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Folate-Targeted pH and Redox Dual StimulationResponsive Nanocarrier for Co-Delivering of Docetaxel and TFPI-2 for Nasopharyngeal Carcinoma Therapy Tao Liu, Shaohua Chen, Xidong Wu, Hong Han, Siyi Zhang, Peina Wu, Xiaomei Su, Ting Wu, Shaobin Yu, and Xiang Cai ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00675 • Publication Date (Web): 29 Apr 2019 Downloaded from http://pubs.acs.org on May 1, 2019

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ACS Applied Bio Materials

Folate-targeted pH and Redox Dual Stimulation-responsive Nanocarrier for Co-Delivering of Docetaxel and TFPI-2 for Nasopharyngeal Carcinoma Therapy Tao Liu*, Shaohua Chen, Xidong Wu, Hong Han, Siyi Zhang, Peina Wu, Xiaomei Su, Ting Wu, Shaobin Yu, Xiang Cai*

T. Liu, Prof. S. Chen, H. Han, S. Zhang, P. Wu, X. Su Department of Otolaryngology-Head and Neck Surgery Guangdong Provincial People's Hospital Guangdong Academy of Medical Sciences No. 106, Zhongshan Second Road, 510080, Guangzhou, P.R. China E-mail: [email protected] X. Wu Department of Pharmacology Jiangxi Testing Center of Medical Instruments No. 181, Nanjing East Road, 330029, Nanchang, P. R. China T. Wu, Prof. X. Cai Department of Light chemical engineering Guangdong Polytechnic No.20, Lanshi 2th Road, Chancheng District, Foshan, P.R. China Prof. S. Yu The no.1 surgery department of no.5 people's hospital of foshan No. 63, xiqiao zhen jiang pu dong road, nanhai district, Foshan 528211, Guangdong province, P.R.China.

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Abstract: Due to the increasing incidence of tumor metastasis and multidrug resistance, even though a combined use of chemotherapy and radiotherapy is introduced, the 5-year’s average survival rate of advanced nasopharyngeal carcinoma (NPC) patient still remains low. Hence, targeted slow-release anti-cancer drugs represent a potential therapy for advanced NPC. In this study, pH and redox dual stimulation-responsive folate-targeted FA-DS-PAAs nanocarriers for co-delivery of docetaxel (DOC) and TFPI-2 for NPC therapy are discussed. Physical and chemical properties, in vitro DOC-release properties, FA-targeting, transfection, Western blotting, DOC and TFPI-2 codelivery, therapeutic properties, targeted inhibition, and biocompatibility, in vivo FA-targeting, toxicity, and therapeutic properties of FA-DS-PAAs/DOC/TFPI2 nanoparticles are evaluated. The results indicate that the 200-nm low-toxicity FA-DS-PAAs/DOC/TFPI2 nanoparticles could enhance TFPI2 gene expression, make cancer cells more sensitive to DOC, induce cell apoptosis, and reduce cell invasion more effectively compared with mono-chemotherapy. With respect to the targeted release of drugs (DOC and TFPI2) in tumor cells, FA-DS-PAAs/DOC/TFPI2 is associated with the slowest growth rate of tumor and the smallest volume of tumor, so this study demonstrates the best synergetic anti-tumor effect. We anticipate that this study is important because it not only provides a potential new therapy approach for NPC

but

also

paves

the

preclinical

way

for

FA-DS-PAAs/DOC/TFPI2. 2 / 38


potential

application

of

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Keywords: nasopharyngeal carcinoma; nanocarrier; dual stimulate-responsive; target; anti-cancer

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1. Introduction In southern China, one of the most common malignant tumors is nasopharyngeal carcinoma (NPC).1 Primary clinical strategy for NPC is chemotherapy combined with radiotherapy. However, due to multidrug resistance of NPC and NPC tumor metastasis, the 5-year’s average survival rate of advanced NPC patient still remains low.2 A targeted codelivery system is a potential choice for NPC patients because it can enhance the anti-tumor effect and reduce drug resistance. Studies have demonstrated that tumor cells (including NPC HNE-1 cells) have high folate receptors (FRs) expression, whereas normal tissues show low FRs expression.3-5 Given the strong binding ability between folate and FRs, nanoparticle modified with folic acid can be internalized by tumor cells more easily.6,7 In most cancers, 6.0–7.0 intracellular pH values and 4.5–5.5 endosomal pH values are lower than 7.4, which are the extracellular pH values.8 This characterization is useful for designing bioresponsive drug carrier to obtain better tumor targeting therapeutic system. So, in previous studies, hydrazone bond is abundant used as drug-carrier conjugate. Because the hydrazone bond is easily cleavable at endosomal environment and exhibits sufficient stability at intracellular environment.9,10 The acid in tumor cell environment will acid splitting the acid-liable hydrazone bond on the tumor targeting therapeutic system, resulting in the rapid targeting drug release in tumor cells.11,12

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On the other hand, with high intracellular glutathione (GSH) concentration, the intracellular space free cysteine (or homocysteine) and oxidizing extracellular environment have differences redox state. This redox state is useful in designing nanosystem for targeted drug delivery in cells.13 In other words, intravenous tumor therapeutic system with disulfide linkage is rapidly degraded in the cytosol but is stable in the bloodstream,14 since the intracellular GSH contents are 2- to 3-fold higher

than

the

extracellular.15

Thus,

disulfide

linkage

contained

(like

N,N’-cystaminebisacrylamide (CBA)) intravenous tumor therapeutic system can potentially provide increase release of drug in cytoplasm. The preparation of sensitive drug vectors based on increased GSH concentration and acid environment in tumor tissues and cells will be advantageous for cancer therapy.

Leamon

constructed

a

folate

conjugate

of

desacetylvinblastine

monohydrazide with a pH sensitive acylhydrazone bond (EC140),16 and Vlahov prepared a novel folate conjugate of desacetylvinblastine monohydrazide containing the redox sensitive disulfide bond (EC145).17-18 EC140 and EC145 both exhibited good molecular targeted anti-tumor effects. EC145 has better tolerated and anti-tumor active than EC140. This finding demonstrates that a disulfide bond is more beneficial to the activity compared with a hydrazine bond. By coupling 7-N-modified mitomycin to folic acid-gamma-cysteine, EC72 is produced through a disulfide bond, demonstrating a good molecular targeted anti-tumor effect.19 However, similar to EC72, Reddy constructed EC110 has not anti-tumor activity, since a more resilient 5 / 38



amide bond EC110 has a folate-mitomycin conjugate constructed without disulfide bond. This finding demonstrates that a cleavable disulfide bond has a great influence on the biological activity of nanomedicines that can effectively release drugs.20 To further investigate the impact of disulfide bonds and pH-sensitive bonds on antitumor activity, a molecule named EC118 (another folate-mitomycin conjugate), composed with an acid-labile hydrazone bond and a reducible disulfide bond, produced significantly increased tumor regression compared with that achieved with EC72. This finding indicates that the rupturing of a greater number of bonds increased the amount of drug released, and induced stronger anti-tumor effects.20 Pérez21 and Lin22 also prepared nanomedicines with a pH and redox dual stimulate-responsive ability, that exhibited good anti-tumor effects. As a serine protease inhibitor, TFPI2 can effectively inhibit the matrix metalloproteinases (MMPs) proteolytic activity, and induce tumor metastasis and invasion.23-25 In the taxoid family, docetaxel (DOC) used as an anticancer agent, promotes tubulin assembly and inhibits tubulin depolymerization. In cancer chemotherapy, DOC can also inhibit tumor growth and induce tumor cell apoptosis, showing a strong anti-tumor effect.26,27 In our previous work, β-CD and L-lysine arms were used to construct a biocompatible CD derivative carrier to codeliver plasmids (MMP-9 siRNA) and DOC for NPC treating, but the gene transfection efficiency of the carrier was not high.28 By using poly(amido amine)s (PAAs), Ma constructed a high biocompatibility 6 / 38


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FA-targeted redox hyperbranched nanocarrier with redox stimulation-responsive ability, and this nanocarrier exhibited had a high gene transfection efficiency in serum, similar to polyetherimide (PEI).29 Based on these studies, in this work, FA-targeted pH and redox dual stimulation-responsive nanocarriers were constructed within the framework of PAAs and β-CD (FA-DS-PAAs) to co-deliver plasmid (TFPI-2) and DOC, and the synergetic anti-advanced NPC effect was performed.

2. Experimental Section 2.2.Synthesis of FA-DS-PAAs Figure 1 displayed the synthesis routes of FA-DS-PAAs. Step 1, carboxylated β-CD: 2 mmol β-CD was added into 10 mL 30% NaOH solution with stirring to dissolve completely. Subsequently, 3 mmol monochloroacetic acid was added and dissolved into 5 mL water, and then the mixed solution was added into the β-CD solution with dropwise. The mixture would react in the dark for four hours. Subsequently, the mixture phase is adjusted with hydrochloric acid to a pH of 5 to 6, and then extracted by methanol and washed by distilled water. Next, by using vacuum drying equipment to remove the organic solvent, carboxylated β-CD was acquired with 91% yield. Step 2, hydrazide-containing β-CD: At room temperature, NHS (1 mmol) and carboxylated β-CD (1 mmol) were dissolved in 10 mL DMF, and reacted for 5 7 / 38



minutes with stirring. Then, in an ice bath, EDC-HCl (1 mmol) was added. Next, 5% hydrazine hydrate solution (10 mL) was added into the mixture and reacted for 24 h. After dialysis and freeze drying, the hydrazide-containing β-CD (CD-CONHNH2) was acquired with 88% yield. Step 3, hyperbranched PAAs: According to previously reported Michael addition polymerization method (one-pot, two-step), bioreducible hyperbranched PAAs were synthesized by AEPZ and CBA.29 Under a nitrogen atmosphere, 0.75 mM AEPZ was added dropwise into the 10 mL CBA (1.5 mM) methanol/water (v/v = 3/1, containing 200 mM calcium chloride) solution with stirring, and then reacted for 30 h at 50°C. Next, the second batch of AEPZ (1.5 mmol) was added dropwise under a nitrogen atmosphere, and reacted for an additional 8 h. After rotatory evaporation removed solvent, the residue was shifted to dialysis bag (MWCO = 3500, USA), and dialyzed for 3 day in distilled water. Next, by using vacuum drying equipment to remove the water, bioreducible hyperbranched PAAs was acquired with 62% yield. Step 4, DS-PAAs: DS-PAAs were prepared according to Michael addition reaction of PAAs and CD-CONHNH2 coupled with CBA. Briefly, 1 mol CD-CONHNH2 and 10 mol CBA were added into the PAAs THF solution (0.1 g/mL), and then reacted for 24 h at 50°C. After rotatory evaporation removed solvent, the residue was shifted to dialysis bag (MWCO = 3000, USA), and dialyzed for 3 day in distilled water. Next, by using vacuum drying equipment to remove the water, DS-PAAs were acquired with 56% yield. 8 / 38


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Step 5, FA-DS-PAAs: 7 mL DMSO was used to dissolve 10 mg FA, and then 10 mg NHS and 20 mg DCC were added. After reacted for 0.5 h, 100 mg DS-PAAs (dissolved in 3 mL DMSO) was dropwise added under stirring. Then, the reaction was introduced in the dark for 24 h at room temperature. After rotatory evaporation removed solvent, the residue was shifted to dialysis bag (MWCO = 5000, USA), and dialyzed for 3 day in distilled water. Next, by using vacuum drying equipment to remove the water, FA-DS-PAAs were acquired with 86% yield. By using a 300 MHz NMR spectrometer (Bruker DPX-300), 1H NMR and 13C NMR were characterized at 25°C to measure the chemical structure of FA-DS-PAAs in DMSO-d6. 2.3.DOC loading For hydrophobic DOC loading, FA-DS-PAAs (50 mg) was first dissolved in water (5 mL), and then 15 mg DOC (dissolved in 5 mL DMF) was added dropwise. Then, reaction was introduced in the dark for 4 h at room temperature. Next, the mixture was shifted to dialysis bag (MWCO = 500, USA), dialyzed for 24 h in distilled water, filtered through a filter (0.45-mm), and lyophilized to obtain FA-DS-PAAs/DOC. The loading amount of DOC in FA-DS-PAAs/DOC was detected by HPLC analysis with a Waters C18 column. The solvent of FA-DS-PAAs/DOC was CH3OH, the mobile phase was purity water and methanol (30/70, v/v), the flow rate was 1.0 mL/min, and the quantified wavelength was monitored at 227 nm with the standard curve (compare the peak areas of DOC).30,31 After calculation, the loading amount of DOC was 19.8 μg/mg. 9 / 38



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2.4.TFPI2 plasmid binding For the complex preparation of FA-DS-PAAs with TFPI-2, an appropriate amount of TFPI-2 and FA-DS-PAAs were first dissolved in the water, and then mixed and gently stirred quarter-hours to obtain FA-DS-PAAs/TFPI2 complexes. Gel electrophoresis was used to confirm the binding ability between TFPI-2 and FA-DS-PAAs. 1.0% agarose gel (w/v) was prepared by using 40 mmol/L trisacetate buffer with 0.25 mg/mL ethidium bromide and 1 mmol/L EDTA. After incubating at room temperature for quarter-hours, all test samples were performed at 70 V by agarose gel electrophoresis for 0.5 h. By using a UV-transilluminator (Fisher Scientific, PA) under a digital imaging suite (Kodak EDAS 290), the image capturing of the sample was accomplished. 2.5.FA-DS-PAAs/DOC/TFPI2 formation, size, morphology, and GSH-response analysis In order to prepare FA-DS-PAAs/DOC/TFPI2, a required concentration of FA-DS-PAAs and DOC were first used to prepare FA-DS-PAAs/DOC micelles. Next, a required concentration of TFPI2 plasmid was added and stirred gently to obtain the DOC and TFPI2-coloaded FA-DS-PAAs/DOC/TFPI2 complexes. ZetaPALS Zeta Potential Analyzer (Brookhaven Instruments Corporation, USA) was used to determine the zeta-potentials and particle sizes of the complexes (diluted with 0.9 wt.%

NaCl

solution).

JEM-2010HR

transmission

electron

microscope

(high-resolution, JEOL, JAPAN ) was used to determine the morphological of the 10 / 38


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complexes (counterstaining with uranyl acetate). By using PEG as the standard, gel permeation chromatography (GPC, 0.8 mol L-1 aqueous sodium nitrate as the eluent, Model 270 RI detector) was used to detect the GSH-response (in 10 or 100 μM GSH aqueous solutions, 2 mg FA-DS-PAAs was used and incubated for 4 h) with a VE 1122 HPLC pump.

3. Result and Discussion 3.1. Synthesis and characterization

Figure 1. Preparation steps for FA-DS-PAAs/DOC/TFPI-2. Preparation of (1) carboxylated β-CD, (2) hydrazide-containing β-CD, (3) hyperbranched PAAs, (4) DS-PAAs, (5) FA-DS-PAAs, and (6) FA-DS-PAAs/DOC/TFPI-2.

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In Figure 1, the preparation steps for FA-DS-PAAs are presented, and the 1H NMR spectrum results of β-CD-CONHNH2 and PAAs are presented in Figure S1 and S2. Moreover,

13C

NMR is further used to confirm that FA-DS-PAAs are obtained

successfully (Figure 2).

Figure 2. 13C NMR spectrum and molecular structure diagram of FA-DS-PAAs (DMSO-d6, 25 ˚C).

In Figure 2, the main signals from FA-DS-PAAs are obviously noted. The peak for C from the amido bond is presented at 175 ppm.28,32 The vinyl groups typical peaks (123–133 ppm) for C from CBA disappear in both cases,15 indicating complete end-capping of DS-PAAs through the Michael addition reaction. All other typical PAAs peaks are well-identified in the spectrum.15,28,32 The new peaks (161 and 165 ppm) for C from FA are found, indicating that the FA-conjugated DS-PAAs were obtained successfully.

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Figure 3. (A) UV-Vis spectra of FA, FA-DS-PAAs and DS-PAAs; (B) Particle size distribution of PAAs and FA-DS-PAAs; (C) Zeta potential distribution of PAAs and FA-DS-PAAs; (D) SEM image of FA-DS-PAAs/DOC/TFPI2; (E) GPC chromatograms of different GSH concentrations treated FA-DS-PAAs at 35 ˚C (0.8 mol·L-1 NaNO3, standard sample was dextran); (F) release behavior of drug from pH/GSH dual responsive FA-DS-PAAs/DOC/TFPI2.

To further study the FA content in FA-DS-PAAs, UV-Vis spectrum analysis is used and shown in Figure 3(A). Peaks at 288 and 365 nm are belonging to FA, but disappear in UV-Vis spectrum of DS-PAAs, which means FA-DS-PAAs are synthesized. After calculation (based on the 288 nm absorbance peak), the FA content is 14.5% in FA-DS-PAAs. For efficient gene delivery of cationic nanoparticles, proper sizes and positive charges are important. Because proper sizes (50 to several hundred nanometers, approximately) are easily uptake by cells for cationic nanoparticles. In present study, for efficient cellular endocytosis, FA-DS-PAAs is expected to form a compact DOC and TFPI2 coloaded nanoparticle with suitable diameter. As shown in Figure 3(B) 13 / 38



and 3(C), with the increased weight ratios from 0 to 30, the particle sizes of PAAs and FA-DS-PAAs firstly decrease from approximately 400 to 200 nanometers, and then remain stable in the range of approximately 200–250 nm. The zeta potentials of PAAs and FA-DS-PAAs increase from approximately -15 to 15 mV with an increase in the weight ratio (from 0 to 30). These results suggest that beyond the weight ratio of 30, FA-DS-PAAs can form compact DOC and TFPI2 coloaded nanoparticle. Furthermore, SEM observation is also used to confirm complex formation. Figure 3(D) is a SEM image of FA-DS-PAAs/DOC/TFPI2 with the weight ratio of 30. This DOC and TFPI2-coloaded nanoparticle (the size is approximately 200 nm) shows a compact spherical shape with good monodispersity. The DOC loading is determined as 19.8 μg/mg.31,32 Similar to other tumors, the concentration of GSH is considerably increased in NPC cells and tissues compared with normal cells and tissues (Figure S3). In present GSH-response study, GPC chromatograms of different GSH concentrations treated FA-DS-PAAs are shown in Figure 3(E). Without GSH incubation, a maximum peak belonging to FA-DS-PAAs exhibits at 8.95 min (Mn/Mw = 1.69, Mn = 31561). With the use of 100 μmol L-1 GSH (incubated for 4 h), the disulfide bonds of FA-DS-PAAs cause an obvious breakage and degradation, and the Mn of FA-DS-PAAs is 2217 (peak shifts to 10.78 min). With the use of 100 mmol L-1 GSH (incubated for 4 h), the Mn of FA-DS-PAAs is 693 (peak further shifts to 10.78 min), suggesting the further degradation of FA-DS-PAAs. Moreover, no obvious difference in the maximum peak 14 / 38


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position is noted upon treatment with 0.1 mmol L-1 and 0.1 mol L-1 GSH. This results indicated that the 100 μmol·L-1 GSH is sufficient for FA-DS-PAA degradation, since the intracellular GSH concentration is mmol level.33 FA-DS-PAAs could bind TFPI2 through the electrostatic interacts in aqueous solutions due to the cationic property and subsequently form the FA-DS-PAAs/TFPI2 complexes. Electrophoretic mobility is used to examine the bind ability of TFPI2 on FA-DS-PAAs. In Figure S4, with high weight ratios (greater than 30), FA-DS-PAAs could bind TFPI2 so tightly that the TFPI2 could not be observed; this is unfavorable for gene release. When the weight ratio is 30, the FA-DS-PAAs could completely impede the electrophoretic mobility of TFPI2, and TFPI2 could be observed. These results indicate a suitable binding ability to TFPI2 in favor of gene release. At the low weight ratios (less than 30), migratory TFPI2 can be observed, since TFPI2 can not bind into FA-DS-PAAs completely. The results indicate that FA-DS-PAAs can bind the hydrophobic drug and gene. This finding highlights their potential to be applied for drug and gene codelivery. 3.2. DOC release in vitro In order to study the GSH and pH dual-responsion of FA-DS-PAAs/DOC/TFPI2 nanoparticles, in vitro release experiments are conducted in 0.15 M NaCl solutions at 37 °C with different pH and GSH concentrations, and the results are present in Figure 3(F). For pH response, a pH of 7.4 or 5.0 is employed. For GSH responses, a GSH concentration of 2 mM or 10mM is employed. First, in pH 7.4 buffer, given that the 15 / 38



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DOCs are encapsulated into the β-CD core of FA-DS-PAAs nanocarriers to form stable inclusions, over a 24-h period, the cumulative DOC release is less than 4% from the FA-DS-PAAs/DOC/TFPI2 nanoparticles, even when the GSH concentration increases to 10 mM. Second, the DOC release from FA-DS-PAAs/DOC/TFPI2 nanoparticles changes markedly in pH 5.0 buffer and is fast for the first 6 h. These results indicate the hydrolytic cleavage of the hydrazone bonds of the FA-DS-PAAs nanocarriers in lower pH environment, resulting the marked release of DOC from FA-DS-PAAs/DOC/TFPI2 nanoparticles. Third, in pH 5.0 buffer, over 24-h period, cumulative DOC release from FA-DS-PAAs/DOC/TFPI2 nanoparticles is ~46% with 2 mM GSH and ~60% with 2 mM GSH. These findings suggest that with the increase of

GSH

concentration,

the

DOC

release

from

FA-DS-PAAs/DOC/TFPI2

nanoparticles is increased. Because incubation with a high GSH concentration solution destabilizes the β-CD of FA-DS-PAAs, which leads to the shedding of macromolecules from the FA-DS-PAAs/DOC/TFPI2 nanoparticles.34 So, the dual-responsive FA-DS-PAAs/DOC/TFPI2 nanoparticles have the targeted release DOC ability in tumor cells. 3.3. FA-targeting assay in vitro

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Figure 4. (A) FA-targeting assay in vitro (image 400×); (B) in vitro gene transfection assay with serum (image 400×); (C) in vitro gene transfection assay without serum (image 400×); (D) Western-blot in vitro gene transfection without serum; (E) Western-blot in vitro gene transfection with serum

Regarding the strong binding ability between FA and FRs, FA-DS-PAAs/DNA complexes are expected to be internalized by HNE-1 cells more easily. In Figure 4(A), with no FR expression, the gene transfection efficiency is lower for CNE-2 cells, and approximately 6% cells are transfected. However, with high FR expression, the gene transfection efficiency considerably increases in HNE-1 cells compared with CNE-2 cells given the molecular targeting phagocytosis effect, and the high amount of FRs on tumor cells obviously increased the endocytosis of FA-DS-PAAs/DNA. After HNE-1 cells are pretreated with FA, the gene transfection efficiency of FA-DS-PAAs/DNA is reduced to less than 10%, because on HNE-1 cells, many FRs are occupied by free FA. The interactions between HNE-1 cells and 17 / 38



FA-DS-PAAs/DNA are affected by the occupation and the subsequently reduced gene uptake and transfection efficiency. 3.4. In vitro transfection In Figure S3, in the absence or the presence of serum, with weight ratio of 30, gene transfection assay is used to evaluate the gene transfection effect of the FA-DS-PAAs/TFPI2 complex in vitro. Since PEI-25k has the highest transfection efficiency, the PEI-25k/TFPI2 complex with N/P ratio of 10 is used as contrast sample.35 In Figure 4(C), for HNE-1 cells, FA-DS-PAAs (weight ratio of 30) and PEI both exhibit good TFPI2-delivering ability with approximately 42% of HNE-1 cells are transfected, and no significant difference is noted between FA-DS-PAAs and PEI. In fact, the cationic complex carriers can interact with proteins in the serum and result in poor transfection efficiency, so in vitro transfection assay of the cationic complex carriers is also performed in the absence of serum.36 As shown in Figure 4(B), HNE-1 cells are transfected only approximately 6% by the PEI/TFPI2 complex in the presence of serum, whereas FA-DS-PAAs transfected approximately 26% of the HNE-1 cells, which is considerably increased compared with of PEI and DS-PAAs. In the presence of serum, FA-DS-PAAs may exhibit good gene delivery ability because FA-DS-PAAs have the ability to target HNE-1 cells and increase cellular uptake. Meanwhile, the zeta potential of FA-DS-PAAs is reduced by FA modification, which weakens the interaction between the proteins and the FA-DS-PAAs. 18 / 38


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3.5. Western blot analysis Western blot analysis is used to evaluate the transfection effect of the complexes into HNE-1 cells at a weight ratio of 30 in HNE-1 cells. As shown in Figure 4(D), compared with the blank FA-DS-PAAs group, PEI mediates an obvious increase in TFPI2 protein in serum-free RPMI-1640, which is slightly increased compared with TFPI2 expression in the FA-DS-PAAs/TFPI2 group. However, no significant difference is noted between both groups. As shown in Figure 4(E), in the presence of serum, PEI cannot mediate the obvious increase of TFPI2 protein expression, which is in contrast to that noted for the FA-DS-PAAs. FA-DS-PAAs/TFPI2 could result in high expression of TFPI2 protein compared with the PEI/TFPI2 group. These results agree with above results, and TFPI2 can effectively deliver into HNE-1 cells by FA-DS-PAAs. 3.6. FA-targeting assay in vivo

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Figure 5. (A) DOC concentrations in tissues after injection at different time with FA-DS-PAAs/DOC/TFPI2; (B) GFP expression in tissues 24 h after injection with FA-DS-PAAs/DOC/TFPI2 (400×); (C) Imaging in vivo 24 h after injection with FA-DS-PAAs/DOC/TFPI2 Both HNE-1 and CNE-2 tumors bearing nude mice are used to evaluate the targeting delivery property of FA-DS-PAAs/DOC/TFPI2. The regression equations between X (DOC concentration) and Y (peak area) in various tissues are as follows: liver (Y=2693.0X-846.12), lung (Y=2581.3X-172.32), kidney (Y=2655.5X+1238.8), spleen (Y=2470.4X+266.92), heart (Y=2608.7X-701.39), brain (Y=2755.0X+1459.2), CNE-2 tumor (Y=2496.8X-272.89), and HNE-1 tumor (Y=2474.3X+799.96). According to these equations, DOC concentrations in tissues were calculated based on intravenous injection (5, 30, and 60 min) with FA-DS-PAAs/DOC/TFPI2 complexes. DOC concentrations analyzed by HPLC in tissues are presented in Figure 5(A). DOC 20 / 38


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is rapidly absorbed and is distributed over a wide range of tissues. In the liver and kidney, DOC has the highest concentration, and then followed by the heart, spleen and lung (P0.05). After FA-DS-PAAs/DOC/TFPI2 injection 5 min, the average DOC concentrations were both 0 μg/g in the HNE-1 and CNE-2 tumors. With prolonging the blood circulation time, DOC concentrations also increase in HNE-1 tumors (4.89 μg/g at 30 min, 4.44 μg/g at 60 min) and CNE-2 tumors (3.20 μg/g at 30 min, 6.90 μg/g at 60 min). In Figure 5(B), after intravenous injection (5, 30, and 60 min) with FA-DS-PAAs/DOC/TFPI2, GFP expression in frozen tissue sections 24 h is presented. GFP is observed in the frozen sections of these assayed tissues except the brain, and much stronger GFP expression is observed in the liver and kidney. The GFP expression in vivo is similiar as the DOC distribution in vivo. HNE-1 tumors have the stronger GFP expression than CNE-2 tumors. This finding agrees with the concentration assay of DOC. In Figure 5(C), in vivo imaging also revealed that the fluorescence area (9338) and integral optical density (IOD, 1643178.6) in HNE-1 tumors are increased compared with CNE-2 tumors (fluorescence area=6758, IOD=837514.3); these parameters were assayed using image pro plus 6.0. 3.7. Codelivery and in vitro therapy

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Figure 6. MTT analysis (A), apoptosis assay (B) by flow cytometry and invasion analysis (C) after incubated HEN-1 cells (400×) with samples (FA-DS-PAAs, FA-DS-PAAs/DOC, FA-DS-PAAs/TFPI2, and FA-DS-PAAs/DOC/TFPI2). FA-DS-PAAs (12.12μg/well), FA-DS-PAAs/DOC (FA-DS-PAAs 12.12μg/well; DOC: 0.24 μg/well, 1.2 μg/mL), FA-DS-PAAs/TFPI2 (FA-DS-PAAs 12.12μg/well; weight ratio=30, TFPI2: 0.40 μg/well, 2 μg/mL) and FA-DS-PAAs/DOC/TFPI2 (FA-DS-PAAs 12.12μg/well; DOC: 0.24 μg/well, 1.2 μg/mL; TFPI2: 0.40 μg/well, 2 μg/mL ), respectively.

Cytotoxic effects of FA-DS-PAAs/DOC/TFPI2 complexes are evaluated by MTT assay in Figure 6(A). The FA-DS-PAAs are nontoxic at the assayed concentration. After loading TFPI2 or DOC, the samples exhibit an obvious cytotoxicity. For FA-DS-PAAs/DOC/TFPI2, the viability of HNE-1 cells is further reduced. To study whether the codelivery system of FA-DS-PAAs/DOC/TFPI2 can more effectively induce cell apoptosis, the apoptosis percentage of HNE-1 cells is estimated by flow cytometry in Figure 6(B). Compared with the control group, HNE-1 cells 22 / 38


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exhibit limited apoptosis after incubation with FA-DS-PAAs for one day. It is equivalent to the observed MTT results of nontoxicity of FA-DS-PAAs. HNE-1 cells treated with FA-DS-PAAs/DOC (32% cell apoptosis), FA-DS-PAAs/TFPI2 (19% cell apoptosis) and FA-DS-PAAs/DOC/TFPI2 (44% cell apoptosis) exhibit increased apoptosis. Since TFPI2 combined with DOC enhances cell apoptosis significantly, the codelivering strategy (FA-DS-PAAs/DOC/TFPI2) is a promising cancer therapy method. To examine whether codelivery system can make HNE-1 cells less invasive, transwell

invasion

experiment

of

FA-DS-PAAs,

FA-DS-PAAs/TFPI2,

FA-DS-PAAs/DOC and FA-DS-PAAs/DOC/TFPI2 is conducted. In contrast to DOC or TFPI2 alone, Figure 6(C) demonstrates that FA-DS-PAAs/DOC/TFPI2 obviously decreased cell invasive ability. In the FA-DS-PAAs/DOC/TFPI2-treated group, the percentages of tumor cells passing through the Matrigel were 45% for TFPI2-treated cells and 59% for DOC-treated cells. The HNE-1 cells invasive ability of FA-DS-PAAs/DOC/TFPI2 significantly decreases. The results demonstrate that the codelivery system could make tumor cells more sensitive to DOC, enhance TFPI2 protein expression, reduce cell invasive ability, and induce tumor cell apoptosis more effectively compared with mono-chemotherapy, which is consistent with the results reported.32 3.8. Targeted inhibitory experiment 23 / 38



Figure 7. (A) Apoptosis assays and (B) invasion analysis of HNE-1 cells (400×) incubated with samples (FA-DS-PAAs, DS-PAAs/DOC/TFPI2 and FA-DS-PAAs/DOC/TFPI2) by targeted inhibitory experiment.

To determine whether the targeted codelivery system could more effectively induce HNE-1 cell apoptosis, after treatment with various formulations, the percentage of apoptotic HNE-1 cells was assayed by flow cytometry. In Figure 7(A), FA-DS-PAAs exhibited limited apoptosis, whereas 9% of cells treated with DS-PAAs/DOC/TFPI2 exhibited apoptosis. For the FA-DS-PAAs/DOC/TFPI2 sample, the HNE-1 cell apoptosis rate reached 17%. The result revealed that FA-DS-PAAs could induce cell apoptosis more effectively to HNE-1 cells by targeted codelivery of TFPI2 and DOC. Next, transwell invasion experiment is used to study whether the targeted codelivery system makes HNE-1 cells less invasive. As shown in Figure 7(B), after treated by FA-DS-PAAs/DOC/TFPI2, the number of HNE-1 cells is only 58% (invade through the Matrigel), while the number of HNE-1 cells is 67% for

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DS-PAAs/DOC/TFPI2-treated cells. So, FA-DS-PAAs/DOC/TFPI2 decreases the HNE-1 cells invasive ability more significantly. 3.9. Biocompatibility

Figure 8. (A) MTT assay of PEI and FA-DS-PAAs on HEN-1 cells; (B) Hemolysis assay of PEI and FA-DS-PAAs with different concentration; (C) Organ histology (400×), top row for control group, bottom row for FA-DS-PAAs injected mice.

The cytotoxicity of FA-DS-PAAs on HNE-1 cells is assayed by MTT. In Figure 8(A), different concentrations of FA-DS-PAAs or PEI are used to culture HNE-1 cells, and the cell viability results are presented. In all experiment concentrations, FA-DS-PAAs exhibited an obviously reduced toxicity compared with PEI, especially in higher 25 / 38



experiment concentrations. For instance, cell viability is greater than 80% when the FA-DS-PAAs concentration reached 200 μg·mL-1, whereas cell viability is less than 5% with 200 μg·mL-1 PEI. So, FA-DS-PAAs exhibit lower cytotoxicity than PEI. As an injectable co-delivery gene and drug system in the blood, the most important factor of the carrier is the stability in therapeutics. Moreover, in the blood, the target ability and bioavailability of complexes can severely diminish by the nonspecific interactions between carriers and proteins.37 Thus, to assess the blood compatibility of FA-DS-PAAs, the hemoglobin released from erythrocytes is detected by spectrophotometric measurement. As shown in Figure 8(B), the FA-DS-PAAs exhibited better blood compatibility compared with PEI at all experiment concentrations. Even incubation with

FA-DS-PAAs (1 mg·mL-1) for one day,

nonhemolytic effect is noted in the sample, and the hemolysis extent is lower than 5% (the permissible level).38 In contrast, PEI (1 mg·mL-1) exhibits obvious hemolysis (greater than 40%). 3.10. In vivo toxicity To further prove the safety, in vivo toxicity studies of FA-DS-PAAs are essential. In Figure 8(C), histological photos of organs are used to evaluate whether FA-DS-PAAs caused tissue inflammation or lesions. Histologically, compared with the control (top row) groups, no visible difference is observed in FA-DS-PAAs groups. A polymer’s chemical structures, the surface and terminal groups, size, location, exposure duration, metabolism, and biodistribution influence its in vivo 26 / 38


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toxicity.39 For FA-DS-PAAs, the molecular weight, type, and generation also influence the in vivo toxicity. FA-DS-PAAs displaying nonobserved toxicity can be attributed to their degradation ability and hyperbranched characteristic molecular structure. First, unlike linear structures of polymers having similar molecular weights of FA-DS-PAAs, the hyperbranched structure of FA-DS-PAAs could reduce the cytotoxicity,40 Second, with the elimination ability from cells and organisms, the biodegradable FA-DS-PAAs can reduce their in vivo toxicity, and enhance their in vivo biocompatibility.

Figure 9. Liver and renal function and HE stain results of nude mice after treatment with FA-DS-PAAs/DOC and Ai Su.

Figure 9 represents a liver function examination, compared with control group, the glutamic oxalacetic transaminase (AST) levels in both the nanomedicine and DOC groups are increased (P0.05). The DOC group exhibited more obvious acute injuries on the liver than FA-DS-PAAs/DOC group, with evident liver cell edema, dilated and congested vessels in the portal area, and infiltration of interstitial inflammatory cells. The nanomedicine and DOC groups exhibited no obvious kidney damage. The glomerular regions exhibited no evident of enlargement, reduction and exudate changes. In addition, no obvious infiltration of interstitial inflammatory cells or expansion of the renal interstitium vessels was observed in the DOC and FA-DS-PAAs/DOC groups. However, the shedding of a small amount of renal tubular epithelial cells was observed in the DOC group. The results of the liver and renal function and HE stain revealed that DOC and nanomedicine are mainly metabolized in the liver and induced hepatotoxicity; however, the hepatotoxicity of the nanomedicine was significantly reduced compared with DOC. 3.11. In vivo therapy

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Figure 10. (A) Treatment with various formulations, the HNE-1 tumor volume of nude mice; (B) Treatment with various formulations after 21th day, the HNE-1 tumor images; (C) At the 21th day, PCNA expression in HNE-1 tumor (400×).

For the anti-tumor test of FA-DS-PAAs/DOC/TFPI2 in vivo, bearing HNE-1 tumors nude mice are used, and Figure 10(A) presents HNE-1 tumor growth curves in 6 groups. During 21 d, the FA-DS-PAAs-treated group has the fast tumor volume increase

speed.

However,

treated

groups

with

the

exception

of

the

FA-DS-PAAs/DOC/TFPI2-treated group exhibit the most obvious HNE-1 tumor inhibition effect. As shown in Figure 10(B), the same results were also obtained. Regarding

the

molecular

stimulation-responsive

targeting

ability

ability

of

the

and

pH

and

FA-DS-PAAs

redox

dual

nanocarrier,

FA-DS-PAAs/DOC/TFPI2 exhibited the best anti-tumor effect with the slowest tumor growth rate and the smallest tumor volume.

29 / 38



PCNA, a cofactor of DNA polymerase δ, is often used as cell proliferation index and DNA replication index.41 In Figure 10(C), PCNA expression in HNE-1 tumors is presented. High PCNA expression is observed in the FA-DS-PAAs group with a PI (positive incidence) of 96.51%. The PI values of the other groups except the DS-PAAs/TFPI2-treated group are reduced compared with the FA-DS-PAAs-treated group. PI in the FA-DS-PAAs/DOC/TFPI2 group is the lowest (only 30.5%) and reveals that FA-DS-PAAs/DOC/TFPI2 has the best anti-tumor effect.

4. Conclusions FA-DS-PAAs/DOC/TFPI2 was successfully prepared, and their chemical structures were confirmed by 13C NMR analysis. UV-Vis spectral analysis revealed that 14.5% FA was contained in FA-DS-PAAs. The loading amount of DOC is 19.8 μg/mg in FA-DS-PAAs/TFPI2 complex (weight ratio of 30), and the particle size is approximately 200 nm. The zeta potential of FA-DS-PAAs increased from approximately -15 to 15 mV with an increase in the weight ratio (from 0 to 30). The FA-DS-PAAs could degrade at the concentration of 100 μmol·L-1 GSH. Electrophoretic mobility analysis indicated that FA-DS-PAAs can bind genes, and load hydrophobic drugs. In vitro release behavior indicated that the dual-responsive FA-DS-PAAs/DOC/TFPI2 nanoparticles had the targeted release DOC ability in tumor cells. In vitro FA-targeting assay revealed that the FA-DS-PAAs facilitated the delivery of FA-DS-PAAs/DNA into HNE-1 cells. In vitro transfection indicated that 30 / 38


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the FA-DS-PAAs transfected approximately 44% of HNE-1 cells, which is considerably increased compared with the percentage of cells transfected by PEI. Western blot analysis confirmed that FA-DS-PAAs FA-DS-PAAs/TFPI2 could result in high expression of TFPI2 protein compared with the PEI/TFPI2 group. In vivo FA-targeting assay revealed a wide DOC distribution and GFP expression in the majority of tissues, with the greatest levels noted in the liver and kidney. Cell apoptosis and transwell invasion assay suggest that codelivery with the aid of FA-DS-PAAs could enhance TFPI2 gene expression, make cancer cells more sensitive to DOC, induce cell apoptosis, and reduce cell invasiveness more effectively compared with mono-chemotherapy. Targeted inhibitory experiments confirmed that the FA-DS-PAAs could target and codeliver DOC and TFPI2 to HNE-1 cells, and then effectively induce HNE-1 cell apoptosis. In vivo toxicity studies demonstrated no visible differences compared to the control; liver and renal function and HE staining showed that DOC and nanomedicine are mainly metabolized in the liver and induced hepatotoxicity. The hepatotoxicity of the nanomedicine was significantly reduced compared with that of DOC. FA-DS-PAAs/DOC/TFPI2 exhibited the best anti-tumor effect with the slowest growth rate and smallest volume in vivo.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 31 / 38



Details of Materials, GSH concentration assay in NPCcells and tissues, DOC release in vitro, FA-targeting assay, In vitro transfection, TFPI-2 protein expression, Cellular toxicity, Apoptosis assay, Cell invasion assay, Targeted inhibitory experiment, In vivo assay, DOC distribution, GFP expression in vivo, Cell viability, Blood compatibility, In vivo toxicity, In vivo tumor inhibition, Statistical analysis, 1H NMR spectrum of β-CD-CONHNH2 (DMSO-d6, 25˚C Figure S1), 1H NMR spectrum of hyperbranched PAAs (DMSO-d6, 25˚C, Figure S2), GSH concentration in NPC cells and tumors (Figure S3) and Electrophoretic mobility for FA-DS-PAAs bind TFPI2 (Figure S4).

Acknowledgements The authors acknowledged financial support from the National Natural Science Foundation of China (81573000, 81260406, 21476052), the Foundation of Science and Technology Projects of Guangdong Province (2016A010103047), the Science and Technology Innovation Platform Project of Foshan City (2016AG100541, 2017AG100092), the Foshan Technology Research Center (2016GA10161), the Talent Introduction Fund of Guangdong Provincial People's Hospital (Y012018142), and the Project supported by GDHVPS (2017).

Data Availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

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