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Albumin binding domain fusing R/K-X-X-R/K sequence for enhancing tumor delivery of doxorubicin Liping Liu, Chun Zhang, Zenglan Li, Chunyue Wang, Jingxiu Bi, Shuang Yin, Qi Wang, Rong Yu, Yong Dong Liu, and Zhiguo Su Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00497 • Publication Date (Web): 27 Sep 2017 Downloaded from http://pubs.acs.org on September 29, 2017

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Molecular Pharmaceutics

Albumin binding domain fusing R/K-X-X-R/K sequence for enhancing tumor delivery of doxorubicin Liping Liu1, 2, *, Chun Zhang1, 2, *, Zenglan Li2, Chunyue Wang1, 2, Jingxiu Bi 3, Shuang Yin1, 2, 3 1

, Qi Wang2, Rong Yu1, †, Yongdong Liu2,†, Zhiguo Su2

Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West

China School of Pharmacy, Sichuan University, Chengdu, 610041, China 2

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese

Academy of Sciences, Beijing 100190, China 3

School of Chemical Engineering, The University of Adelaide, Adelaide SA, 5005, Australia

† Correspondences: [email protected] (Y. Liu); Tel/Fax: +86-010-82545028. No. 1, Zhongguancun Beiertiao, Haidian District, Beijing, China; [email protected] (R. Yu); Tel/Fax: +86-028-85503012. Southern Renmin Road, No. 17, Section 3, Chengdu, China. * These authors contributed equally to this work.

Abstract Graphic:

Abstract For the purpose of improving the tumor delivery of doxorubicin (DOX), a kind of peptide-DOXO conjugate was designed and prepared, in which the peptide composed of an albumin-binding domain (ABD) and a tumor-specific internalizing sequence (RGDK or RPARPAR) was conjugated to a (6-maleimidocaproyl) hydrazone derivative of doxorubicin (DOXO-EMCH). The doxorubicin uptake by lung cancer cell line of A549 evidenced that the

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conjugates are capable of being internalized through a tumor-specific sequence mediated manner, and the intracellular imaging of distribution in A549 cell demonstrated that the conjugated doxorubicin can be delivered to the cell nucleus. The A549 cell cytotoxicity of peptide-DOXO conjugates was presented with IC50 values and shown in the range of about 9 11 µM. Pharmacokinetics study revealed that both conjugates exhibited nearly 5.5 times longer half-time than DOX, and about 4 times than DOXO-EMCH. The in vivo growth inhibitions of the two peptide-DOXO conjugates on BALB/c nude mice bearing A549 tumor (47.78% for ABD-RGDK-DOXO and 47.09% for ABD-RPARPAR-DOXO) were much stronger than that of doxorubicin and DOXO-EMCH (24.28% and 25.67% respectively) at a doxorubicin equivalent dose. Besides, the in vivo fluorescence imaging study confirmed that the peptide markedly increased the payload accumulation in tumor tissues and indicated that albumin binding domain fusing tumor-specific sequence effectively enhanced the tumor delivery of doxorubicin and thus improved its therapeutic potency. Key words: albumin binding domain; RGDK; RPARPAR; doxorubicin; peptide-drug conjugate. Abbreviations: DOX, doxorubicin; ABD, albumin binding domain; DOXO-EMCH, (6-maleimidocaproyl)-hydrazone derivative of doxorubicin; ADC, antibody-drug conjugate; PDC, peptide-drug conjugate; TCEP, tris (2-carboxyethyl) phosphine; MALDI-TOF MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; HSA, human serum albumin; AUC, area under curve; ROI, region of interest.


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1. INTRODUCITON Chemotherapy with cytotoxic drugs represents one of the major categories of therapeutic regimens for cancer. However, the therapeutic activities of most anti-cancer drugs in clinic use are limited by their concomitant side effects because these cytotoxic drugs always lack the capability of discriminating the tumor cells from normal counterparts 1-4. The cytotoxic drugs are mostly small molecules with a relatively short half-life in body. Administration of high dose of cytotoxic drugs is generally necessitated for killing tumor cells, but it would aggravate the systemic toxicities that mainly involve in heart, hair follicles, skin, bone marrow, digestive tract and reproductive system etc. 5, 6. Therefore, extending circulation time in blood and improving drug tumor delivery are the two most important factors that should be addressed for enhancing the therapeutic efficacy of cytotoxic drugs. Several strategies have been developed to solve these problems, including nanoparticle delivery platforms as well as antibody-drug-conjugates (ADCs)

7-9

. Among these strategies,

the innovative tumor targeting delivery approach of ADCs which utilizes tumor cell-specific monoclonal antibodies as a vehicle to selectively deliver cytotoxic drugs to tumor tissues has been widely investigated. Up to now, two ADCs of Adcetris® and Kadcyla®, respectively targeting the cell surface protein CD30 and the HER2 receptor, have been approved by FDA, inspiring numerous ADCs in development to treat many types of cancer

10

. The enhanced

pharmacokinetic behavior and tumor specificity of ADCs endowed by antibodies are the two most important elements for reducing systemic toxicity and enhancing therapeutic benefits. However, ADCs are still plagued by some deficiencies including extremely low cargo/carrier ratio, poor tissue penetration, cumbersome downstream process and unaffordable spending for patients etc. 11.



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Recently, peptide-drug-conjugates (PDCs) are emerging as another important class of therapeutic agents that combine a short peptide, which is capable of binding to receptors overexpressed on tumors, with a cargo of cytotoxic moiety through a conditional cleavage linker 4. These short targeting peptides are always composed of tens of amino acids that can be chemically synthesized and more conveniently produced than antibodies

12

. Small

molecular size benefits substantially high cargo/carrier ratio and penetrating tumor tissues, as well as the preparation process, however PDCs, on the other hand, still suffer short half-life in blood when compared to ADCs. Human serum albumin (HSA), as the most abundant plasma protein, is highly soluble, very stable and exhibits an extraordinarily long circulatory time of about 19 days in blood mainly because of its relatively large molecular size and pH-dependent FcRn-mediated recycling pathway that prevents intracellular degradation

13

. To take full advantage of the

benefits of PDCs and meanwhile overcome their drawbacks, we created a new kind of E. coli expressed peptide which was composed of an albumin binding domain (ABD) and a short tumor-specific internalizing sequence (RGDK or RPARPAR) in C-terminus with a total molecular weight of nearly 7 kDa (Table 1). The albumin binding domain is derived from the bacteria surface-exposed protein with extremely high affinity to human serum albumin

14, 15

.

The tumor-specific internalizing motif follows an amino acid sequence of R/K-X-X-R/K that can specifically bind to neuropilin-1 (NRP-1)

16

, an over-expressed cell surface receptor in

many types of cancers including breast and ovarian carcinoma, sarcoma, lung cancers and many other solid tumors

17

, and subsequently mediate cell internalization. RPARPAR

sequence is a divalent formula of R/K-X-X-R/K and has been validated to show the highest


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binding affinity with NRP-1. By contrast, RGDK sequence also contains a RGD motif that shows tumor targeting ability via integrin and is a di-specific ligand for many kinds of tumor cells 18. In this study, we designed a new kind of peptide-drug conjugate as an alternative to ADCs (molecular formula was shown in Table 1). Two types of peptide (ABD-RGDK and ABD-RPARPAR) and an R/K-X-X-R/K motif negative control peptide (ABD-GGDK) were highly expressed in E. coli bacteria in soluble form and easily purified from the lysate supernatant with a two-step chromatography scheme. Both of the peptides showed high capability and specificity of binding to human serum albumin. The fusion peptides were then coupled with a model antitumor agent of DOXO-EMCH, a (6-maleimidocaproyl)-hydrazone derivative of doxorubicin (DOX) which is one of the most widely used antitumor drugs because of its broad spectrum of antitumor activity 19. The physicochemical properties of the two ABD fusing peptide-doxorubicin conjugates (named ABD-RGDK-DOXO and ABD-RPARPAR-DOXO, respectively) were characterized and validated by spectral analysis and mass spectrometry. The in vitro cytotoxic activities and the role of tumor-specific internalizing sequence were evaluated in lung cancer A549 cell experiment. Pharmacokinetics of the two conjugates was evaluated in SD rats, and the anti-tumor potencies were assessed using BALB/c nude mice bearing A549 xenograft tumors. Besides, the in vivo fluorescent imaging of tumor-bearing mice was also conducted through intravenously injecting cyanine-5 (sulfo-Cy5-maleimide) labeled peptides to assess the tumor targeting ability of the fusion peptides.

2. MATERIALS AND METHODS

2.1 Materials



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Recombinant Escherichia coli (E. coli BL21) containing the DNA sequences encoding the ABD-RGDK, ABD-RPARPAR and ABD-GGDK hybrid peptides was designed and constructed in our laboratory (amino acid sequences were shown in Table 1). The (6-maleimidocaproyl)-hydrazone derivative of doxorubicin (DOXO-EMCH) was obtained from MedChem Express (Princeton, NJ, USA) with purity of above 95%. Sulfo-Cyanine 5 maleimide was obtained from Lumiprobe Corp (Florida, USA). Short peptides of RGDK, RPARPAR and GGDK were chemically synthesized by GL Biochem (Shanghai, China); Dulbecco’s

Modified

Eagle

Media

(DMEM),

fetal

bovine

serum

(FBS),

Penicillin-Streptomycin solution (100×) and 0.25% trypsin-EDTA (1×) solution were supplied by Mediatech (Manassas, VA). Trypan blue, Cell Counting Kit (CCK-8), paraformaldehyde (4% PFA) and Hoechst 33258 were all from Sigma (USA). The human lung cancer A549 cells were provided by Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). The athymic Nude Mice (BALB/c, males) and the Sprague-Dawley (SD) mice (males) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). 2.2 Expression and purification of the fusion peptides The recombinant E.coli BL21 was grown at 37 °C in LB medium supplemented with 100 µg/mL kanamycin and induced with 1 mM isopropyl-D-thiogalactopyranoside (IPTG) when cell density reached the mid-exponential phase. After fermentation, the bacterial cells were harvested by centrifugation at 4,000 rpm for 30 min at 4 °C. The pellets were suspended in 20 mM PB (pH 6.0) buffer containing 1 mM EDTA and then the cell suspensions were disrupted by high-pressure homogenizer (APV, Germany) for 3 cycles with a pressure of 800 - 900 bar.


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After centrifugation at 10,000 rpm for 30 min at 4 °C, the supernatant was collected and diluted 3 times in 20 mM PB, pH 6.0 to be purified through two chromatographic steps. Firstly, it was loaded to a CM Fast Flow column (XK 16×100 mm ID, GE Healthcare), equilibrated by 20 mM PB, pH 6.0, washed with the same buffer and then eluted with 25% elution buffer (20 mM PB, 1 M NaCl, pH 6.0). Secondly, the eluted peptide was further purified by a Superdex 75 column (XK 26×600 mm ID, GE Healthcare), equilibrated and eluted with 20 mM PB, 0.1 M Na2SO4, pH 7.0. The purified peptide was further desalted by a Sephadex G-25 column (XK 26×100 mm ID, GE Healthcare) in the buffer of 1 mM PB, pH 8.0 and ultimately stored in the form of lyophilized powder. The purity of the final product was determined by 12% SDS-PAGE. 2.3 Assessment of the binding capacity to human serum albumin in vitro To verify the binding affinity of the fusion peptides to serum albumin, we mixed the ABD-RGDK and ABD-RPARPAR fusion peptides with human serum albumin and transferrin in different molar ratios (1:0, 1:1, 1:2, 1:5, 1:10), separately. The concentration of fusion peptides in different groups should remain consistent. After mixing for 5 minutes, 500 µL of mixture was analyzed by a Superdex 75 HR column (10×300 mm ID, GE Healthcare) which was equilibrated and eluted with 20 mM PB, 0.1 M Na2SO4, pH 7.0. The peak heights of fusion peptides in various conditions were measured and compared to validate its binding affinity and the specificity to HSA. 2.4 Preparation of the peptide-DOXO conjugates DOXO-EMCH was used to synthesize the peptide-drug-conjugates. The maleimide moiety in DOXO-EMCH could react rapidly and specifically with the free cysteine at the N



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terminal of peptides in a Michael addition way. Briefly, to the fusion peptides (0.8 mM) in a 0.1 M sodium carbonate (pH 9.2) buffer solution, a 3-fold molar excess of tris (2-carboxyethyl) phosphine (TCEP) was added to reduce the disulfide bonds for 20 min at room temperature. Then DOXO-EMCH (7.2 mM) in dimethylformamide (DMF) was freshly prepared and added drop-wise to the above solution. The mixture was incubated with gentle stirring for 2 h at 25 °C in the dark room. Notably, the molar ratio of the peptide to DOXO-EMCH was 1: 6 and the concentration of DMF was strictly controlled below 40%. The final conjugates were purified by gel-filtration on a Sephadex G25 column, equilibrated and eluted with 0.1 M NH4HCO3, pH 8. The final peptide-DOXO product was lyophilized and stored at -70 °C. The coupling rate was determined by spectrophotometry 20. The molar concentration of DOX (C dox), was obtained through a standard curve according to its absorbance value at 495 nm. The molar concentration of peptide (C

peptide),

was measured by Lowry method. The

coupling rate was calculated using the following equation:

R = C dox / C peptide

(1)

2.5 Characterization of the peptide-DOXO conjugate Matrix-assisted

laser

desorption/ionization

time

of

flight

mass

spectrometry

(MALDI-TOF MS) analysis was performed on the Autoflex III (Bruker, USA) with α-cyano-4-hydroxycinnamic acid as the matrix. The operation mode was reflective mode with positive ion detection. The accelerating voltage was 19 000 V, and the delayed extraction time was 200 ns. Samples (0.2 mg/mL) were dissolved in water and then mixed with saturated


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matrix running buffer with a volume ratio of 1:1. High performance size exclusion chromatography: HP-SEC was performed on ÄKTA purifier (GE Health, USA) using Superdex Peptide column (10×30mm). 500 µL peptide-DOXO conjugates was loaded into the column and was eluted by 50 mM PB, 0.1M Na2SO4, pH7.4 buffer with a flow rate of 0.5 ml/min. The elution was detected by a UV detector at 280 nm and 480 nm. Circular dichroism: CD spectroscopy was measured on J-810 spectrometer (Jasco, Japan) at room temperature using a 1.0 mm path length quartz cuvette. The spectrum was obtained from 260 nm to 190 nm and the scanning rate was 500 nm per minute. The average of five scans of each sample was presented. Intrinsic fluorescence analysis: The intrinsic fluorescence analysis of the doxorubicin in the conjugates was performed on F-4500 fluorescence spectrophotometer (Hitachi, Japan). The excitation wavelength was 480 nm and the emission wavelength was recorded from 500 nm to 650 nm with a scanning rate of 1200 nm/min. 1.0 cm path length cuvette was utilized. Each sample was also subjected to scan for three times. 2.6 Acid-hydrolysis stability of the peptide-DOXO conjugates To evaluate the stability of the peptide-DOXO conjugates at several representative pH conditions, the conjugates were dissolved in 0.1 M sodium acetate buffer (pH 5.0) or 0.1 M PB buffer (pH 6.0 and 7.4) and incubated at 37 °C at the desired time points. These pH levels could separately represent the physiological environment of normal tissue (pH 7.4) and the acid condition of tumor microenvironment (pH 4-6). A 500 µL volume of each sample was analyzed by Superdex Peptide (300×10 mm ID, GE Healthcare), equilibrated and eluted with



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0.1 M PB, 0.15 M Na2SO4, pH 7.0 at a flow rate of 0.6 mL/min. The released DOX ratio at each time point was calculated according to the following equation: R = (A0 – Ax) / A0

(2)

(R: released DOX ratio; A0: peak area of the conjugate at zero time point; Ax: peak area of the conjugate at other time point). 2.7 In vitro study 2.7.1 Cell culture The human lung cancer A549 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% (v/v) fetal bovine serum, 10 U/mL penicillin and 100 µg/mL streptomycin at 37 °C in a fully humidified atmosphere of 5% CO2. For experiments, cells were digested with 0.25% trypsin-EDTA (1×) and counted using trypan blue as dyeing agent. 2.7.2 Cytotoxicity study The cytotoxicity of peptide-DOXO conjugates was determined by CCK-8 assay. NRP-1 overexpressed cells (A549) were seeded at 1×104 cells/well in 96-well plates and incubated for 24 h at 37 °C in 5% CO2. The medium was then removed and serial dilutions of free DOX, DOXO-EMCH and peptide-DOXO conjugates were added to the cells at various concentrations (0.01-70 µM DOX equiv.) for 60 h. After treatment, the agents were removed and the cells were washed twice with PBS and then returned to 100 µL complete growth media. 10 µL of the CCK-8 reagent was later added to each well and incubated with the cells for another 2 h. Thereafter, the absorbance was determined at 450 nm and 630 nm on a microplate reader. The absolute half maximal inhibitory concentration (IC50) of each


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treatment was calculated using GraphPad Prism v5.0 software. The results were the average of 3 experiments. 2.7.3 Cellular uptake assay A549 cells in the exponential growth phase were seeded in 96-well plates at a density of 2×105 cells/well and cultured for 24 h. Then, the media was replaced by fresh media containing peptide-DOXO conjugates (20 µM DOX equiv.) with or without the presence of RGDK or RPARPAR, GGDK short peptides (0.5 mM). The cells were further incubated at 37 °C for 1 h, 2 h and 4 h, respectively. After that, the cells were washed twice with PBS and subsequently disintegrated by cell lysis buffer. Finally, the cellular fluorescence intensity of the cell lysate was recorded at λex = 480 nm and λem = 580 nm using a fluorescence microplate reader. The results were the average of 3 experiments. 2.7.4 Intracellular distribution study In order to validate that whether the peptide-DOXO conjugate can diffuse into cell nucleus like free DOX, A549 cells were plated on glass coverslips at 4×105 cells/well and incubated for 24 h. The media was then removed and cells were treated with fresh media containing the conjugates and free DOX (20 µM DOX equiv.) in 1 mL/well for 3 h. Then the drugs were removed and the cells were incubated with fresh complete media for another 24 h. After treatment, the coverslips were washed three times with PBS and then cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. After washing with PBS, the cells were stained with 0.5 µg/mL Hoechst 33258 for 5 minutes and visualized under a confocal laser scanning microscope. All images were then captured with a Leica camera system.



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2.8 In vivo study 2.8.1 Pharmacokinetic studies Male SD rats (6-8 weeks old) were used to evaluate the pharmacokinetic profiles of the conjugates. These rats were randomly divided into 5 groups (three rats in each group) and then intravenously injected through the tail vein with free DOX, DOXO-EMCH and the peptide-DOXO conjugates at the dose of 5 mg/kg DOX equivalent, respectively. PBS was used as negative control. The rats were anesthetized and blood samples were collected from the retro-orbital sinus at the desired time points. The serum was then obtained by centrifugation at 6,000 rpm for 20 min after blood coagulation. Fluorescence intensity of drug in each serum sample was measured via a fluorescence microplate reader at λex = 480 nm and λem = 580 nm. Drug concentration was then calculated according to a standard curve of DOX which was detected as the above method. Pharmacokinetic parameters were fitted and calculated using DAS 2.0 software. 2.8.2 Growth inhibitory effect on tumors in vivo 4-week nude mice (BALB/c, males) were housed in specific-pathogen free caging under a 12 h light-dark rhythm with standard temperature and humidity conditions. The tumor-bearing mice were established through subcutaneous injection of A549 cells (1×106 cells in 0.1 mL PBS) into the right forelimb armpit of each mouse. When the tumor size reached a volume of approximately 100-150 mm3, the mice were randomly divided into 5 groups with at least 6 mice in each group. Then the mice were subcutaneously injected with PBS, free DOX, free DOXO-EMCH and the peptide-DOXO conjugates at the dose of 3 mg/kg DOX equivalent every four days for four times. The growth inhibitory effect on tumors was evaluated by


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monitoring animal weight and tumor size every 2 days. Tumor volume was calculated by the following equation: Volume = (Length×Width2) / 2. The mice were sacrificed on day 20, and then the volume and weight of excised tumor tissue were measured and photographed. 2.8.3 In vivo fluorescence imaging ABD-RGDK and ABD-RPARPAR peptides were firstly labeled by maleimide-Cy5 with a molar ratio of 1:3 (peptide to Cy5) and the uncoupled Cy5 was eliminated by gel filtration. The subcutaneous implantation mice model of A549 tumor was established as mentioned previously. When the diameter of the tumor reached about 5-6 mm, a 200 µL sample of Cy5 and ABD-RGDK/RPARPAR-Cy5 conjugates (0.2 mg/kg MAL-Cy5 equiv.) were separately and intravenously injected into the tumor-bearing nude mice via tail vein. After treatment, the mice were anesthetized using isoflurane at 4, 8, 12, 24 and 48 h and fluorescence images were taken under λex = 650 nm and λem = 700 nm using FX Pro in-vivo imaging system. All animal experiments were performed with the approval of Institute of Process Engineering, Chinese Academy of Sciences (IPE, CAS). 2.9 Statistical analysis All data were presented as mean ± standard deviation (SD). The statistical significance was assessed via unpaired T test or one-way ANOVA and was defined as *p < 0.05, **p < 0.01, ***p < 0.001. The parameters in the pharmacokinetic assay were calculated using DAS 2.0 software. The IC50 values in the CCK-8 assay were calculated by GraphPad Prism v5.0.

3. RESULTS AND DISCUSSIONS

3.1 Expression, purification and characterization of the hybrid peptide



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The hybrid peptides were highly expressed in E. coli in soluble form, which could be simply purified from the disrupted supernatant through a two-step chromatography scheme with a final purity above 95% (Figure 1A, B, C). After being incubated with HSA, the height of the elution peak for the peptide decreased or even vanished with increased HSA ratio when subjected to SEC analysis (Figure 2A, C), but the peak height increased when peptides were incubated with human transferrin (Figure 2B, D). The increased absorbance at the peptide position was due to the retarded elution for transferrin, indicating that the hybrid peptides are capable of binding to HSA specifically relative to transferrin. The binding process could be completed in several minutes and the hybrid peptides were completely bound to HSA at a low molar ratio of about 2.0 (HSA to peptide), indicating that both ABD-RGDK and ABD-RPARPAR fusion peptides remain the potent binding ability with HSA. 3.2 Synthesis and characterization of the peptide-DOX conjugate DOXO-EMCH was efficiently conjugated with the hybrid peptides in a semi-organic phase with a coupling rate of about 90%. To characterize the two peptide-drug conjugates, MALDI-TOF MS was used to analyze the molecular weights and showed that the ABD-RGDK-DOXO and ABD-RPARPAR-DOXO were approximately 7487 and 7835 Da, as shown in the Peak 3 (Figure 3A, B). It should be noted that the acidic matrix of α-cyano-4-hydroxycinnamic acid could lead to the release of doxorubicin from the conjugates during this analysis, as shown in the peak 2

21

. High performance size exclusion

chromatography of Superdex Peptide was adopted to further identify the conjugates and the results demonstrated that the absorption signal of the conjugates could be detected in both 280 nm and 480 nm, indicating that DOXO-EMCH was successfully coupled to the two peptides


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(Figure 3C, D). This result was further validated by the fluorescence analysis (Figure 4A, B). Moreover, circular dichroism showed that the conjugation of DOXO-EMCH could not significantly undermine the secondary structures of the fusion peptides (Figure 4C, D). Releasing DOX from the conjugates is necessary to maximally maintain the cytotoxic potency

3, 22

. To confirm the cleavage behavior of hydrazone in the conjugate, we evaluated

the in vitro pH-induced release of doxorubicin from the conjugate at pH 5.0, 6.0 and 7.4 (Figure 5A, B). The DOX-release percentages of ABD-RGDK-DOXO conjugate after 48 h were approximately 30% (pH7.4), 75% (pH6.0), and 80% (pH5.0), respectively. Similar results were observed in the ABD-RPARPAR-DOXO conjugate and indicated that both conjugates are sensitive to the change of pH condition. Fast release of DOX from the conjugates at acid condition in tumor was favorable for its diffusing into the cell nucleus to exert the damaging effects on DNA 23. 3.3 In vitro cell cytotoxicity To evaluate the cytotoxic activities of the peptide-DOXO conjugates, we tested their inhibiting efficacy against A549 cell line which was validated to overexpress NRP-1

24

, as

shown in Figure 6A and Table 2. The conjugates could inhibit the growth of A549 cells in a dose-dependent manner and displayed lower cytotoxicity than free DOX and DOXO-EMCH. With a static co-culture of cells and drugs, it might be reasonable that free doxorubicin (IC50, 0.31 ± 0.19 µM) and DOXO-EMCH (IC50, 0.47 ± 0.25 µM) both exhibited a more potent cytotoxicity than the conjugates. The cellular uptake of DOX is mainly through a passive diffusion transport mechanism 25, whereas conjugation of a peptide would hinder the passive diffusion route, resulting in higher IC50 values for the peptide-DOX-conjugates (9.27 ± 3.56



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µM for ABD-RGDK-DOXO and 11.21 ± 4.54 µM for ABD-RPARPAR-DOXO, respectively). This phenomenon was also observed for some other peptide-drug-conjugates

23, 26

. It should

be noted that both ABD-RGDK-DOXO and ABD-RPARPAR-DOXO showed a similar IC50 value of about 10 µM, but ABD-GGDK-DOXO demonstrated a relatively higher IC50 value of approximately 40 µM, almost 4 times higher than that of ABD-RGDK/RPARPAR-DOXO conjugates. The silence of R/K-X-X-R/K motif in ABD-GGDK-DOXO resulted in a much higher IC50 value, indicating that the R/K-X-X-R/K motif played an important role for ABD-RGDK/RPARPAR-DOXO conjugates in inhibiting the growth of A549 cells. 3.4 Cellular uptake and intracellular distribution To further validate the roles of the R/K-X-X-R/K motif in transporting across tumor cell membrane, the cellular uptake of the peptide-DOXO conjugates were investigated through co-culture with A549 cells with or without the presence of the chemically synthesized RGDK or RPARPAR, GGDK peptides (Figure 6B). Increased intracellular accumulation of doxorubicin

was

observed

with

increasing

incubation

time

for

the

ABD-RGDK/RPARPAR-DOXO conjugates. This kind of accumulation rate was notably inhibited when the cells were cultured in the presence of RGDK or RPARPAR (p < 0.01), indicating that the R/K-X-X-R/K motif in the fusion peptides could mediate the conjugates to penetrate into the A549 cell in a neuropilin-1dependent manner. In contrast, no significant difference was observed in ABD-GGDK-DOXO groups with or without the presence of the GGDK

peptide.

Besides,

significant

difference

(p

K Sequence for

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