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Multifunctional nanoparticles loading with docetaxel and GDC0941 for reversing multidrug resistance mediated by PI3K/Akt signal pathway Yiyue Wang, Jing Li, Jing Jing Chen, Xuan Gao, Zun Huang, and Qi Shen Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b01045 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 17, 2017

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Molecular Pharmaceutics

Multifunctional nanoparticles loading with docetaxel and GDC0941 for reversing multidrug resistance mediated by PI3K / Akt signal pathway

Yiyue Wang†, Jing Li†, Jing Jing Chen, Xuan Gao, Zun Huang, Qi Shen* School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China †They have the same contribution to this work.

To whom correspondence should be addressed Qi Shen School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China Tel: +86-21-34204049 Fax: +86-21-34204049 E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT The poly lactic-co-glycolic acid polyethylene glycol conjugated with cell penetrating

peptide

R7 (PLGA-PEG-R7)/

poly

sulfadimethoxine-Folate

nanoparticles loading with Docetaxel (DTX) and GDC0941 (R7/PSD-Fol NPs) were prepared to overcome multidrug resistance (MDR) and enhance the antitumor activity. First, poly sulfadimethoxine-Folate was synthesized to construct the R7 / PSD-Fol NPs. The R7/PSD-Fol NPs were prepared with the ability that effective entrapment and drug loading. Due to the pH-sensitive effect of PSD-Folate, the releasing of DTX and GDC0941 from the R7/PSD-Fol NPs was lower in pH7.4 buffer solution with that in pH 5.0 buffer solution. The half maximal inhibitory concentration (IC50) of MCF-7 and resistant to doxorubicin (MCF-7/Adr) cells illustrated the cytotoxicity of R7/PSD-Fol nanoparticles by using the MTT method. The uptake of R7/PSD-Fol NPs was visualized by using the fluorescence of Rh-123 to detect the targeting effect of folate on the surface of R7/PSD-Fol NPs. The results of the cell apoptosis and the depolarization of mitochondrial membrane potential (MMP) were adopted to show the cytotoxicity of the R7/PSD-Fol NPs on MCF-7/Adr cells. The western blot revealed the inhibition of PI3K/Akt pathway in MCF-7/Adr cells induced by R7 / PSD-Fol NPs. Finally, both in vivo distribution and in vivo antitumor showed the R7 / PSD-Fol NPs displayed the better distribution at tumor site and the stronger suppression of tumor growth in the tumor bearing nude mice compared with 2


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control group. It was concluded that R7/PSD-Fol NPs loaded with DTX and GDC0941 could overcome multidrug and enhance the antitumor effect further.

Keywords: nanoparticles, multidrug resistance, DTX, Folate, p-Akt

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1. Introduction Multidrug resistance (MDR) had been commonly recognized as one of the mainly reasons chemotherapy failure in the antitumor clinic therapy. Among the series of studies on the MDR related proteins, the p-gp and ATP-binding cassette (ABC) transporter were accepted as the protein to induce the drug efflux.1-3 The over expression or activation of glutathione-S-transferase-π4 and the decrease or denaturation of topoisomeraseⅡ 5 were also the other factors which MDR occurred in intrinsic breast cancer. Phosphatidylinositol-3-kinase / protein kinase B (PI3K /Akt) signal path was a kind of tyrosine kinase signaling pathway, playing a very important role in regulating cell proliferation and inhibiting apoptosis. For the abnormal regulation of PI3K/Akt in cancer cells, hyperplasia, angiogenesis, MDR often occurred in tumors.6 Several substrate were mediated by PI3K/Akt such as caspase-97, Bcl-28, m-TOR9. The activation of PI3K/Akt signal path could promote cell proliferation and the tumor cell self-protection function that named MDR. GDC0941 was a selective inhibitor for PI3K / Akt signal path.Ⅰphase of clinical trial data suggests it had good antitumor activity, and was well tolerated with patients with breast cancer, ovarian cancer and melanoma.10 Up to now, the drug delivery systems using ligand modified nanoparticles have displayed their site-specific targeting capacity.11-13 The protein-derived and chemical cell penetrating peptides (CPPs) have been widely used to deliver the 4


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protein and oligonucleotides.14 R7 was a kind of CPPs that made of seven arginine. In our design, it facilitated the nanoparticles internalize into tumor cells by endocytosis and accelerated drug accumulate in cytoplasm through endosomal esacpe15. Folate has a high affinity for cell surface folate receptor on tumor cell. So, it was a practical significance that folate conjugated to the surface of nano-sized polymeric drug delivery system to promote target tumor cell and endocytosis via folate receptor on tumor cell membrane mediated.16 The derivatives of Sulfadimethoxypyrimidine (SD) was used as monomer to synthesize the copolymer PSD. This polymer was modified with folate to form the pH sensitive material and provide the target group.17 The nanoparticles modified with pH sensitive PSD-Folate polymer could active target tumor cell and hide the positive charge of R7. Hence,

we

prepared

the nanoparticles that were made of poly

sulfadimethoxine-folate (PSD-Folate) as well as lactic-co-glycolic acid polyethylene

glycol

conjugating

with

cell

penetrating

peptide

R7

(PLGA-PEG-R7) to co-deliver the DTX and GDC0941. In vitro releasing was applied to explore characteristic of pH-triggered drug release. The nanoparticles loading with the fluorescent dye, Rh-123 or DiR, were used to detect the in vitro and in vivo targeting effects. To prove the function of reversing the MDR, cell apoptosis, mitochondrial membrane potential (MMP) and western blot were conducted using the MCF-7 and MCF-7/Adr cells treated with the drug loaded 5



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in nanoparticles. The cytotoxicity and in vivo antitumor were carried out among different formations of DTX to compare the killing ability on drug resistant cell line or tumor.

Figure 1. Schematic illustration of the targeted delivery of DTX and GDC0941 using multifunctional nanoparticles to overcome multidrug resistance.

2. Materials and Methods 2.1 Materials PLGA15,000-mPEG2,000 (Jinan Daigang Biology Tech Co. Ltd., Shandong Province, China), Polyving akohol (PVA, Aladdin Reagent Co. Ltd., (Shanghai, China), 6


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GDC0941 (Hanxiang Biotechnology, Wuhan, China), DTX (Anticancer Phytochemistry

Co.

Ltd.,

Shanghai,

China),

Folate

and

sulfadimethoxine(Aladdin Reagent Co. Ltd., Shanghai, China), were of analytical grade. Methanol and acetonitrile (J&K Chemical Scientific Co. Ltd., Shanghai, China) for HPLC analysis were chromatogram grade. RPMI 1640 medium (Gibco, Carlsbad, CA, USA), Fetal bovine serum (FBS) and Phosphate buffered solution (PBS)(Biosun Biotechnology Co. Ltd., Shanghai, China), 0.25% trypsin-0.02% EDTA (Beyotime), tetrasodium and penicillin streptomycin (Thermofisher Scientific Co. Ltd., Shanghai, China) were used for cells culture. MTT, Rh-123 and DiR (Sigma Aldrich, St. Louis, MO, USA). R7-NH2 (RRRRRRR-NH2) was

synthesized by GL Biochem Ltd. (Shanghai City, China). And it was a peptide consist of seven arginine which terminal carboxyl group was ammoniated. The human breast carcinoma MCF-7 cancer cell lines (MCF-7) and that resistant to doxorubicin (MCF-7/Adr) obtained from Institute of Biochemistry and Cell Biology; Shanghai Institutes for Life Science, China. BALB/c nude mice (female, 5-6 weeks old, 20 ± 2 g) were taken good care under specific pathogen-free (SPF) conditions in compliance with the protocol evaluated and approved by the ethics committee of Shanghai Jiao Tong University.

2.2 The Synthesis polymer blocks of PSD-Folate and PLGA-PEG-R7 7



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The pH-sensitive PSD-Folate was synthesized according to the route shown in Figure 2. And the synthesize route of PSD has been developed by previous researches.17 NaOH (5 mmol, 0.30 g), sulfadimethoxine (5 mmol, 1.50 g), methacryloyl chloride (5 mmol, 0.5 mL) were added into mixed solvents acetone / water (5 mL:5 mL) with magnetic stirring for 3 h in an ice bath. The methacryloyl chloride was evaporated under vacuum. The VSDM was prepared after vacuum freezing drying. Next, the VSDM (white loose powder) after drying (4 mmol, 1.5 g), AIBN (0.15 mmol, 0.0116 g), cysteamine (2 mmol, 0.0013 g) were dissolved in DMSO (10 mL) by step, then reacting for 24 h using magnetic stirrer at 70 °C under nitrogen without water. The reaction mixtures were poured into water to get the white precipitation. The raw polymers were purified by dialyzing for 3 days (Mcro 1,000) and the white powder PSD was obtained by lyophilization. To prepare the PSD-Folate, folate (0.3 mol, 0.13 g), DIC (0.6 mol, 93 µL) was dissolved in dry DMSO. The mixture was stirred for 3 h under nitrogen and the NHS was added into the solution to keep stirring for another 24 h. Then the PSD (0.2 g) was added in the solution stirring for 72h at room temperature. The reaction solution was dropped into pure water (100 mL) to precipitate the PSD-Folate. The precipitation was collected by filtration, washed with distill water and dried under reduced pressure to give product. Then the targeted product was purified by dialyzing for 48 h (Mcro1,400).

8


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Figure 2. The Synthesis scheme of PSD-Folate: (1) the synthesis of VSDM, (2) the synthesis of PSD ploymer (3) the modification with folate to compose the PSD-Folate.

2.3 Determination of the molecular weight and the pKa of PSD and characterization of PSD-Folate The dried PSD (8 mg) was dissolved in the DMF to detect the molecular weight by the gel permeation chromatography. Potentiometric titration was used to detect the pKa of the PSD 5 mg PSD was dissolved in NaOH (0.01 M, 25 mL), titrated by HCL (0.01 M). The consuming of HCL (0.01 M) and the pH value was recorded to draw the V-pH chart. The knee points were pKa1 and pKa2. The pKa of 9



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PSD was mean value of pKa1 and pKa2.The FT-IR and 1H-NMR (400 M) method were used to determine intermediate and final product in the synthesis of PSD-Folate.

2.4 Preparation of R7/PSD-Fol NPs The R7/PSD-Folate NPs loading with DTX and GDC0941 were formed through the emulsion-solvent evaporation method. First, DTX (2 mg), GDC0941 (1 mg) PLGA-mPEG (10 mg) , PLGA-PEG-R7 (5 mg) were dissolved in the methylene chloride (2 ml) , then the methylene chloride solution was added into the 1% cooled PVA solution (20 mL) at 0 °C and a ultrasonic probe was used to sonicate for 4 min at 140 W. Then, the emulsion was evaporated at the room temperature under reduced pressure to withdraw the organic solvent and solidify the NPs. The R7 NPs (PSD-Folate 0 mg) were prepared. For R7/PSD-Fol NPs, the PSD-Folate (1mg) was added in the above solvent. After removing the uncombined material using centrifugal machine, the upper solution was centrifuged at the rate of 12,000 rpm for 30 min. And then the precipitation was redissolved by double distill water. The white powder of nanoparticles was collected after lyophilizing. And the preparation process was optimized according the amount of DTX and GDC0941 single factor on R7/PSD-Fol NPs. The NPs that were used to investigate the cellular internalization and distribution in vivo, the blank R7 NPs, blank R7/PSD-Fol NPs, Rh-123-R7 NPs, Rh-123-R7/RSD-Fol NPs and DiR-R7 10


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NPs, DiR-R7/RSD-Fol NPs, were prepared using the forementioned method but the Rh-123 or DiR was substitution for chemical drugs.

2.5 Characterization of R7/PSD-Fol NPs The suspension of nanoparticles was diluted and measured by a Dynamic Light Scattering (DLS) to determine particle size and zeta potential. The morphological examination was performed by transmission electron microscopy (TEM). R7/PSD-Fol NPs were diluted with distilled water and placed onto a copper sheet. The sheet was dried at 40 °C under reduced pressure for TEM analysis.

2.6 Evaluation of drug content The encapsulation efficiency (EE) (%) and drug loading (DL) (%) capacity of DTX and GDC0941 was measured by HPLC. The chromatography system was composed of a Shimadzu LC-20AT chromatographic system (Shimadzu, Kyoto, Japan) with a LC-20AT binary pump and a SPD-20A UV-vis detector. Data processing was performed with a LC Solution program. Analysis was carried out on a Dikma Dimonsil C18 column (200 mm × 4.6 mm, 5 µm, Dikma Technologies). The mobile phase of DTX detection was composed of pure water -acetonitrile (4:6, v/v) and GDC0941 was 0.02 M sodium dihydrogen phosphate-acetonitrile (4:6, v/v), and the flow rate was 1.0 mL/min. The column temperature was maintained at 25°C, the UV-Vis detection wavelengths of DTX 11



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and GDC0941 was set at 249 nm and 254 nm, and the injection volume was 20 µL. Standard solutions (1 mg/mL) of DTX in methanol were prepared and stored at 4°C. The working solutions of DTX with 0.3125, 0.625, 1.25, 2.5, 5, 10, 20 µg/mL were prepared by serial dilution of DTX stock solution. Determination of each concentration of the peak area parallel to three samples, the average peak area was calculated. With an average peak area, A versus DTX of the concentration of c (µg/mL) plot using linear regression to create the HPLC standard curve of DTX in vitro. Standard solutions (0.1 mg/mL) of GDC0941 in methanol were prepared and stored at 4°C. The working solutions of GDC0941 with 0.1, 0.3, 5, 10, 12.5, 20, 25 µg/mL were prepared by serial dilution of GDC0941 stock solution. The same method was used to create the HPLC standard curve of GDC0941 in vitro. The NPs powers were dissolved in the ultrapure water, then solution was centrifuged at 2,000 rpm for 30 min. The 200 µL supernatant was placed in the ultrafiltration device (Mcro1,000) to centrifugalize at 4,000 rpm for 30min. The lower liqud in 400 µL methanol was analyzed by HPLC to detect the Wfree of DTX and GDC0941. The same NPs solution was centrifuged at 2,000 rpm. The 200 µL supernatant was mixed with 800 µL acetonitrile. The ultrasonic emulsion breaking solvent was centrifuged at 2,000 rpm to collecting the supernatant using for detecting the Wtotal by HPLC. The WNPs of dried nanoparticles was dissolved in 1mL ultrapure water. The 200 µL solvent was mixed with 800 µL acetonitrile by votex and ultrasonic. After 12


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centrifuged at 12,000 rpm, the 200 µL supernatant was collected to detect the Wentapped by HPLC. The drug encapsulation efficiency and drug loading were calculated by the following equations: (1) EE (%)=(Wtotal-Wfree)/ Wtotal*100% (2) DL (%)=(Wentapped/WNPs)*100%

2.7 In vitro Drug Release The release rate of the DTX and GDC0941 in R7/PSD-Fol NPs at different pH conditions was measured by the dialysis bag method. 2 mL PBS (pH=7.4) suspension of R7/PSD-Fol NPs, which contain theoretic DTX (2 mg) and GDC0941 (1 mg) was added in the dialysis bag (Mcro 2,700) and placed into the release medium (30 mL). The experiment was performed in the shaking bath at the rate of 100 rpm at the 37 ± 0.5 °C. 1% SDS was added in the release medium of PBS buffer to promote the releasing of DTX. 1 mL medium was collected at the preset point in time, and fresh release medium was replenished. All the collecting samples were detected by the known HPLC conditions. The release of R7/PSD-Fol NPs in Acetic acid-ammonium acetate buffered solution (ACS) (pH=5.0) was followed the same procedure.

2.8 In vitro Cytotoxicity Assays by MTT Multiscan Spectrum (HERMO Varioskan Flash) was used to detect the 13



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absorbance value of MTT to evaluate the cytotoxicity of R7/PSD-Fol NPs. Both MCF-7 and MCF-7/Adr cells were seeded into 96-well plates (5×104 cells/mL, 100 µL), then cultivated at 37 °C in 5% CO2 atmosphere for overnight. For DTX, DTX+GDC0941, R7 NPs and R7/PSD-Fol NPs groups, the medium was replaced by 100 µL fresh DMEM containing different formations of 50 ng/mL DTX relatively. For the free folate+R7/PSD-Fol NPs group, the free folate (1 mg/mL) was added to incubate for 1h before replacing with R7/PSD-Fol NPs. After incubating for 48 h, the cells were incubated with the PBS containing 5 mg/mL MTT and keep in dark for another 4 h at 37 ± 0.5 °C followed by dissolving formazan with 100 µL DMSO. Then the cell viability was determined through a microplate reader. In addition, each DTX, DTX+GDC0941, R7 NPs, R7/PSD-Fol NPs groups containing 100, 200, 400, 800 ng/mL of DTX tested on MCF-7 cells as well as 0.05, 0.4, 1.5, 3.6 µg/mL of DTX tested on MCF-7/Adr cells were set up to detect and contrast the cytotoxicity of DTX in four forms quantitatively. The 100, 200, 300, 400, 500 µg/mL of blank R7 NPs and blank R7/PSD-Fol NPs tested on the MCF-7 and MCF-7/Adr cells were carried out by the same way. Four parallel samples for every groups were tested to eliminate error.

2.9 Cellular Uptake The Intracellular distribution of R7/PSD-Fol NPs was conducted by loading a small amount of the Rh-123 fluorescent dyes instead of DTX and GDC0941. 14


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Confocal laser scanning microscopy (CLSM) provided the visualization uptake of MCF-7 or MCF-7/Adr cells and flow cytometry (FACS) was applied to contrast the fluorescent intensity. The cells were spread out across coverslips in the 6 well culture plates after culturing for overnight. For Rh-123, Rh-123-R7 NPs, Rh-123-R7/PSD-Fol NPs groups, free folate+Rh-123-R7/PSD-Fol NPs (the 1 mg/mL free folate was incubated with the MCF-7 or MCF-7/Adr cells for 1h before replacing with the Rh-123-R7/PSD-Fol NPs), the medium was replaced by DEME containing different formulations at the same 5 µg/mL Rh-123 concentration. After incubating for another 4 h, the medium was removed slightly. To remove extracellular fluorescent material left on the stained coverslips, cold PBS was used to clean the surface slightly for three times. Then, the coverslips was steeped in 4% paraformaldehyde for 5 min and observed by CLSM. As for the FACS assays, the cells were treated with the same way and transferred to flow cytometery tube. The PE-Texas Red A was chosen as fluorescent light channel.

2.10 Cell apoptosis assay Annexin V has an affinity for phosphatidylserine18, which is used to detect the early apoptotic cells. Propidium Iodide (PI) is a fluorescent dye to mark the cell nucleus of later apoptotic and dead cells. The apoptotic stages of MCF-7/Adr cells inducted by R7 / PSD-Fol NPs was analyzed by measuring the fluorescence 15



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strength of Annexin V-FITC and PI. Briefly, the cells (1×106 cells per well) were seeded into 6 well culture plates to adherence for overnight. For control, DTX, (DTX+GDC0941), R7 NPs, R7 / PSD-Fol NPs groups, the medium were replaced by DEME containing different formulations at the same 300 ng/mL DTX concentration. After collecting the washed cells, the concentration was adjusted to 5× 105 cells/mL in 400 µL Binding Buffer and incubated with the dyes at 0 °C away from light. And the cells in tubes were immediately analyzed by FACS in 30 min. The percentages of cells in different apoptotic stages were determined using Cell Quest software (Becton Dickinson).

2.11 Measurement of mitochondrial membrane potential Rh-123 is a fluorescent dye acted as the indicator of mitochondrial membrane. To verify the MCF-7 and MCF-7/Adr cells apoptosis induced by R7/PSD-Fol NPs, the fluorescent intensity was measured by FACS. For the control, DTX+GDC0941, R7 NPs, R7/PSD-Fol NPs groups, the 1×106 cells per wells were seeded into 6-well culture plates for overnight, the medium were replaced by DEME containing different formulations of DTX containing the same concentration of 300 ng/mL, incubating for 24 h. Then the Rh-123 (10 µg/mL) was added into each wells to stain the cells at constant temperature incubator for 30 min. And 1×104 cells per sample were analyzed by Flow cytometry. Among the stained cells, the fluorescence intensity more than 50% was calculated as 16


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index of the level of MMP.

2.12 Western blot analysis of PI3K/Akt SDS-polyacrylamide gel electrophoresis, protein transferring and immunoassay method19 were conducted to detect the expression of p-Akt and Akt in MCF-7/Adr cells. The 1×106 cells per well was incubated for overnight. Then the designed groups were respectively treated with control, DTX, DTX+GDC0941, R7 NPs, R7/PSD-Fol NPs for 24 h, containing 300 ng/mL concentration of DTX. 160 µL cold RIPA buffer was added into the well to break the washed cells and the plates was to be rocked occasionally for 10 min at 0 °C. The centrifuge tubes were used to collect lysate. The insoluble part was discarded after centrifuged at 4 °C. The equivalent supernatant was diluted with 4 fold loading buffer and boiled for 10 min. The 6 µL color dye protein and 20 µL protein sample were added to 10% SDS-PAGE and a nitrocellulose membrane was used to transfer the separated proteins. The membranes were blocked in a solution using confining liquid under continuous rocking at room temperature for 1 h. For immune blot, Akt Antibody and Phospho-Akt (S473) Antibody (Cell Signaling) was applied for overnight at 4 °C with constant agitation. To incubate with the secondary antibody, the membranes were placed in the prepared solution for 1 h at 37 ± 0.5 °C, under the condition of constant agitation. The washed membranes were measured by light exposure apparatus (LI-COR). 17



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2.13 In vivo Imaging To establish subcutaneous solid tumor in the BALB/c mice, approximately 1×106 MCF-7/Adr cells (200 µL) were injected into the subsurface of the right front legs of each mice. When the tumor volume reached to 50 mm3 (about 10-20 days later), DiR, DiR-R7 NPs, DiR-R7/PSD-Fol NPs were injected (200 µL) through a tail vein of model mice at the same dose of DiR (25 µg/mL).Then, the treated mice were anesthetized with gas to take photos at the predetermined time points by small animals living imager (IVIS Lumina, Caliper).

2.14 In vivo Antitumor study Tumor model in mice were made by the same above mentioned method. Two weeks after the model establishing, the mice were randomly assigned to three groups, control, DTX+GDC0941, R7/PSD-Fol NPs, five mice per group. For the three groups，200 µL solution were given intravenously to each mouse through the tail vein. And the free DTX and that loaded in the nanoparticles were the same 10 mg/kg for mice. While the treated period, those mice were taken good care with drug treating and the longest diameter (a) and shortest diameter (b) of solid tumor as well as body weight were kept a record every three days for six times. The volume (V = π/6 ×a ×b2) was the index of tumor size. After that, the 18


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peeled tumors were needed to record weight and take photo.

3. Results and Discussion 3.1 Synthesis and characterization of PSD-Folate The targeted product of PSD-Folate synthesis was characterized by FT-IR in Figure 3.A. In the infrared spectrum of the monomer VSDM compared with SD, the bands 3486 cm-1, 3308 cm-1 corresponds to the primary amine (-NH2) was replaced by the bands 3270 cm-1 corresponds to second amine (-NH-) and the band 1667 cm-1corresponded to the C = C stretching vibrations characterized the peak of olefin in propylene. -NH2 characteristic peak disappeared, the characteristic peak of olefins and amide linkages presence indicated the synthesizing of VSDM was achieved. The 1H-NMR spectrum of PSD in DMSO-d6 (Figure 3.C) revealed the characteristic PSD peaks appeared at δ2.609 ppm, which the signal was assigned to the H in the group of -CH2-S- at the end of polymer. The signal appearing at δ2.040 ppm was assigned to the H in the group of -CH2CH2-S- at the repeat part. At the branch chain, the signal appearing at δ6.579 ppm, δ7.145 ppm and δ4.068 ppm was assigned to the H in the groups of benzene ring and in the methoxy group of pyridine ring. The success of the conjugation of folate to PSD (Figure 3.D) was confirmed by the appeared protons peaks of folate containing δ2.069 ppm (-CH2CH2CO-), δ3.958 ppm (-NH2 in the pterin of folate), δ7.864 ppm (H in the pterin of folate) and the signals at δ3.472 19



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ppm, δ6.351 ppm, δ7.310 ppm were assigned to the groups of PSD structure. In the Infrared spectrum of PSD-Folate (Figure 3.B), the bonds 3386.45 cm-1 (NH stretching vibration), the bonds 1677.06 cm-1 (C=O stretching vibration), the bonds 1524.48 cm-1 (NH bending vibration and C-N stretching vibration), the bonds 1210.47 cm-1 (C-N stretching vibration and NH bending vibration) were the characterization of the second amine in PSD-Folate compared with PSD and folate. The typical peaks of primary amine in PSD were replaced by the signals of second amine in PSD-Folate compared with folate. Thus, the synthesis of PSD-Folate was reached. The ploymer PSD-Folate was consisted of a pH sensitive part PSD and folate as an active targeting group to modify the nanoparticles and protect the R7 peptide. The synthesize of PSD was a radical polymerization and 2-aminoethanethiol was chosen as a chain transfer agent.17 Folate, which was activated by DIC, was combined with PSD by condensation. The copolymer of folate and polymer through covalent bond could be used as materials to form drug carriers.20, 21 GPC technique was used to measure the molecular weight and the molecular weight distribution of PSD (Figure 4.A). The symmetry narrow chromatographic peak indicated the well distribution of molecular weight. The molecular weight of Mn = 1,014±37, Mw = 1,213±41. The degree of polymerization was 4 after calculation. Compared with the reported 10 polymerization degree, it can be concluded that the removal of oxygen environment and solvent made an effect 20


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on reaction degree of radical polymerization. The pKa1 and pKa2 in potentiometric titration curve of PSD (Figure 4.B) was 9.21, 4.75. The pH sensitive PSD (pKa= 6.98) could dissociate as anion in the agent (pH>7.0). This faintly acid may indicate the more effectively targeting the tumor microenvironment than that using carboxyl groups as the general pH sensitive materials. The pH-sensitive copolymer (stearoyl-PEG-poly SD, SD monomer, SD) was made up of average seven methacryloyl sulfadimethoxines per molecule and an apparent pKa of 7.2 was testified.22 The different back bone both containing poly SD shows different polymerization degree and pKa. It could be speculated the molecular weight and polarity of groups affect ionization of PSD.

21



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Figure 3. Characterization for intermediate and final product in the pathway of synthesis: (A) FI-TR of VSDM and SDM (B) FI-TR of folate, PSD and PSD-Folate (C) 1H-NMR spectra of PSD (D) 1H-NMR spectra of PSD-Folate.

Figure 4. Characterization for polymer PSD: (A) GPC of PSD (B) potentiometric titration curve of PSD. Data are presented as mean ± SD (n = 3).

3.2 Preparation and Characterization of R7/ PSD-Fol NPs The TEM image of the R7/ PSD-Fol NPs was shown in Figure 5.A. The dispersive spherical shapes were averagely less than 200 nm. The zeta potential of R7/ PSD-Fol NPs in pure water was shown in Figure 5.B. And the average of particle size was 151 nm with the PDI 0.163 (Figure 4.C). For R7/ PSD-Fol NPs and R7 NPs in Table 1, the average size of particle was similar with PDI < 0.2 22


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and different zeta potential. The standard curve equation of DTX and

GDC0941 was y = 26345x + 4806.5,r =0.9996 and y = 41603x + 5451.8，r = 0.9998. Considering the EE (%) and DL (%) of DTX and GDC0941, DTX had higher EE (%) and DL (%) than GDC0941. A certain amount of DTX and GDC0941 were dissolved in selective oil phase and added to PVA solution. The structure of nanoparticles was formed because the certain amount amphipathy PLGA-PEG-R7 and PLGA-mPEG blocks directionally distributed in the PVA solution for emulsion. And it was solidified after removing the oil phase at suitable rate. The R7 would expose and display the penetrate function once the depolarization of PSD-Folate in the certain pH solution.23-25 The negative PSD-Folate combined with positive R7 though electrostatic force and the shielding effect was reversible in the environment of faintly acid. The size of particles and the stability in solution made it possible to deliver the slightly solubility drugs to certain positions. And the optimized nanoparticles were able to package the drugs effectively and avoid the leakage.

R7/PSD-Fol NPs contained both DTX and GDC0941. When the encapsulation efficiency of DTX and GDC0941 was increased, the drug loading was reduced. Keeping the other conditions unchanged, with the amount of GDC0941 reduced, the drug loading and entrapment rate of DTX has a certain increase. The amount of DTX and GDC0941 were guaranteed to be 2 mg, 1 mg for the half inhibitory concentration (IC50 = 23



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479 nM or 0.291 mg / mL) requirements, based on the above results, select the amount of DTX and GDC0941 were 2 mg, 1 mg.

Figure 5. Properties of the nanoparticles. (A) TEM images of R7/ PSD-Fol NPs (B) the zeta potential of R7/ PSD-Fol NPs (C) the particle size and PDI of R7/ PSD-Fol NPs. Data are presented as mean ± SD (n = 3). Table 1 Physicochemical characteristics of the R7 NPs and R7/PSD-Fol NPs (Mean ± SD, n = 3) Particle Group

EE(%)

Zeta

DL(%)

PDI size(nm)

R7 NPs

potential(mV)

DTX

GDC0941

DTX

GDC0941

150.1±6.8

0.17

+32.1±1.52

90.5±6.32

87.1±9.21

1.73±0.23

0.99±0.08

R7/PSD-Fol NPs 151.8±9.5

0.16

-31.1±2.14

90.0±6.32

86.4±7.35

1.72±0.23

0.98±0.11

Table 2 Effect of drug amount single factor on R7/PSD-Fol NPs (Mean ± SD, n = 3) Particle Size Drug(mg)

EE (%)

(nm)

DTX

DL (%)

PDI DTX

GDC0941

DTX

GDC0941

2

175.1±12.4

0.175

85.76±6.88

84.89±7.67

1.79±0.21

0.84±0.08

1

151.8±9.5

0.163

90.05±6.32

86.38±7.35

1.72±0.23

0.98±0.11

0.5

143.8±7.9

0.171

92.31±7.57

87.52±7.17

1.01±0.13

1.12±0.10

24


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2

176.5±9.9

0.182

85.47±6.77

85.34±8.59

1.69±0.21

1.58±0.15

1

151.8±9.5

0.163

90.05±6.32

86.38±7.35

1.72±0.23

0.98±0.11

0.5

149.1±8.7

0.165

88.79±7.19

92.65±6.92

1.78±0.22

0.79±0.08

GDC 0941

3.3 In vitro Drug Release The DTX and GDC0941 releasing from R7/PSD-Fol NPs in two kinds of buffer in 72 h was displayed in Figure 5. The cumulative release of DTX and GDC0941in the pH 7.4 solution was 30.2% and 29.0% during the first 2 h with no burst release. And the following time points revealed a continued pattern at slower rate. No more than 77.5% of the total DTX and 75.0% of the total GDC0941 were released after 72 h. And the curves kept closely horizontal from 48 h to 72 h. On the other hand, it was noticeable that R7/PSD-Fol NPs displayed a differently relative rapid phase release behaviors were in the pH 5.0 solution. The cumulative release of DTX and GDC0941 was respectively 36.2% and 34.4 % in 2 h. After 48h, the 88.75% DTX and 82.8% GDC0941 were released. The releasing of DTX and GDC0941 reached nearly 89.3% and 85.0 % at 72 h. In pH 5.0 and pH 7.4 releasing medium, there was a statistical significance for the release of DTX and GDC0941 after 12h. According to the results, it could be concluded that the release of DTX and GDC0941 were improved in the subacidity environment. The pH-sensitive property of R7/PSD-Fol NPs was contributed by the electrostatic attraction between pH-sensitive PSD-Folate and PLGA-PEG-R726.The PSD-Folate was separated from the nanoparticles surface in pH 5.0 medium, and the electropositive R7 was exposed. This transformation lead 25



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to the nanoparticle structure unstable and accelerated the drug release. Thus, the release rate was improved in pH 5.0 medium.

Figure 6. In vitro release profile of (A) DTX and (B) GDC0941 from R7/PSD-Fol NPs in PBS (pH 7.4) and ACS (pH 5.0) at 37 ± 0.5 °C. Data are presented as mean ± SD (n = 3). * p DTX + GDC0941 >R7 NPs > R7/PSD-Fol NPs. And the order of the expression levels of Akt was reversed. The R7/PSD-Fol NPs showed the strongest inhibition of phosphorylation of Akt. These results indicated that the PI3K/Akt pathway was inhibited effectively by the R7/PSD-Fol NPs. The less p-Akt would reduce the phosphorylation of downstream substrates and induce the cell apoptosis. The co-conveying of GDC0941 and DTX using the ligand modified nanoparticles could promote the programmed dead of MCF-7/Adr cells and increase the cytotoxicity of DTX.

Figure 10. p-Akt and Akt in MCF-7/Adr cells treated with Control, DTX, DTX+GDC0941, R7 NPs, R7/PSD-Fol NPs respectively (A) and the relative expression of the targeted protein (B) p-Akt/Akt compared with control. The data were represented as mean ± SD (n = 3). * p DiR-R7/PSD-Fol NPs> DiR at 4 h, DiR-R7/PSD-Fol NPs> DiR-R7 NPs> DiR at 12 h, 24 h and 48 h. Thus, the DiR-R7/PSD-Fol NPs and DiR-R7 NPs could target the tumor site efficiently. DiR-R7/PSD-Fol NPs resided in vivo for the longest time and released the encapsulated fluorescent dyes gradually. This can be explained that the PSD-Folate helped the DiR-R7/PSD-Fol NPs escape from the reticuloendothelial system and avoid the destruction of mononuclear macrophages.34-36 And it was reported that the arginine-rich CPPs could be enriched at tumor site.37 DiR-R7/PSD-Fol NPs could target the tumor more effectively through the pH sensitive PSD-Folate.

.

3.10 In vivo antitumor effect The antitumor effect of R7/PSD-Fol NPs was decided by observing the tumor 37



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size while the whole administration period. According to the curve of tumor volume to time, the different growth of tumor was displayed in Figure 11.B. The R7/PSD-Fol NPs group appeared the slowest growth rate compared with DTX+GDC0941 and control group. The relative tumor increment rate of R7/PSD-Fol NPs and DTX+GDC0941 group were 35.6% and 62.5% respectively. The stripped tumors of each group were shown in Figure 11.D. There was not obvious difference in tumor size in the same group. And the size of tumor was the smallest in R7/PSD-Fol NPs group compared with other groups. The weight of tumor tissue among groups followed the order: control > DTX+GDC0941> R7/PSD-Fol NPs (Figure 11.E). The tumor weight of control group was 1.8 times than DTX+GDC0941 group. The DTX+GDC0941 group was 2.5 times the weight of tumor than the R7/PSD-Fol NPs group. The outcomes indicated the DTX combined with GDC0941 could inhibit tumor growth effectively. Due to the GDC0941 induced the apoptosis of drug resistant cells by blocking PI3K/Akt pathway. And the R7/PSD-Fol NPs could produce better antitumor therapeutic effect. Because the nanoparticles co-delivery the DTX and GDC0941 to tumor site efficiently. PLGA-PEG-R7 and PSD-Folate not only stabilized the NPs in the circulatory system, but also promote the NPs penetrate into targeted tumor cells. The R7/PSD-Fol NPs drug delivery system accumulated in the tumor site of nude mice through blood circulation after injection, then released the DTX and GDC0941 by acid environment stimulation. 38


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The pH-sensitive PSD-Folate enhanced the target effect of NPs at tumors site and receptor mediated endocytosis.21, 38 The results also showed agreement with the in vivo imaging. The systemic toxicity was evaluated by measuring the body weight during the treatment (Figure 11.C). The systemic toxicity occurred due to the cytotoxicity of DTX.39 The gradually increase of body weight in control group was the result of tumor weight and no drug effect. While the body weight declined, there was no significant difference between R7/PSD-Fol NPs group and DTX+GDC0941 group. Therefore, R7 /PSD-Fol NPs showed the better antitumor and lower side effect.

39



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Figure 11. (A) In vivo imaging of mice bearing MCF-7/Adr tumors which were intravenously injected with DiR solution, DiR-R7 NPs and DiR-R7/PSD-Fol NPs, respectively at predetermined time points. The arrow indicates the MCF-7/Adr tumor. (B) Tumor volume records of nude mice bearing MCF-7/Adr tumors 40


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treated regularly with Control, DTX+GDC0941 and R7/PSD-Fol NPs respectively. (C) Body weight changes of nude mice bearing MCF-7/Adr tumors. (D) The peeled tumors from of the relevant experimental groups. (E) Tumor weight at the end of the experiment. Each value represents the mean ± SD (n=5). *p < 0.05, **p < 0.01 versus the control group.

4. Conclusion In the end, the copolymers of pH-sensitive PSD-Folate and PLGA-PEG-R7 to compose the R7/PSD-Fol NPs were successfully synthesized. The results of in vitro and in vivo evaluation provided the proof that multifunctional nanoparticles could target to tumor model precisely and inhibit the tumor growth. The enhanced endocytosis and intracellular drug release trigged by acid microenvironment accelerated the apoptosis of MCF-7/Adr cells. The decreased expression of p-Akt and the increased expression of Akt induced by the R7/PSD-Fol NPs indicated the inhibition of PI3K/Akt pathway to promote apoptosis and reverse multidrug resistant. It could be concluded that R7/PSD-Fol NPs emerged as effective drug carrier to play a multifunctional role in inhibiting tumor growth and enhancing therapeutic efficacy.

AUTHOR INFORMATION Corresponding Author *Tel: +86-21-34204049. Fax: +86-21-34204049. 41



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E-mail: [email protected].

Notes The authors declare no competing financial interest.

REFERENCES

1. Zhao, S.; Tan, S.; Guo, Y.; Huang, J.; Chu, M.; Liu, H.; Zhang, Z. pH-sensitive docetaxel-loaded d-α-tocopheryl polyethylene glycol succinate–poly (β-amino ester) copolymer nanoparticles for overcoming multidrug resistance. Biomacromolecules 2013, 14, (8), 2636-2646. 2. Roy, A.; Murakami, M.; Ernsting, M. J.; Hoang, B.; Undzys, E.; Li, S.-D. Carboxymethylcellulose-based and docetaxel-loaded nanoparticles circumvent P-glycoprotein-mediated multidrug resistance. Mol.pharmaceutics 2014, 11, (8), 2592-2599. 3. Liang, D.; Wang, A.-t.; Yang, Z.-z.; Liu, Y.-j.; Qi, X.-r. Enhance cancer cell recognition and overcome drug resistance using hyaluronic acid and α-tocopheryl succinate based multifunctional nanoparticles. Mol.pharmaceutics 2015, 12, (6), 2189-2202. 4. Soubeyran, I.; Quenel, N.; Mauriac, L.; Durand, M.; Bonichon, F.; Coindre, J. M. Variation of hormonal receptor, pS2, c-erbB-2 and GSTπ contents in breast carcinomas under tamoxifen: a study of 74 cases. Br.J Cancer 1996, 73, (6), 735-743. 5. Mack, L.; Kerkvliet, N.; D oig, G.; O'Malley, F. P. Relationship of a new histological categorization of ductal carcinoma in situ of the breast with size and the immunohistochemical expression of p53, c-erb B2, bcl-2, and ki-67. Hum. Pathlogy 1997, 28, (8), 974-9. 6. Shaw, R. J.; Cantley, L. C. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 2006, 441, (7092), 424-430. 7. Duguay, D.; deBlois, D. Differential regulation of Akt, caspases and MAP kinases underlies smooth muscle cell apoptosis during aortic remodelling in SHR treated with amlodipine. Br. J. Pharmacol 2007, 151, (8), 1315-23. 42


Page 42 of 47

Page 43 of 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8. Henshall, D. C.; Araki, T.; Schindler, C. K.; Lan, J. Q.; Tiekoter, K. L.; Taki, W.; Simon, R. P. Activation of Bcl-2-associated death protein and counter-response of Akt within cell populations during seizure-induced neuronal death. J.Neurosci 2002, 22, (19), 8458-65. 9. Babchia, N.; Calipel, A.; Mouriaux, F.; Faussat, A. M.; Mascarelli, F. The PI3K/Akt and mTOR/P70S6K signaling pathways in human uveal melanoma cells: interaction with B-Raf/ERK. Invest. Ophthalmol. Visual Sci 2010, 51, (1), 421-9. 10. D’Angelo, N. D.; Kim, T.-S.; Andrews, K.; Booker, S. K.; Caenepeel, S.; Chen, K.; D’Amico, D.; Freeman, D.; Jiang, J.; Liu, L. Discovery and optimization of a series of benzothiazole phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitors. J. Med. Chem 2011, 54, (6), 1789-1811. 11. Das, M.; Mohanty, C.; Sahoo, S. K. Ligand-based targeted therapy for cancer tissue. Expert Opin. Drug Delivery 2009, 6, (3), 285-304. 12. Purtov, K.; Petunin, A.; Burov, A.; Puzyr, A.; Bondar, V. Nanodiamonds as carriers for address delivery of biologically active substances. Nanoscale Res. Lett. 2010, 5, (3), 631. 13. Poon, Z.; Lee, J. A.; Huang, S.; Prevost, R. J.; Hammond, P. T. Highly stable, ligand-clustered “patchy” micelle nanocarriers for systemic tumor targeting. Mol. Biol. Nanomed. 2011, 7, (2), 201-209. 14. Lindgren, M.; Hällbrink, M.; Prochiantz, A.; Langel, Ü. Cell-penetrating peptides. Trends Pharmacol. Sci. 2000, 21, (3), 99-103. 15. Erazooliveras, A.; Muthukrishnan, N.; Baker, R.; Wang, T. Y.; Pellois, J. P. Improving the endosomal escape of cell-penetrating peptides and their cargos: strategies and challenges. Pharmaceuticals 2012, 5, (11), 1177-1209. 16. Salazar, M. D. A.; Ratnam, M. The folate receptor: what does it promise in tissue-targeted therapeutics? Cancer Metastasis Rev. 2007, 26, (1), 141-152. 17. Han, S. K.; Na, K.; Bae, Y. H. Sulfonamide based pH-sensitive polymeric micelles: physicochemical characteristics and pH-dependent aggregation. 43



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Colloids and Surfaces 2003, 214, (1), 49-59. 18. Sun, L.; Guo, C.; Liu, D.; Zhao, Y.; Zhang, Y.; Song, Z.; Han, H.; Chen, D.; Zhao, Y. PROTECTIVE EFFECTS OF BONE MORPHOGENETIC PROTEIN 7 AGAINST AMYLOID-BETA INDUCED NEUROTOXICITY IN PC12 CELLS. Neuroscience 2011, 184, 151-163. 19. Duan, J. H.; Mansour, H. M.; Zhang, Y. D.; Deng, X. M.; Chen, Y. X.; Wang, J. W.; Pan, Y. F.; Zhao, J. F. Reversion of multidrug resistance by co-encapsulation of doxorubicin and curcumin in chitosan/poly(butyl cyanoacrylate) nanoparticles. Int. J. Pharm. 2012, 426, (1-2), 193-201. 20. Bersani, S.; Vila-Caballer, M.; Brazzale, C.; Barattin, M.; Salmaso, S. pH-sensitive stearoyl-PEG-poly (methacryloyl sulfadimethoxine) decorated liposomes for the delivery of gemcitabine to cancer cells. Eur. J. Pharm. Biopharm. 2014, 88, (3), 670-682. 21. Yoo, H. S.; Park, T. G. Folate-receptor-targeted delivery of doxorubicin nano-aggregates stabilized by doxorubicin-PEG-folate conjugate. J.Controlled Release 2004, 100, (2), 247-256. 22.Chen, S. Z.; Yang, K. N.; Tuguntaev, R. G.; Mozhi, A.; Zhang, J. C.; Wang, P. C.; Liang, X. J. Targeting tumor microenvironment with PEG-based amphiphilic nanoparticles to overcome chemoresistance. Biotechnol., Mol. Biol. Nanomed. 2016, 12, (2), 269-286. 23. Clawson, C.; Ton, L.; Aryal, S.; Fu, V.; Esener, S.; Zhang, L. F. Synthesis and Characterization of Lipid-Polymer Hybrid Nanoparticles with pH-Triggered Poly(ethylene glycol) Shedding. Langmuir 2011, 27, (17), 10556-10561. 24.Chen, W.; Zhong, P.; Meng, F. H.; Cheng, R.; Deng, C.; Feijen, J.; Zhong, Z. Y. Redox and pH-responsive degradable micelles for dually activated intracellular anticancer drug release. J. Controlled Release 2013, 169, (3), 171-179. 25. Sethuraman, V. A.; Bae, Y. H. TAT peptide-based micelle system for potential active targeting of anti-cancer agents to acidic solid tumors. J. Controlled Release 2007, 118, (2), 216-224. 26. Chen, J.; Li, S.; Shen, Q. Folic acid and cell-penetrating peptide 44


Page 44 of 47

Page 45 of 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


conjugated PLGA–PEG bifunctional nanoparticles for vincristine sulfate delivery. Eur. J. Pharm. Sci 2012, 47, (2), 430-443. 27. Jain, S.; Mathur, R.; Das, M.; Swarnakar, N. K.; Mishra, A. K. Synthesis, pharmacoscintigraphic evaluation and antitumor efficacy of methotrexate-loaded, folate-conjugated, stealth albumin nanoparticles. Nanomedicine 2011, 6, (10), 1733-1754. 28. Shishido, T.; Yonezawa, D.; Iwata, K.; Tanaka, T.; Ogino, C.; Fukuda, H.; Kondo, A. Construction of arginine-rich peptide displaying bionanocapsules. Med. Chem. Lett. 2009, 19, (5), 1473-1476. 29. Nakase, I.; Akita, H.; Kogure, K.; Graslund, A.; Langel, U.; Harashima, H.; Futaki, S. Efficient Intracellular Delivery of Nucleic Acid Pharmaceuticals Using Cell-Penetrating Peptides. Acc. Chem. Res. 2012, 45, (7), 1132-1139. 30. Hamberg, P.; Verweij, J.; Seynaeve, C. Cytotoxic therapy for the elderly with metastatic breast cancer: a review on safety, pharmacokinetics and efficacy. European Journal of Cancer 2007, 43, (10), 1514-1528. 31. Tazzari, P.; Cappellini, A.; Ricci, F.; Evangelisti, C.; Papa, V.; Grafone, T.; Martinelli, G.; Conte, R.; Cocco, L.; McCubrey, J. Multidrug resistance-associated protein 1 expression is under the control of the phosphoinositide 3 kinase/Akt signal transduction network in human acute myelogenous leukemia blasts. Leukemia 2007, 21, (3), 427-438. 32. Baracca, A.; Sgarbi, G.; Solaini, G.; Lenaz, G. Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F-0 during ATP synthesis. Biochim. Biophys. Acta, Bioenerg. 2003, 1606, (1-3), 137-146. 33.Han, Z.; Hong, L.; Han, Y.; Wu, K.; Han, S.; Shen, H.; Li, C.; Yao, L.; Qiao, T.; Fan, D. Phospho Akt mediates multidrug resistance of gastric cancer cells through regulation of P-gp, Bcl-2 and Bax. J. Exp. Clin. Cancer Res.CR 2007, 26, (2), 261-268. 34. Xu, Z.; Chen, L.; Gu, W.; Gao, Y.; Lin, L.; Zhang, Z.; Xi, Y.; Li, Y. The performance of docetaxel-loaded solid lipid nanoparticles targeted to hepatocellular carcinoma. Biomaterials 2009, 30, (2), 226-232. 35. Maeda, H.; Nakamura, H.; Fang, J. The EPR effect for macromolecular 45



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Adv. Drug Delivery Rev 2013, 65, (1), 71-79. 36. Liu, H.; Liao, G.; Yan, Z. Gynecomastia during imatinib mesylate treatment for gastrointestinal stromal tumor: a rare adverse event. BMC gastroenterology 2011, 11, (1), 1. 37. Nakase, I.; Konishi, Y.; Ueda, M.; Saji, H.; Futaki, S. Accumulation of arginine-rich cell-penetrating peptides in tumors and the potential for anticancer drug delivery in vivo. J. controlled release 2012, 159, (2), 181-188. 38. Yoo, H. S.; Park, T. G. Folate receptor targeted biodegradable polymeric doxorubicin micelles. J. controlled release 2004, 96, (2), 273-283. 39. Gao, W.; Xiang, B.; Meng, T. T.; Liu, F.; Qi, X. R. Chemotherapeutic drug delivery to cancer cells using a combination of folate targeting and tumor microenvironment-sensitive polypeptides. Biomaterials 2013, 34, (16), 4137-4149.

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