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Tandem peptide based on structural modification of poly-arginine for enhancing tumor targeting efficiency and therapeutic effect Yayuan Liu, Zhengze Lu, Ling Mei, Qianwen Yu, Xiaowei Tai, Yang Wang, Kairong Shi, Zhirong Zhang, and Qin He ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12611 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on December 31, 2016

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ACS Applied Materials & Interfaces

Tandem peptide based on structural modification of poly-arginine for enhancing tumor targeting efficiency and therapeutic effect Yayuan Liu1, a, b, Zhengze Lu1, a , Ling Meia , Qianwen Yua , Xiaowei Taia , Yang Wanga , Kairong Shi a , Zhirong Zhang a , Qin He*, a

a

Key Laboratory of Drug Targeting and Drug Delivery Systems, West China School of Pharmacy,

Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, China b

Haisco Pharmaceutical Group Co.,Ltd, Baili road No.136, Cross-Straits IT Industry Development

Zone, Wenjiang, Chengdu 611130, China 1

These authors contributed equally to this work.

* Corresponding author. Tel. /fax: +86 28 85502532. E-mail addresses: [email protected] (Q. He).

ABSTRACT The non-selectivity of cell penetrating peptides had greatly limited the application in systemic administration. By conjugating a dGR motif to the C-terminal of octa-arginine, the formed tandem peptide R8-dGR had been proved to specifically recognize both integrin αvβ3 and neuropilin-1 receptors. However, the positive charge of poly-arginine would still inevitably lead to rapid clearance in the circulation system. Therefore in this study, we tried to reduce the positive charge of poly-arginine by decreasing the number of arginine, thus to achieve improved tumor targeting efficiency. We had designed several different R X-dGR peptides (X = 4, 6, 8) modified liposomes, and investigated the tumor targeting and penetrating properties both in vitro and in vivo. Among all the liposomes, R6-dGR modified liposomes exhibited similar long circulation time as PEGylated liposomes while remained strong penetrating ability into both tumor cells and tumor ti ssues, thus had displayed the most superior tumor targeting efficiency. Then paclitaxel and indocyanine green co-loaded liposomes were prepared, and R6-dGR modified co-loaded liposomes also exhibited enhanced anti-tumor effect on C6 xenograft tumor bearing mice. Therefore, we suggested R6-dGR as a potential tumor targeting ligand with both strong penetrating ability and improved

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pharmacokinetic behavior, which could be further used for efficient anti-tumor therapy.

KETWORDS Tandem peptide, cell penetrating peptide, poly-arginine, photothermal therapy, combination therapy

1. INTRODUCTION In recent years, cell penetrating peptides (CPPs) had drawn great attention of researchers as they could show efficient penetrating abilities through cellular membranes 1. Since the discovery of the first CPP more than two decades ago2-3, CPPs had started their magic journey in the field of drug delivery, especially in carrying drug delivery systems to overcome bio-barriers 4-5. As is commonly believed, solid tumors exhibited a series of specific microenvironment including enhanced cell density, irregular vascular system and increased interstitial fluid pressure 6-7. These factors formed physiological barriers that prevented drugs or drug delivery systems from entering the core region of solid tumors, thus resulting in limited therapeutic effects. And CPPs were reported to kind of solve this intratumoral drug delivery problem due to their efficient penetrating and cell internalization activities 8. However, the utilization of CPP modified drug carriers in systemic administration was still strongly limited because of two main drawbacks. Most cell penetrating peptides were positively charged and achieved cellular entry through adsorptive mediated pathway. Thus these peptides could easily bind to negatively charged plasma proteins in vivo, and underwent a relative rapid clearance through the recognition of mononuclear phagocyte system9. On the other hand, the non-selectivity of CPPs would lead to uncontrollable distribution of drug carriers in vivo10-11, which turned into a potential threaten to those non-target tissues, especially in the application of cytotoxic anti-tumor agents. Therefore, more and more researches had been focused on the reform of CPP-based drug delivery systems, aiming at achieving elevated tumor targeting efficiency and reduced side effects 12-14. In the previous study of our group, we had designed a dual receptor recognizing cell penetrating peptide, R8-dGR. The dGR motif was a retro-inverso isomer of RGD peptide that had been proved to specifically bind to integrin αvβ 315. Moreover, by leading dGR motif in the C-terminal of octa-arginine, an exposed -RXXR sequence was formed in the C-terminal of the peptide, which


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was reported as C-end Rule and could selectively recognize neuropilin-116-17. Therefore, the tandem peptide R8-dGR could bind to both integrin αvβ 3 and neuropilin-1 receptors, thus achieved an enhanced specific targeting activity of poly-arginine18. However, the positive electricity still remained in R8-dGR and would inevitably lead to a quick elimination process in the circulation system. Considering this, in this study we planned to decrease the number of arginine on the basis of R8-dGR, so that to further reduce the positive charge of poly-arginine. It was reported that short poly-arginine sequences like R5 were already unable to transport into living cells19, but the detailed relationship between the penetration activity and the sequence length of poly-arginine was still not clear. Thus in this study, we had designed R4-dGR, R6-dGR and R8-dGR. By decorating these tandem peptides on liposomes respectively, we had evaluated the in vitro cellular uptake and tumor spheroids penetration activity, and the in vivo pharmacokinetics and biodistribution behavior of all the modified liposomes. And the main purpose of this study was to identify one of these three RX-dGR peptides (X = 4, 6, 8) which could exhibit both efficient penetrating capability and superior pharmacokinetic property. Meanwhile, combined therapeutic strategies with both chemotherapy and photothermal therapy (PTT) had been widely explored recently20-22. The addition of PTT agents could efficiently reduce the dosage of chemotherapeutics, and achieve a synergistic anti-tumor therapeutic effect. Indocyanine green (ICG) is one of FDA approved near infrared imaging agents that had high photothermal conversion efficiency 20. Therefore, after investigating the tumor targeting and penetrating capabilities of each RX-dGR, we also prepared paclitaxel (PTX) and ICG co-loaded liposomal systems in this study, and had evaluated both in vitro and in vivo anti-tumor efficiency of co-loaded liposomes modified with different R X-dGR (P-I-RX-dGR-Lip).

2. MATERIALS AND METHODS 2.1. Materials and Animals. Peptides with N-terminal cysteine residues including Cys-R4 (Cys-RRRR), Cys-R4-dGR (Cys-RRRR-dGR), Cys-R6 (Cys-RRRRRR) and Cys-R6-dGR (Cys-RRRRRR-dGR) were synthesized by China Peptides Co. Ltd. (Shanghai, China) through standard solid-phase peptide synthesis. DSPE-PEG2000 and DSPE-PEG2000-Mal were obtained from Shanghai Advanced Vehicle Technology (AVT) LTD Company (Shanghai, China). Soybean phospholipid (SPC) was purchased from Shanghai Taiwei Chemical Company (Shanghai, China) and cholesterol was purchased from Chengdu Kelong Chemical Company (Chengdu, China). Paclitaxel (PTX) and indocyanine green (ICG) were



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purchased from AP Pharmaceutical Co. Ltd. (Chongqing, China) and Tokyo Chemical Industry Co. Ltd. (Japan) respectively. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) (CFPE) was

obtained

from

Avanti

Polar

Lipids.

4’-6-diamidino-2-pheylindole

(DAPI)

and

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Beyotime Institute Biotechnology (Haimen, China). Coumarin-6 and coumarin-7 were both purchased from Sigma-Aldrich (USA). All other chemicals were obtained from commercial sources. Murine glioma C6 cells were cultured in RPMI-1640 medium (Gibco) supplement with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin under 37 °C in a humidified 5% CO 2 atmosphere. Balb/c mice were purchased from West China animal center of Sichuan University (Sichuan, China). All animal experiments were approved by the Experiment Animal Administrative Committee of Sichuan University.

2.2. Synthesis of Lipid Materials. The synthesis of peptide modified DSPE-PEG2000 materials was carried out following a previously reported method in our group18. In brief, the peptide with N-terminal cysteine (Cys-R4, Cys-R4-dGR, Cys-R6 and Cys-R6-dGR) and DSPE-PEG2000-Mal were dissolved in a mixed solvent of chloroform and methanol (v/v = 2:1) with a molar ratio of 1:1.5. The reaction system was stirred for 24 h under room temperature in darkness with triethylamine as catalyst. After DSPE-PEG2000-Mal was confirmed to be disappeared through thin layer chromatography identification, the mixture was evaporated to remove the organic solvent. Then the residue was dissolved in chloroform again and the solution was filtered to remove unreacted peptides. Finally the filtrate was evaporated by rotary evaporation and the obtained production was stored under -20 °C. The existence of DSPE-PEG2000-R4, DSPE-PEG2000-R4-dGR, DSPE-PEG2000-R6 and DSPE-PEG2000 -R6-dGR was confirmed by mass spectrometry (autoflex III smartbeam, Bruker, USA). DSPE-PEG2000-R8 and DSPE-PEG2000-R8-dGR were obtained from the previously reported work18.

2.3. Preparation of Liposomes. All the liposomes including PEGylated liposomes (PEG-Lip), R4 modified liposomes (R4-Lip), R4-dGR modified liposomes (R4-dGR-Lip), R6 modified liposomes (R6-Lip), R6-dGR modified liposomes (R6-dGR-Lip), R8 modified liposomes (R8-Lip) and R8-dGR modified liposomes (R8-dGR-Lip) were prepared through thin-film hydration method. Briefly, SPC, cholesterol and DSPE-PEG2000 were dissolved in chloroform with a molar ratio of 62:33:5, then the


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organic solvent was evaporated to form lipid film. After being kept in vacuum overnight, the lipid film was hydrated in 5% glucose solution for 20 min under 37 °C. And the obtained lipid solution was intermittently sonicated by a probe sonicator at 80 W for 80 s to form PEG-Lip. The peptide modified liposomes were prepared by the same method with 0.8% of DSPE-PEG2000 in PEG-Lip being replaced by equal amounts of homologous peptide conjugated DSPE-PEG2000 materials. Fluorescence labeled or drug loaded liposomes were prepared through the similar method as described above. To prepare CFPE-labeled, coumarin-6 loaded and PTX-loaded liposomes, appropriate amount of CFPE, coumarin-6 or PTX was added in the lipid organic solution before the lipid film formed. To prepare ICG-loaded liposomes, 5% glucose solution containing indocyanine green was used as hydration solution. The drug-lipid ratios of paclitaxel and indocyanine green were both 1:30 (w/w) and the free drug was removed via size exclusion chromatography using Sephadex G-50. 2.4. Characterization of liposomes. 2.4.1 Size distribution and zeta potential. We used ultrapure water to dilute the prepared liposomes to the appropriate concentration. 1.5 mL sample was put in quartz colorimetric utensil each time. Size distribution and zeta potential of different liposomes were determined using Malvern Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK). 2.4.2 Entrapment efficiency of PTX. The prepared PTX-loaded liposomes were divided into group A and B. Group A was centrifuged (4°C, 10,000 rpm, 20 min) and the supernate was collected as sample A. Group B was directly collected as sample B. Equal volume of sample A and B was mixed with equal volume of methanol separately, vortex vibrated for 5 min and centrifuged (4°C, 10,000 rpm, 10 min). The supernate was collected and analysed by HPLC (Agilent 1200, USA) separately. The mobile phase was a 60:40 (V/V) mixture of acetonitrile and water (C18 chromatographic column, flow rate 1 mL/min, column temperature 30 °C). The samples were detected at 227 nm. The entrapment efficiency (EE%) was calculated by the formula : EE% = AA/AB × 100% (AA and AB represent the PTX peak area of sample A and B, respectively). 2.4.3 Entrapment efficiency of ICG. The ICG-loaded liposomes were collected as sample A before purified by size exclusion chromatography. The purified ICG-loaded liposomes were collected as sample B. Equal volume of methanol was mixed with equal volume of sample A and B separately to make the approximate concentration of ICG in both samples 5 μg/mL. After 5 min of vortex



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vibration, the absorbance value of ICG was determined using ultraviolet-visible spectrophotometry at the wavelength of 779 nm. The exact concentration of ICG was calculated using standard curve. The entrapment efficiency (EE%) was calculated by the formula : EE% = CB/CA × 100% (C A and C B represent the ICG concentration of sample A and B, respectively). 2.4.4 Serum stability. To evaluate the serum stability of different liposomes, the samples were mixed with equal volume of FBS, incubated under 37 °C with gently oscillating for 48 h. The transmittance of liposomes was measured at 750 nm using a microplate reader (Thermo Scientific Varioskan Flash, USA) and the size distribution of liposomes was measured using Malvern Zetasizer Nano ZS90 (Malvern Instruments Ltd., UK) at different time points. 2.4.5 Drug release behavior. The drug release behavior of PTX-ICG co-loaded liposomes was investigated through a dialysis method. Different modified P-I-Lip or free drug solution was added in dialysis tubes (MWCO 8-14 kDa), the dialysis tubes were then placed into 40 mL release media (PBS with 0.1% Tween 80 (v/v), pH=7.4) and incubated under 37 °C with gently oscillating for 48 h. 0.5 mL release media was sampled and replaced with equal volume of fresh media at predetermined time points. These collected samples were finally analyzed by HPLC to detect the concentration of paclitaxel and by ultraviolet-visible spectrophotometry for the measurement of indocyanine green. (Measuring methods listed in 2.4.2 and 2.4.3) 2.5. Cellular Uptake Study. The uptake of different modified liposomes was evaluated on C6 glioma cells. For quantitative analysis, C6 cells were plated in six-well plates at a density of 5 × 105 cells per well and cultured for 24 h. CFPE-labeled liposomes were added into the plates for 4 h incubation, then the cells were washed and collected, and finally analyzed using a flow cytometer (Cytomics FC 500, Beckman Coulter, USA). Confocal laser scanning microscopy was utilized for qualitative investigation. C6 cells were seeded on cover slip in 6-well plates and cultured for 24 h. After 4 h incubation with CFPE-labeled liposomes, the cells were washed, fixed with 4% paraformaldehyde, stained with DAPI under room temperature, and finally imaged using confocal microscopy (FV1000, Olympus, USA).

2.6. Tumor Spheroids Penetration Study. C6 cells were plated in low melting point agarose coated 96-well plates at a density of 5 × 103. When the tumor spheroids formed, CFPE-labeled liposomes were added for 4 h incubation. Then the C6 tumor spheroids were washed with cold PBS,


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fixed in 4% paraformaldehyde, and imaged using a confocal laser scanning microscopy (FV1000, Olympus, USA).

2.7. Pharmacokinetics and Quantitative Biodistribution Study. C6 xenograft tumor bearing mice models were established as follows. Balb/c mice weighing 20-25 g were anesthetized using 5% chloral hydrate and inoculated subcutaneously with 5 × 10 6 C6 cells in the left flank. The mice were then raised under standard condition and used for study when the tumor volume reached about 100 mm3. For pharmacokinetics study of different modified liposomes, 21 C6 xenograft tumor bearing mice were randomly divided into 7 groups. Coumarin-6 loaded PEG-Lip, R4-Lip, R4-dGR-Lip, R6-Lip, R6-dGR-Lip, R8-Lip and R8-dGR-Lip were intravenously administered through tail vein at a coumarin-6 dose of 0.15 mg/kg. At 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after the injection, blood samples were collected using capillary tubes through orbit. After being centrifuged at 10000 rpm for 10 min, the plasma samples were collected and comarin-7 was added as internal standard. For quantitative distribution study, 84 C6 xenograft tumor bearing mice were randomly divided into 7 groups. Seven different groups of coumarin-6 loaded liposomes described above were intravenously administered through tail vein at the same dosage. At 1 h, 4 h, 8 h and 24 h, the mice were sacrificed after heart perfusion and the livers and tumors of each mouse were sampled. All the tissues were homogenized with triple amount of water and conmarin-7 was added in the organ homogenate as internal standard. All the plasma and organ homogenate samples were extracted with N-hexane, dried under air stream and finally redissolved in methanol for HPLC analysis (excitation at 465 nm and emission at 502 nm). The concentrations of coumarin-6 in all the samples were determined using internal standard method.

2.8. In Vivo Imaging and Tumor Distribution. ICG-loaded liposomes were injected to C6 xenograft tumor bearing mice at a dose of 250 μg ICG/kg. The mice were imaged using IVI Spectrum system (Caliper, Hopkington, MA, USA) at 4 h and 24 h after the injection. Then the mice were sacrificed after heart perfusion at these time points. Hearts, livers, spleens, lungs, kidneys and tumors were collected and imaged as well. For the tumor distribution study of different liposomes, the tumors of 24 h post-injection mice



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described above were fixed in 4% paraformaldehyde, dehydrated in sucrose solution and sectioned at a thickness of 10 μm. The tumor sections were then stained with DAPI and imaged using confocal microscopy (FV1000, Olympus, USA).

2.9. Cytotoxicity Study in Vitro. MTT study was carried out to evaluate the cytotoxicity of drug loaded liposomes. 2 × 103 C6 cells were plated in 96-well plates and cultured for 24 h. PTX-loaded liposomes were added at pre-determined concentration and cultured for another 24 h. 20 μL MTT (5 mg/mL) was then added for 4 h incubation. Finally, the culture medium was removed and cells were dissolved in dimethyl sulfoxide. The absorbance was measured at 490 nm using a microplate reader (Thermo Scientific Varioskan Flash, USA) and the cell viability of each group was calculated. For PTX-ICG co-loaded liposomes, the concentrations of PTX and ICG in the plates were both 1 μg/mL. The cells incubated with co-loaded liposomes were divided into 3 groups and had undergone three kinds of different treatments. The first group did not suffer infrared laser irradiation; the second group was irradiated with 808 nm laser at 1 W/cm2 for 5 min immediately after co-loaded liposomes were added, while the last group was irradiated under the same condition 12 h after the liposomes were added. The total incubation time of these three groups of cells with different PTX-ICG formulations was all 24 h. Then the cells were treated with MTT solution and detected as described above.

2.10. Anti-tumor Efficacy. 48 C6 xenograft tumor bearing mice were randomly divided into 8 groups including 5% glucose solution, free PTX and ICG mixture with laser irradiation, P-I-R4-dGR-Lip with laser irradiation, P-I-R6-dGR-Lip with laser irradiation, P-I-R8-dGR-Lip with laser irradiation, P-I-R6-dGR-Lip without laser irradiation, ICG-R6-dGR-Lip with laser irradiation and PTX-R6-dGR-Lip. The dosages of PTX and ICG were about 2.2 mg/kg and 2 mg/kg respectively. The different formulations were intravenously injected at the fifth day and ninth day after implantation, and the tumors of laser treatment groups were irradiated with 808 nm lasers at 1 W/cm 2 for 5 min at the sixth day and tenth day after implantation. 24 h after the second irradiation was finished, one mouse of each group was sacrificed and all the eight tumors were collected for paraffin sections. Hematoxylin-eosin (HE) staining and TUNEL staining were performed according to the standard protocols. The tumor volumes of the rest mice were detected and recorded every three days since


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the third day after tumor implantation (tumor volume = 0.52 × length × width2). All the mice were sacrificed at the 30th day after tumor implantation and the tumors were excised and pictured.

2.11. Statistical Analysis. All the data were presented as mean ± standard deviation. Statistical comparisons were performed by one-way ANOVA for multiple groups, and p values of < 0.05 and < 0.01 were considered indications of statistical difference and statistically significant dif ference, respectively.

3. RESULTS 3.1. Characterization of Liposomes. The results of mass spectrometry of different peptide modified DSPE-PEG2000 materials were shown in Figure S1-S4. The HPLC spectra of different peptide modified DSPE-PEG2000 materials were shown in Figure S5. The theoretic molecular weights of DSPE-PEG2000 -R4, DSPE-PEG2000-R4-dGR, DSPE-PEG2000-R6 and DSPE-PEG2000 -R6-dGR were 2921, 3353, 3234 and 3665, which were coincide with the mass spectrums separately. Table S2 showed the characterization of different modified PTX and ICG co-loaded liposomes. All the liposomes were uniformly distributed with particle sizes around 110 nm. Both drugs could be successfully loaded into liposomes with high entrapment efficiencies (EE%) of about 90% for paclitaxel and 80% for indocyanine green. The zeta potentials of co-loaded liposomes were all about -20 mV, which were much lower than commonly prepared PEGylated liposomes. This negatively charged property might be due to the sulfo groups from indocyanine green. Thus we further prepared corresponding liposomes without ICG, and particle sizes and zeta potentials were exhibited in Table S1. According to the data, these PTX-loaded liposomes displayed zeta potentials of about -8 mV. It was notably that the liposomes modified with R8 or R8-dGR became less negatively charged significantly, which had kind of proved the strong electropositivity of octa-arginine. The in vitro serum stability of different modified liposomes was shown in Figure S6. The transmittance and the size of all the liposomes didn’t show obvious change within 48 h incubation with FBS, implying that no aggregation or sediment was appeared. As for the drug release behavior, we had evaluated the release profile of both drugs from co-loaded liposomes. Both PTX (Figure S6C) and ICG (Figure S6D) had undergone a sustained release process with a total release amount of about 60% within 48 h. And there’s no significant difference among these different groups of



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liposomes.

3.2. Cellular Uptake and Tumor Spheroids Penetration Study. The quantitative evaluation of the cellular uptake on C6 cells was shown in Figure 1A. For liposomes modified with poly-arginine, it could be first observed that R8 could efficiently enhance the liposomal internalization into C6 cells. However, the cellular uptake of R4-Lip and R6-Lip didn’t show significant difference compared to PEGylated liposomes. Thus it could be inferred that poly-arginine would lose the penetration activity when the number of arginine decreased to six or less. On the other hand, when dGR motif was conjugated to the C-terminal of poly-arginine, all these different RX-dGR-Lip exhibited significantly enhanced cellular uptake on C6 cells compared to their corresponding RX modified liposomes. It could be seen that the cellular uptake achieved about 1.3-fold increasing when dGR motif was decorated on the C-terminal of R4 and R8. However, when the same situation occurred on R6, it was worth noting that the uptake of R6-dGR-Lip was almost 7-fold higher than R6-Lip. The internalization ability of R6-dGR was almost as strong as R8. Meanwhile, we had qualitatively evaluated the cellular uptake of liposomes through confocal images of C6 cells and the results were similar to the quantitative data. The green fluorescence signal of R6-dGR-Lip was significantly stronger than that of R6-Lip. Then the tumor spheroids uptake study was carried out to mimic the tumor tissue penetration ability of different RX-dGR (X = 4, 6, 8) in vitro (Figure 2). The fluorescence signal could hardly be observed in PEG-Lip, R4-Lip, R4-dGR-Lip and R6-Lip groups, indicating their weak penetrating abilities. R8, as a commonly used CPP, had displayed an obvious penetration behavior into C6 tumor spheroid, and R8-dGR was stronger than R8. As for R6-dGR-Lip, the intratumoral penetrating depth was much more remarkable than R6-Lip, and had even exceeded R8-Lip to 200 μm, similar to R8-dGR-Lip.


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Figure 1. (A) Cellular uptake of CFPE-labeled liposomes on C6 cells detected by a flow cytometer (n = 3, mean ± SD), * and *** indicate p < 0.05 and p < 0.001 respectively. (B) Qualitative cellular uptake evaluation of CFPE-labeled liposomes on C6 cells imaged by a confocal microscopy. Scale bar represents 30 μm.



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Figure 2. The uptake of CFPE-labeled liposomes on C6 tumor spheroids within different depth imaged by a confocal microscopy. Scale bar represents 200 μm.

3.3. Pharmacokinetics and Quantitative Biodistribution Study. The pharmacokinetic study was carried out on C6 xenograft tumor bearing mice. The plasma concentration-time curves of different coumarin-6 loaded liposomes were shown in Figure 3A. It could be seen that the plasma concentrations of R8-Lip and R8-dGR-Lip were significantly lower than PEGylated liposomes since 8 h after the injection. The pharmacokinetic parameters of liposomes were also listed in Table 1. R8-Lip and R8-dGR-Lip both displayed obviously shortened half-life period and decreased AUC compared to PEG-Lip, and the clearance rate of these two groups of liposomes was significantly enhanced. These data proved that liposomes decorated with octa-arginine contained peptides would undergo a relative rapider clearance. However, when the number of arginine decreased, R4-Lip, R4-dGR-Lip, R6-Lip and R6-dGR-Lip all exhibited similar in vivo pharmacokinetic behavior to PEGylated liposomes.


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The biodistribution of liposomes in the tumors and livers of C6 xenograft tumor bearing mice was quantitatively detected and the results were shown in Figure 3B and Figure 3C. R8-Lip and R8-dGR-Lip displayed rapider distribution in the liver ever since 1 h after the injection of liposomes, and the accumulation of them in the liver remained significantly higher than PEG-Lip till 4 h and 8 h after the injection. The liver accumulation of R4-Lip, R4-dGR-Lip, R6-Lip and R6-dGR-Lip showed no significant difference compared to PEG-Lip. Besides, the concentration of coumarin-6 24 h after the injection was low in all the liposomal groups, indicating that coumarin-6 in the liver was almost metabolized. On the other hand, R8-Lip and R8-dGR-Lip exhibited efficient accumulation in the tumor within a short time, and R8-dGR showed a stronger tumor targeting ability than R8 due to the dGR motif (Figure 3C). However, the accumulation of R6-dGR-Lip in the tumor kept increasing because of its long circulation time (Figure 3A), and finally achieved the most remarkable tumor targeting efficiency 24 h after the injection. Meanwhile, the distribution ratio of target tissue and non-target tissue was commonly utilized to evaluate the targeting efficiency of drug delivery systems. Thus in this study, we further calculated the tumor / liver distribution ratio of liposomes, in order to analyze the tumor targeting efficiency of different peptides (Figure 3D). According to the data, R8-Lip and R8-dGR-Lip displayed higher accumulation in the tumor at 1 h and 4 h post-injection (Figure 3C), but their accumulation in the liver was also much stronger than any other groups (Figure 3B). Therefore, the tumor / liver distribution ratio of R8-Lip and R8-dGR-Lip was significantly lower than R6-dGR-Lip, and R6-dGR had exhibited the most superior tumor targeting efficiency.

Table 1. The pharmacokinetic parameters of coumarin-6 loaded liposomes in C6 xenograft tumor bearing mice. (n = 3, * represents p < 0.05 versus PEG-Lip). Groups

T

1/2

(h)

AUC

(0→24)

(μg/L*h)

AUC

(0→∞)

(μg/L*h)

CL (L/h/kg)

PEG-Lip

3.081±0.814

94.589±4.879

138.708±6.381

1.081±0.036

R4-Lip R4-dGR-Lip

3.336±0.513 3.725±0.323

91.907±5.243 86.034±3.395

156.712±9.647 166.803±7.353

0.957±0.075 0.899±0.132

R6-Lip R6-dGR-Lip

3.195±0.138 2.897±0.161

86.298±4.411 97.040±5.207

110.658±15.688 142.513±10.773

1.356±0.053 1.053±0.061

R8-Lip R8-dGR-Lip

1.620±0.329* 1.281±0.452*

59.741±4.215* 56.968±4.533*

73.942±8.010* 72.095±8.354*

2.029±0.232* 2.081±0.041*



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Figure 3. (A) The plasma concentration-time curve of coumarin-6 loaded liposomes in C6 xenograft tumor bearing mice. The biodistribution of coumarin-6 loaded liposomes in the liver (B) and in the tumor (C) of C6 xenograft tumor bearing mice. (D) The tumor / liver distribution ratio of coumarin-6 loaded liposomes in C6 xenograft tumor bearing mice. n = 3, mean ± SD, * represents p < 0.05.

3.4. In Vivo Imaging and Tumor Biodistribution. ICG-loaded liposomes were prepared to qualitatively investigate the biodistribution in C6 xenograft tumor bearing mice. In vivo imaging and images of ex vivo tumors in Figure 4A showed that R8-dGR-Lip could achieve the strongest tumor accumulation at 4 h after systemic administration. However, the fluorescence signal of R6-dGR-Lip kept increasing and finally exhibited a higher accumulation in the tumor at 24 h after the administration. Figure 4B displayed the ex vivo images of other main organs, in which R8-Lip and R8-dGR-Lip both showed significantly increased liver distribution, while R4-Lip, R4-dGR-Lip, R6-Lip and R6-dGR-Lip showed no significant difference compared to PEG-Lip. These results were consistent with the quantitative data in Figure 3. Then the excised tumors were sectioned and the confocal images were displayed in Figure 5. Besides, we also quantitatively compared the tumor-site biodistribution (See Supporting Information Figure S7 and S8).R6-dGR-Lip showed the most widely distributed red fluorescence signal around the tumor cells, suggesting that R6-dGR


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could achieve the strongest tumor targeting and intratumoral penetrating efficiency.

Figure 4. (A) In vivo images and ex vivo images of tumors of C6 xenograft tumor bearing mice at 4 h and 24 h after systemic administration of ICG-loaded liposomes. (B) Ex vivo images of the main organs of C6 xenograft tumor bearing mice at 4 h and 24 h after systemic administration of ICG-loaded liposomes.

Figure 5. Confocal images of tumor sections from C6 xenograft tumor bearing mice 24 h after systemic administration of ICG-loaded liposomes (red). Nuclei were stained with DAPI (blue). Scale bar represents 50 μm.

3.5. Cytotoxicity Study. In order to compare the cytotoxicity of drug loaded liposomes modified with different peptides, we first prepared paclitaxel loaded liposomes (PTX-Lip). The



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anti-proliferation effect of PTX-R4-Lip and PTX-R6-Lip both didn’t show significant difference compared to PTX-PEG-Lip, while PTX-R8-Lip achieved significantly increased cytotoxicity (Figure 6A). As was certified in the cellular uptake study, the penetration ability of poly-arginine would disappear when the number of arginine decreased to six or less (Figure 1), and the MTT assay here had further proved this point. R4-dGR still didn’t improve the cytotoxicity, while PTX-Lip modified with R6-dGR and R8-dGR had displayed significantly increased C6 cells inhibition effect compared to their corresponding PTX-R6-Lip and PTX-R8-Lip. Thus R6-dGR and R8-dGR could both achieve more superior anti-proliferation effect on the basis of improved cellular internalization properties. To investigate the cytotoxicity of PTX-ICG co-loaded liposomes (P-I-Lip), the C6 cells incubated with different modified P-I-Lip were divided into 3 groups, and the cytotoxicity of each corresponding PTX-Lip was used as controlled group. As shown in Figure 6B, all the co-loaded liposome groups without laser irradiation (green columns) didn’t show obvious improvement on cytotoxicity, indicating photothermal therapy agents could only exert cytotoxic effect under appropriate irradiation. On the other hand, we further studied the cytotoxicity of co-loaded liposomes with laser irradiation at different time points. 0 h incubation groups (Red columns) represented the cells were irradiated immediately after the liposomes were added, while 12 h incubation groups (red columns with shadows) represented the irradiation was applied 12 h after the liposomes were added. For P-I-PEG-Lip, P-I-R4-Lip, P-I-R4-dGR-Lip and P-I-R6-Lip, the incubation time before irradiation didn’t influence the cytotoxicity obviously. However, for P-I-R6-dGR-Lip, P-I-R8-Lip, P-I-R8-dGR-Lip and free drug group, the viability of cells which were irradiated after 12 h incubation with drugs was significantly decreased compared to those without pre-incubation. These results were due to the different cell internalization abilities of different liposomes. Liposomes modified with R6-dGR, R8 and R8-dGR could achieve efficient intracellular delivery, thus more ICG could exert photothermal therapeutic effect in the cytoplasm after 12 h incubation. However, PEG-Lip, R4-Lip, R4-dGR-Lip and R6-Lip which only weakly penetrate into cells couldn’t achieve this effect.


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Figure 6. (A) The cytotoxicity study of PTX-loaded liposomes and free PTX on C6 cells (n = 3, mean ± SD), * represents p < 0.05 and N.S. indicates none significant difference. (B) The cytotoxicity study of PTX-loaded liposomes and PTX-ICG co-loaded liposomes on C6 cells, without or with near infrared laser irradiation at different time points (n = 3, mean ± SD), * represents p < 0.05.

3.6. Anti-tumor Efficacy. The photographs of the tumors and the tumor growth curves were shown in Figure 7A and Figure 7B separately. We also measured the tumor-site temperature during laser irradiation (See Supporting Information Figure S9).Compared to the negative control, all the drug contained formulations had inhibited the growth of C6 xenograft tumors in different degrees. Among all the co-loaded liposomal groups with laser irradiation, P-I-R6-dGR-Lip exhibited the most efficient anti-tumor effect. Meanwhile, the tumor inhibition rate of P-I-R6-dGR-Lip with laser irradiation was also higher than PTX-R6-dGR-Lip and ICG-R6-dGR-Lip. The HE staining and TUNEL staining of tumor sections were shown in Figure 7C and Figure 7D separately. The HE staining of 5% glucose group exhibited a large crowd of tumor cells shown in dark purple and with high density. In the picture of P-I-R6-dGR-Lip group with laser irradiation, large areas of cell nucleus failed to be stained, indicating the most obvious cell necrosis areas. TUNEL staining was commonly utilized to



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detect cell apoptosis, and a large crowd of apoptosis cells shown in brown could be observed in P-I-R6-dGR-Lip group with laser irradiation. All these results had proved that R6-dGR modified PTX-ICG co-loaded liposomes could achieve the most efficient anti-tumor effect in vivo.

Figure 7. (A) Photographs of tumors of C6 xenograft tumor bearing mice treated with different PTX and ICG formulations. (B) Tumor growth curves of C6 xenograft tumor bearing mice treated with different PTX and ICG formulations (n = 5, mean ± SD). * represents p < 0.05, green arrows indicate the times of treatment and red arrows indicate the times of irradiation. The tumors were dissected 30 days after implatation. Hematoxylin-eosin staining (C) and TUNEL staining (D) of tumor sections from C6 xenograft tumor bearing mice. Scale bars represent 400 μm.

4. DISCUSSION Among all the CPPs, poly-arginine had exhibited effective penetrating ability in tumor targeting drug delivery23-24. The penetration activity of poly-arginine mainly came from the abundant guanidinium groups, and the realization of successful penetrating had been proved to be highly related to the fatty acid in cell membranes and the pH gradient between cytoplasm and


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extracellular matrix19, 25. Deprotonated fatty acids in the cell exterior would interact with positively charged guanidinium groups, leading to a transient membrane channel that facilitates the transport of poly-arginine toward into the cytoplasm. Then the fatty acids turned protonated in the cytoplasm, and released the combined polyarginine peptides 19. Therefore, the penetration activity of poly-arginine was mainly determined by the number of guanidinium group, which was actually the number of arginine in the sequence. The most frequently used poly-arginine peptides in drug delivery systems were R8 and R926-27, and too long or too short poly-arginine chains would both influence the penetrating ability. According to the cellular uptake and tumor spheroid penetration study in vitro (Figure 1 and Figure 2), the uptake of R4-Lip and R6-Lip was as weak as PEGylated liposomes, implying that the penetration activity was lost when the number of arginine decreased to six or less. In our former work, we designed a dual receptor recognizing cell penetrating peptide herein with a sequence of RRRRRRRRdGR (R8-dGR, lower case letter represents D-amino acid residue). Our previous study had proved that dGR motif in the C-terminal could endow dual receptor recognizing ability to poly-arginine18. By connecting an RGD reverse sequence dGR to a CPP poly-arginine, the peptide was endued with selective targeting property by recognizing both integrin αvβ 3 and NRP-1 receptors, and could undergo a CendR based intratumoral penetrating process. Thus R4-dGR-Lip and R8-dGR-Lip both showed a 1.3-fold increase on cellular uptake compared to R4-Lip and R6-Lip respectively. Interestingly, R6-dGR-Lip had exhibited a 7-fold significantly enhanced penetration behavior compared to R6-Lip. The sequence length of R6 might be just in the balance point when the penetration activity was disappearing, while the addition of dGR had activated the restoring of penetration activity. As a result, R6-dGR could achieve successful penetration across cell membrane and into tumor spheroids. The main purpose of this study was to screen out a poly-arginine based tandem peptide which could achieve both prolonged circulation time and enhanced tumor targeting efficiency. PEG coating of liposomes could inhibit RES-mediated clearance directly and indirectly through prevention of opsonization. We conjugated the R X-dGR (X = 4, 6, 8) peptides at the end of DSPE-PEG2000 and used them to prepare R X-dGR-Lip (X = 4, 6, 8). Thus, the circulation, clearance and half-life stability in plasma of our R X-dGR-Lip (X = 4, 6, 8) could be improved. We prepared different groups of RX-Lip and RX-dGR-Lip (X = 4, 6, 8) and investigated their characteristics. Both PTX and ICG could be successfully loaded into liposomes with high entrapment efficiencies (EE%) of about 90% and 80%



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respectively. The transmittance and size distribution of liposomes didn’t show obvious change within 48 h incubation with FBS, indicating the satisfactory serum stability of different liposomes. The release results showed that co-loaded liposomes exhibited a sustained drug release behavior over 48 h while free drug carried out a rapid release in the media within 8 h. No significant difference was observed among all groups of liposomes. The following in vivo study had proved our view. R8-Lip and R8-dGR-Lip both exhibited rapider clearance because of the positive charge (Figure 3A), while the pharmacokinetic behaviors of R4-Lip, R4-dGR-Lip, R6-Lip and R6-dGR-Lip showed no significant difference to PEG-Lip. Positively charged CPPs would possibly interact with plasma proteins in the circulation system and undergo accelerated clearance by the reticuloendothelial system12, 28-29, thus R8-Lip and R8-dGR-Lip both showed remarkable accumulation in the liver (Figure 3B). However, the enhanced liver accumulation was also avoided when the number of arginine reduced. Meanwhile, as R6-dGR could achieve both long circulation time and strong penetration activity, R6-dGR-Lip had displayed the most superior tumor targeting and intratumoral penetration efficiency (Figure 3D and Figure 5). Therefore, on the basis of the targeting study all above, we had analyzed the in vivo process of different R X-dGR-Lip (X = 4, 6, 8). As shown in Figure 8, when different modified liposomes were injected into the blood vessel, R8-dGR-Lip would undergo a relative rapider clearance while the tissue penetration of R4-dGR-Lip was weak. Only R6-dGR-Lip could first efficiently accumulated in the tumor tissue on the basis of long circulation time, and then achieve strong penetration both into tumor tissues and tumor cells. Photothermal therapy was considered as a relative safe anti-tumor treatment as the irradiation could be controlled spatiotemporally30-31. By transforming optical energy into heat directly at the tumor foci, the unwanted side effects to non-targeted tissues could be avoided32. An ideal PTT agent should achieve heat conversion with high efficiency, and be excited by near infrared laser which could easily penetrate into deeper tumor tissues 33. Indocyanine green is one of the most commonly used PTT agents which had displayed anti-tumor potential in recent researches. However the therapeutic effect of heat was still poor compared to chemotherapy, and the application of photothermal therapy was kind of limited in clinical32. Therefore, many researchers had combined chemotherapy or gene therapy to photothermal therapy, aiming at an enhanced anti -tumor effect21-22,

34

. We had prepared paclitaxel and indocyanine green co-loaded liposomes. The

combination of PTX and ICG could improve the anti-proliferation effect against C6 cells in vitro, and


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the in vivo anti-tumor data had proved that the co-loaded liposomal system could also achieve a reduced drug dose. The administration dose of PTX was about 2.2 mg/kg in this study, while the P-I-R6-dGR-Lip group still exhibited superior anti-tumor effect with a tumor inhibition rate of 72.6%. And the decreasing on drug dose would benefit to minimal side effects, especially for chemotherapeutics.

Figure 8. Schematic illustration of P-I-Rx-dGR-Lip. P-I-R8-dGR-Lip would undergo a rapider clearance process and P-I-R4-dGR-Lip displayed weak penetration property. Only P-I-R6-dGR-Lip could achieve the strongest tumor accumulation and tumor cell internalization. When the tumor tissue was irradiated by near infrared laser, the released ICG and PTX would exert a combined anti-tumor effect of both photothermal therapy and chemotherapy.

5. CONCLUSIONS In this study, we have designed several poly-arginine based tandem peptides, and have evaluated the tumor targeting efficiency of all these R X-dGR peptides (X = 4, 6, 8). According to both in vitro and in vivo results, R6-dGR-Lip exhibited similar pharmacokinetic properties as PEGylated liposomes while remained strong tissue and cell penetration ability, thus had achieved the most superior tumor targeting efficiency. When paclitaxel and indocyanine green were co-loaded in liposomes, P-I-R6-dGR-Lip also displayed the most efficient anti-tumor effect on the basis of the combination of chemotherapy and photothermal therapy. Therefore, we suggested R6-dGR-Lip as a



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potential tumor targeting drug delivery system with both strong penetrating ability and long circulation time.

SUPPORTING INFORMATION The mass spectrum of DSPE-PEG2000-R4, DSPE-PEG2000-R4-dGR, DSPE-PEG2000 -R6 and DSPE-PEG2000-R6-dGR. The HPLC spectra of DSPE-PEG2000-peptide conjugates. The serum stability and release profiles of free drug mixture and different modified co-loaded liposomes over 48 h. The semi-quantitative results on tumor-site distribution of ICG-loaded R6-dGR liposomes. The semi-quantitative results of the confocal images of glioma sections of C6 xenograft tumor bearing mice 24 h after systemic administration of ICG-loaded liposomes. The tumor-site temperature during laser irradiation. Particle sizes and zeta potentials of different modified PTX-loaded liposomes. Particle sizes, zeta potentials and the drug entrapment efficiency of different modified co-loaded liposomes.

ACKNOWLEDGEMENTS The work was funded by the National Basic Research Program of China (973 Program, 2013CB932504) and the National Natural Science Foundation of China (81373337). REFERENCES (1) Milletti, F. Cell-Penetrating Peptides: Classes, Origin, and Current Landscape. Drug Discovery Today 2012, 17 (15), 850-860. (2) Lee, S. H.; Castagner, B.; Leroux, J. C. Is There a Future for Cell-Penetrating Peptides in Oligonucleotide Delivery? Eur. J. Pharm. Biopharm. 2013, 85 (1), 5-11. (3) Bechara, C.; Sagan, S. Cell-Penetrating Peptides: 20 Years Later, Where Do We Stand? FEBS lett. 2013, 587 (12), 1693-1702. (4) Dietz, G. P.; Bähr, M. Peptide-Enhanced Cellular Internalization of Proteins in Neuroscience. Brain Res. Bull. 2005, 68 (1), 103-114.


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Tandem Peptide Based on Structural Modification ... - ACS Publications

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