significance was defined as **p < 0.01.

3. RESULTS AND DISSCUSSION


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3.1. Synthesis and Characterization of Functionalized Phospholipid. We conjugated T7, D-SP5, Pep-1 and Angiopep-2 to the distal end of DSPE-PEG3400-Mal by Michael addition of cysteine residue and maleimide, respectively. Similarly, c(RGDfK) and Peptide-22 were conjugated to the distal end of DSPE-PEG3400-NHS respectively by the reaction of amino groups with active groups NHS. The results of MALDI-TOF mass spectrometry (see Supporting Information Figure S1) confirmed the successful synthesis of all the six products, with the observed molecular weight around 5099 Da (DSPE-PEG3400-T7), 5689 Da (DSPE-PEG3400-DSP5), 5648 Da (DSPE-PEG3400-Pep-1), 6797 Da (DSPE-PEG3400-Angiopep-2), 5180 Da (DSPEPEG3400-Peptide-22) and 5096 Da (DSPE-PEG3400-c(RGDfK)), which were close to the theoretical molecular weights.

3.2. Evaluation of BBB/BBTB Targeting Ability of Different Ligands Modified Liposomes. We successfully prepared different ligands modified FAM loaded liposomes with 1%, 3% and 5% DSPE-PEG3400-ligand. The mean particle sizes of various formulations ranged from 100 nm to 125 nm, PDI were less than 0.3 and zeta potentials were between -15 mV and 30 mV. The BBB targeting ability of Angiopep-2-LP, T7-LP and Pep-22-LP was evaluated on brain capillary endothelial cells (BCECs), which was widely used as a model for mimicking BBB.30, 36 As shown in Figure 1A, the cellular uptake of liposomes was enhanced by ligand modification, which was respectively owning to the specific binding to low-density lipoprotein receptor-related protein-1 (LRP1), transferrin receptors (TfR) and low-density lipoprotein receptor (LDLR) highly expressed on BCECs and then initiated RMT process.24, 29, 36 However, fluorescence intensity of different ligands decorated liposomes differed from each other. We speculated that it was correlated with the expression level of corresponding receptor on BCECs,


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the ligand-receptor binding affinity and the cellular uptake mechanism. Herein, the cellular uptake of Pep-22-LP was significantly higher than those of Angiopep-2-LP and T7-LP (p < 0.001), indicating the strongest potency of Peptide-22 in transporting liposomes across BBB. Peptide-22, generated by phage display biopanning, is a cyclic peptide with properties of good stability, easy synthesis at a relative low cost and lack of immunogenicity.29 Previous studies have revealed that Peptide-22 exhibits special affinity for LDLR but without competition with endogenous LDL and could be served as a LDLR-targeting moiety for CNS targeting and delivery.25, 29 Furthermore, Pep-22-LP displayed a peptide density-dependent manner. With the increased amount of DSPE-PEG3400-Peptide-22 in the lipid constitution, the cellular uptake was improved as well. Compared with 1% modification ratio, the cellular uptake of 3% DSPEPEG3400-Peptide-22 modified liposomes was significantly higher (p < 0.001). Nevertheless, there was no obvious difference in fluorescence intensity between 3% and 5% modification ratio of Peptide-22, which might be due to the receptor saturation. Considering that large amount of DSPE-PEG3400-ligand would influence the integrity and the stability of liposomes in serum, 3% DSPE-PEG3400-Peptide-22 was opted for the following studies. Similarly, the BBTB targeting capacity of c(RGDfK)-LP, D-SP5-LP and Pep-1-LP was evaluated on human umbilical vascular endothelial cells (HUVECs), which was the commonly used model for mimicking BBTB.37-39 As shown in Figure 1B, compared with unmodified liposomes, the cellular uptake of ligand decorated liposomes was enhanced, among which c(RGDfK)-LP was significantly higher than those of DSP-5-LP and Pep-1-LP (p < 0.001). c(RGDfK) is the specific ligand of integrin αvβ3/αvβ5, which is overexpressed on tumor neovasculature (BBTB). Thus, c(RGDfK) could mediate endocytosis to improve the cellular uptake of neovascular endothelial cells.16 Analogously, Pep-1 could also increase cellular


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internalization through interleukin 13 receptor α2 (IL-13Rα2) mediated endocytosis.40 However, it has been found that the expression level of IL-13Rα2 was lower than that of αvβ3 on HUVECs.39 D-SP5 is a novel retro-inverso analogue of L-SP5.27 Although the binding mechanism of D-SP5 is not much clear, it has been reported that the targeting efficiency of DSP5 might be lower on inactivated HUVECs than VEGF stimulated HUVECs.41 Moreover, from the fluorescence intensity of different density of DSPE-PEG3400-c(RGDfK) modified liposomes, we could find that 3% modification ratio showed significant increase than 1% (p < 0.001), but there was no difference between 3% and 5%. Therefore, 3% DSPE-PEG3400-c(RGDfK) was chosen for the following studies.

Figure 1. Cellular uptake of liposomes modified with different DSPE-PEG3400-ligands on BCECs (A) and HUVECs (B) measured by a flow cytometer. Cells were incubated with 5 µM


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FAM loaded liposomes at 37 °C for 4 h (n = 3, mean ± SD). The horizontal ordinate suggested the ratio of DSPE-PEG3400-ligand in total lipid of the liposomes. *** indicates p < 0.001 and N.S. indicates there is no significant difference.

3.3.

Preparation

and

Characterization

of

Dual

Ligands-modified

Liposomes.

Conventional chemotherapy of glioma has been hampered due to ineffective drug delivery across BBB/BBTB.12, 42 In most cases, BBB and BBTB exist simultaneously when glioma is diagnosed. Based on the cellular uptake results on BCECs and HUVECs (Figure 1A and B), 3% c(RGDfK) and 3% Peptide-22 were selected to construct a dual-modified liposomal drug delivery system, c(RGDfK)/Pep-22-DOX-LP, in which Peptide-22 and c(RGDfK) could enhance the targeting and permeation of the liposomes of the BBB and the BBTB, respectively. Moreover, both of the ligands could bind with the specific receptors on the tumor cells further and then target the liposomes to glioma, which was of great significance for the specific tumor distribution of the system in the brain. Appropriate particle size and uniform distribution of nanoparticles were required for BBB/BBTB permeation and brain tumor targeting.13,

43

The size distribution graph of

c(RGDfK)/Pep-22-DOX-LP was shown in Figure 2A. The mean particle sizes, zeta potentials, DOX encapsulation efficiency and loading efficiency of different formulations were listed in Table 1. For all the liposomes with or without ligand modification, the mean particle sizes ranged from 95 nm to 105 nm, PDIs were less than 0.1 and the DOX encapsulation efficiency and loading efficiency were about 95% and 5.5%, respectively. The results indicated that the incorporation of DSPE-PEG3400-c(RGDfK) and DSPE-PEG3400-Peptide-22 did not affect the physical properties of liposomes significantly. The morphology of c(RGDfK)/Pep-22-DOX-LP


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obtained by TEM revealed that the liposomes were homogeneously spheroids with moderate dispersivity (Figure 2B). No significant change of particle size and PDI of different DOX loaded liposomes was observed in 10% FBS-contained medium over 24 h, suggesting good stability of liposomes in serum-contained medium (see Supporting Information Figure S2). The average size of different formulations maintained approximately 110 nm with a narrow distribution within 14 days, revealing good stability under the storage condition (Data not shown).

Figure 2. Particle size distribution of c(RGDfK)/Pep-22-DOX-LP (A) determined by DLS. Morphology of c(RGDfK)/Pep-22-DOX-LP (B) obtained by TEM after negative staining with 2% sodium phosphotungstate solution. Bar: 100 nm.


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Table 1. Characterization of different doxorubicin liposomes. Data represented mean ± SD (n =3)

-28.57 ± 0.75

Encapsulation efficiency of DOX (%) 97.54 ± 1.13

Loading efficiency of DOX (%) 5.68 ± 0.21

0.094 ± 0.005

-17.45 ± 1.25

95.26 ± 3.19

5.54 ± 0.32

101.33 ± 0.40

0.091 ± 0.008

-16.57 ± 0.61

94.04 ± 0.70

5.16 ± 0.15

103.43 ± 0.92

0.098 ± 0.010

-17.40 ± 1.15

96.65 ± 3.13

5.39 ± 0.27

Liposomes

Particle Size (nm)

PDI

Zeta potential (mV)

DOX-LP

97.56 ± 0.71

0.058 ± 0.007

c(RGDfK)-DOX-LP

99.59 ± 0.87

Pep-22-DOX-LP c(RGDfK)/Pep-22-DOX-LP

3.4. In Vitro DOX Release. The in vitro release of DOX from liposomes was performed in PBS (0.01 M, pH 7.4). As shown in Figure 3, compared with the rapid release of free DOX, all the DOX loaded liposomes exhibited sustained release behaviours that the cumulative DOX release from liposomes was less than 40% within 48 h and no burst initial release was detected. Similar release patterns were observed among DOX-LP, c(RGDfK)-DOX-LP, Pep-22-DOX-LP and c(RGDfK)/Pep-22-DOX-LP, implying that the modification of ligands didn’t affect the release behaviour of DOX obviously.


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Figure 3. In vitro release curves of DOX from solution and liposomes (DOX-LP, c(RGDfK)DOX-LP, Pep-22-DOX-LP, c(RGDfK)/Pep-22-DOX-LP) in PBS (0.01 M, pH 7.4) at 37°C (n = 3, mean ± SD). 3.5. Cellular Uptake on U87 cells. The targeting effect of FAM loaded dual-modified liposomes was investigated by the cellular uptake on U87 cells through fluorescent microscopy (Figure 4A) and flow cytometry (Figure 4B) in vitro. As shown in Figure 4A, compared with unmodified liposomes (LP), c(RGDfK)-LP and Pep-22-LP exhibited stronger green fluorescence signals inside U87 cells, which indicated that both c(RGDfK) and Peptide-22 modification could effectively increase the uptake of liposomes by U87 cells. This result was consistent with previous reports. For instance, Kim et al. demonstrated that the cellular uptake of DOX in cRGD-modified liposomes into U87MG was 8-fold higher than that of unmodified liposomes.44 Zhang et al. found that the fluorescence intensity of C6 cells treated with coumarin-6-labelled peptide-22-decorated nanoparticles were obviously higher than those treated with unmodified NPs.29 Furthermore, the liposomes modified with c(RGDfK) and Peptide-22 simultaneously resulted in the strongest fluorescence signal among four groups, indicating that the synergetic recognition of integrin αvβ3 and LDLR on U87 cells could significantly improve the internalization of liposomes. Similar results were obtained from flow cytometry (Figure 4B). Compared with LP, the cellular uptake of liposomes modified with ligands was significantly increased on U87 cells. Moreover, the fluorescence intensity of c(RGDfK)/Pep-22-LP was 7.57-fold and 1.58-fold higher than those of c(RGDfK)-LP and Pep-22-LP. These data illustrated that dual-modification could not only combine the transcytosis capability of c(RGDfK) and Peptide-22 themselves, but also exerted a synergetic effect on tumor cell uptake. The synergetic effect was realized by


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expanding the number of the recognized receptors (binding sites) and strengthening the binding affinity via conjugations of two different ligands to the corresponding receptors on the same cell surface, which made the process of internalization more easily.

Figure 4. Qualitative (A) and qualitative (B) cellular uptake of 5 µM FAM loaded liposomes on U87 cells for 4 h. Original magnification = 100 ×. Data are represented as mean ± SD (n = 3). ***indicates statistically significant difference (p < 0.001).

3.6. In Vivo Imaging Study. During the occurrence and development of glioma, BBB is gradually impaired and BBTB emerges as the new obstacle for the transport of nanoparticles into tumor.45 Therefore, it is essential to construct a novel system to realize BBB/BBTB dualcrossing and brain tumor targeting simultaneously. In our study, we evaluated the in vivo glioma targeting efficiency of different liposomes containing near infrared dye DiR in nude mice bearing intracranial glioma. The liposomes were administrated fifteen days after the inoculation of tumor cells, at the time the BBB and the BBTB exist simultaneously. A real-time distribution of liposomes was observed under IVIS Spectrum system. As shown in Figure 5A, c(RGDfK)/Pep-22-LP group exhibited the strongest fluorescence signal in the glioma region at all-time points when compared with that of unmodified or single-modified groups, indicating


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that dual-modification of Peptide-22 and c(RGDfK) could significantly facilitate liposomes to traverse the BBB/BBTB and enhance the accumulation of liposomes in the tumor site via the synergetic effect of integrin αvβ3 and LDLR mediated endocytosis. The efficient BBB/BBTB transporting capability and precise glioma targeting ability of c(RGDfK)/Pep-22-LP were further verified by ex vivo imaging of the brains after 24 h post-injection (Figure 5C). Excised organs were also harvested and imaged (Figure 5B). Intensive fluorescence signal was observed in liver and spleen, which was corresponding to the main clearance route of liposomes.46 Moreover, no perspicuously difference in fluorescence signal of peripheric organs was observed among groups, suggesting that c(RGDfK) and Peptide-22 modification would not change the non-specific distribution behavior of liposomes.


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Figure 5. In vivo imaging of intracranial glioma-bearing mice at 2 h, 6 h, 12 h and 24 h after i.v. injection with DiR loaded liposomes (A). Ex vivo imaging of excised organs (B) and brains (C) after 24 h post-injection.

3.7. In Vitro Cytotoxicity Study. In vitro cytotoxicity of free DOX and different DOX loaded liposomes on U87 cells was measured by MTT assay (Figure 6 and Table 2). It could be concluded from the results that both free DOX and DOX loaded liposomes could inhibit the proliferation of U87 cells in a concentration-dependent manner, proving the anticancer effect of DOX on such kind of brain tumors. As shown in Table 2, free DOX (IC50 = 0.325 µg/mL) exhibited stronger inhibitory effect to the proliferation of U87 cells than DOX-LP (IC50 = 0.747 µg/mL), c(RGDfK)-DOX-LP (IC50 = 0.504 µg/mL) and Pep-22-DOX-LP (IC50 = 0.389 µg/mL). This phenomenon was also observed in other reports,32, 47 which was because free DOX could be quickly transported into cells directly via passive diffusion without the drug release process, while liposomes were internalized through endocytosis which cost time and energy, and then probably underwent a sustained-release process. Moreover, the PEGylation of liposomes might retard the interaction between liposomes and cells, resulting in the lower cytotoxicity of unmodified or single-modified liposomes. However, c(RGDfK)/Pep-22-DOX-LP displayed the strongest cytostatic activity among the groups, whose IC50 value (IC50 = 0.164 µg/mL) was only half of the free DOX. The effect was ascribed to the tremendously enhanced cellular uptake mediated by the specific binding between c(RGDfK)/αvβ3 and Peptide-22/LDLR. Targeting more than one receptors in the same cell surface could increase the number of binding sites, strengthen the binding affinity, and subsequently deliver more anticancer drugs into tumor cells, thus leading to the enhanced cytotoxicity.48


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Table 2. IC50 Values of free DOX and different DOX liposomes against U87 cells. Formulations Free DOX DOX-LP c(RGDfK)-DOX-LP Pep-22-DOX-LP c(RGDfK)/Pep-22-DOX-LP

IC50（µg/mL）） 0.325 0.747 0.504 0.389 0.164

Figure 6. Cytotoxicity of free DOX and different DOX liposomes against U87 cells after 48h incubation at 37°C (n = 4, mean ± SD).

3.8. In Vivo Anti-glioma Effect. To investigate the in vivo anti-glioma effect of functionalized liposomes, intracranial glioma-bearing mice were injected with different DOX formulations on day 7, 14 and 21. Figure 7A showed the body weight changes of mice during the experiment. Compared with other groups, the mice treated with free DOX exhibited a rapidest


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loss in body weight, which was caused by the severe systemic toxicity of free DOX.49 The gentler loss of body weight was observed in the groups administrated with DOX loaded liposomes, indicating the reduction of systemic side effect by liposome encapsulation. As shown in Figure 7B, the modification of c(RGDfK) or Peptide-22 could improve the anti-glioma effect of DOX-loaded liposomes, the median survival time of mice were notably prolonged. Among all the groups, the mice treated with c(RGDfK)/Pep-22-DOX-LP, i.e., 39.5 days, was significantly longer than that of mice treated with saline (27 days, p < 0.001), free DOX (30.5 days, p < 0.001), DOX-LP (31.5 days, p < 0.001), c(RGDfK)-DOX-LP (34.5 days, p < 0.01) and Pep-22DOX-LP (36 days, p < 0.05), respectively. The results demonstrated that c(RGDfK) and Peptide22 dual-modified liposomes exhibited a significant improvement in anti-glioma effect than those decorated with any of the single ligand, indicating the synergetic effect of two ligands in treating brain tumors. Nanocarries functionalized with targeting ligands were frequently reported for overcoming BBB and targeting the tumor cells in the treatment of glioma.17, 47, 50 But there were only several reports about modifying ligands to the surface of nanoparticles to realize simultaneous BBB/BBTB crossing and brain tumor targeting. This study was the first one to construct c(RGDfK) and Peptide-22 co-modified liposomes loaded with anti-tumor drugs, which could cross the BBB/BBTB simultaneously and deliver drugs into the tumor.


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Figure 7. Anti-glioma effect of different DOX formulations after intravenous administration to intracranial glioma-bearing mice. (A) Body weight change and (B) Kaplan−Meier survival curves of model mice (n = 6). 3.9 Cardiotoxicity Evaluation. DOX, one of the most effective chemotherapeutic agents, is used as a first-line drug in numerous types of cancer. However, it exhibits serious adverse effects, especially lethal cardiotoxicity.51 To further evaluate the safety of different formulations, sections of heart were stained with hematoxylin and eosin. As shown in Figure 8, myocardial hypertrophy was obviously observed in the heart section of free DOX treated group. However, there was no injury was found in the samples of all the liposome treated groups, which indicated that the liposomes could decrease the cardiac toxicity of DOX and had good in vivo safety.


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Figure 8. Histochemistry inspection of heart sections stained with hematoxylin and eosin of intracranial glioma-bearing mice after i.v. administration of different DOX formulations. Red arrows indicate the myocardial hypertrophy. Original magnification: 200×.

4. CONCLUSIONS In this study, we investigated the BBB targeting capacity of Angiopep-2-LP, T7-LP and Peptide22-LP and the BBTB targeting ability of c(RGDfK)-LP, D-SP5-LP and Pep-1-LP, respectively. Based on the cellular uptake results on BCECs and HUVECs, Peptide-22 and c(RGDfK) were picked out to develop a BBB/BBTB dual-crossing, glioma targeting liposomal drug delivery system c(RGDfK)/Pep-22-DOX-LP. Compared with the modification of a single peptide, the cooperative effect of integrin αvβ3 and LDLR mediated recognition significantly improved the internalization and cytotoxicity of DOX loaded liposomes on U87 cells. In vivo biodistribution and anti-glioma effects further verified that c(RGDfK)/Pep-22-DOX-LP could efficiently transport across BBB/BBTB and specifically accumulate in the glioma site, and then prolong the survival time of intracranial glioma-bearing mice. Taking these together, we claimed c(RGDfK) and Peptide-22 dual-modified liposomes could serve as a promising platform for glioma chemotherapy in different progression stages.

SUPPORTING INFORMATION The MALDI-TOF mass spectrum of DSPE-PEG3400-T7, DSPE-PEG3400-D-SP5, DSPE-PEG3400Pep-1, DSPE-PEG3400-Angiopep-2, DSPE-PEG3400-Peptide-22 and DSPE-PEG3400-c(RGDfK). The serum stability of different DOX-loaded liposomes over 24 h. The information is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author *Tel: +86-21-3127-0810. E-mail: [email protected] *Tel: +86-21-5198-0088. E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the grant from the National Basic Research Program of China (No. 2013CB 932500) and National Natural Science Foundation of China (No. 81361140344).

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Peptide-22 and Cyclic RGD Functionalized ... - ACS Publications

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