Enhanced Lysosomal Escape of pH-Responsive Polyethylenimine

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Biological and Medical Applications of Materials and Interfaces

Enhanced Lysosomal Escape of pH-Responsive PEI-Betaine Functionalized Carbon Nanotube for the Co-delivery of Survivin siRNA and Doxorubicin Yue Cao, Hao-Yan Huang, Li-Qing Chen, Huan-Huan Du, JingHao Cui, Leshuai W. Zhang, Beom-Jin Lee, and Qing-Ri Cao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20810 • Publication Date (Web): 18 Feb 2019 Downloaded from http://pubs.acs.org on February 20, 2019

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ACS Applied Materials & Interfaces

Enhanced Lysosomal Escape of pH-Responsive PEI-Betaine Functionalized Carbon Nanotube for the Co-delivery of Survivin siRNA and Doxorubicin

Yue Caob,1, Hao-Yan Huanga,1, Li-Qing Chena, Huan-Huan Dua, Jing-Hao Cuia, Leshuai W. Zhangc,*, Beom-Jin Leed, Qing-Ri Caoa,**

aCollege

of Pharmaceutical Sciences, Soochow University, Suzhou 215123,People’s Republic of China

bDepartment

of Pharmacy, Beijing Health Vocational College, Beijing 100053, People’s Republic of

China cSchool

for Radiological and Interdisciplinary Sciences (RAD-X), State Key Laboratory of Radiation

Medicine and Protection, School of Radiation Medicine and Protection, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou 215123, People’s Republic of China d College

of Pharmacy, Ajou University, Suwon, 16499, Republic of Korea

Corresponding authors: *Leshuai

W. Zhang, E-mail: [email protected], Tel: 86-13913193240.

**Qing-Ri

1 These

Cao, E-mail: [email protected], Tel: 86-512-69564123.

authors contributed equally to this work.

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ABSTRACT The combination of gene therapy and chemotherapy has recently received considerable attention for cancer treatment. However, low transfection efficiency and the poor endosomal escape of genes from nanocarriers strongly limit the success of the clinical use of small interfering RNA (siRNA). In this study, a novel pH-responsive surface modified single-walled carbon nanotube (SWCNT) was designed for the co-delivery of doxorubicin (DOX) and survivin siRNA. Polyetherimide (PEI) was covalently conjugated with betaine, and the resulting PEI–betaine (PB) was further synthesized with the oxidized SWCNT to form SWCNT–PB (SPB), which exhibits an excellent pH-responsive lysosomal escape of siRNA. SPB was modified with the targeting and penetrating peptide BR2 (SPBB), thereby achieving considerably higher uptake of siRNA than SWCNT-PEI (SP) or SPB. Furthermore, SPBB–siRNA presented substantially lower survivin expression and higher apoptotic index than Lipofectamine 2000. DOX and survivin siRNA were adsorbed onto SPB to form DOX–SPBB–siRNA, and siRNA/DOX was released into the cytoplasm and nuclei of adenocarcinomic human alveolar basal epithelial (A549) cells without lysosomal retention. Compared with SPBB–siRNA or DOX–SPBB treatment alone, DOX–SPBB–siRNA significantly reduced tumor volume in A549 cell-bearing nude mice, demonstrating the synergistic effects of DOX and survivin siRNA. Pathological analysis also indicated the potential therapeutic effects of DOX–SPBB–siRNA on tumors without distinct damages to normal tissues. In conclusion, the novel functionalized SWCNT loaded with DOX and survivin siRNA was successfully synthesized and the nanocomplex exhibited effective antitumor effects both in vitro and in vivo, thereby providing an alternative strategy for the co-delivery of antitumor drugs and genes.

KEY WORDS: SWCNT, PEI, Betaines, survivin siRNA, DOX, BR2


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1.

INTRODUCTION

Traditional chemotherapy exhibits limited antitumor efficacy after repeated treatments of cancer patients, probably due to the resistance development to chemotherapeutic drugs.1 To overcome this problem, molecular-targeted therapy by using signal transduction inhibitors and anti-angiogenesis agents has been applied in clinical trials. In the past two decades, the use of small interfering RNA (siRNA) has been a useful tool for inhibiting protein expression; proteins involved in drug resistance, detoxification, cell survival, apoptosis inhibition, and DNA repair have been selected, targeted, and inhibited by siRNA to efficiently reduce drug resistance.2 Therefore, strategies for inhibiting targeted proteins through the transcription level by using siRNA have been widely accepted and combined with cisplatin, doxorubicin (DOX), or paclitaxel to enhance the chemosensitivity of tumor cells.3 Although RNA interference technology has been recognized to specifically and effectively induce gene silence, its in vivo and clinical applications are extremely limited because of the rapid degradation in the circulation, low transfection efficiency, and endosomal retention of siRNA.4,5 Although siRNA has exhibited certain effects on the treatment of various disorders through protein knockdown, its efficacy remain unsatisfying due to the low efficiency of siRNA in crossing cell membranes and its inability to avoid RNAase degradation. Different types of transfection agents have been applied to overcome these problems; however, severe adverse effects are extremely common.6 Alternatively, a co-delivery system loading multi-agents has been developed not only to protect siRNA from degradation but also to allow different types of agents to be localized in the same tumor site, thereby exhibiting synergistic antitumor effects.7,8 Polyethylenimine (PEI) has been recognized as one of the most efficent gene carriers since 19959,10 due largely to the efficient escape of chemicals from lysosomal sequestration by the protein sponge mechanism.11 The high densities of cationic charge in the polymeric chains of PEI result in tight DNA binding and efficient cellular uptake in vitro. However, PEI also causes some severe problems, such as high cytotoxicity attributed to membrane disruptive properties and decreased serum stability of the polyplexes due to the strong interaction with negatively charged proteins.12 Therefore, chemically modified PEI with additional functions or enhanced performance has been used in different ways to improve gene delivery. Kazuhiko et al. created a series of zwitterionic polymers, showing reduced blood clotting.13 Liu’s group designed a novel copolymer with polysulfobetaine, which exhibited high gene transfection efficiency and efficent DNA condensation ability in a serum-containing medium.14



Among PEI modification approaches, the conjugation of zwitterion monomer with PEI is superior to zwitterion polymer due to its simplicity and efficiency.15 Betaine monomer-conjugated PEI exhibits remakable gene delivery efficacy, excellent anticoagulant property and serum tolerance, along with minimal cytotoxicity and protein adsorption. In addition, the overall performance of betaine monomer-modified PEI in terms of gene transfection has been demonstrated to be superior to those of other PEI derivatives.16,17 Therefore, the conjugation of zwitterion monomer with PEI can reduce the positive zeta potential of PEI, thereby decreasing the cytotoxicity of the PEI conjugate and promoting siRNA escape into the cytoplasm through the proton sponge effect. Carbon nanotubes (CNTs) have been recognized as promising carriers for efficient drug and biomolecule delivery due to their unique structural properties.18 CNTs can be carried with anticancer drugs via different interaction methods.19 Chemotherapeutic agents with extended π structures tend to bind CNTs noncovalently through strong π–π stacking. 20Although siRNA delivery by CNT has been previously reported in vitro, extremely few reports are available at the therapeutic level.21 In addition, anticancer drug (or siRNA)-CNT conjugates modified with tumor-targeting ligands will be desirable because it will endow the entire nanoformulation with tumor-specific recognition and internalization efficiency, thereby minimizing adverse effects.22 In the present study, a novel tumor microenvironment-responsive functionalized single-walled CNT (SWCNT) was designed to increase drug loading and gene transfection for the co-delivery of survivin siRNA and DOX (Scheme 1). PEI was covalently conjugated with betaine, and the resulting PEI– betaine (PB) was further interacted with the oxidized SWCNT (O-SWCNT) to form SWCNT–PB (SPB), which was expected to have pH-responsive lysosomal escape ability. DOX and survivin siRNA were noncovalently adsorbed onto SPB, which was further modified with the targeting and penetrating peptide BR2 (SPBB)23 to form DOX–SPBB–siRNA (Scheme 2). The in vitro physicochemical properties, cytotoxicity, cellular uptake, intracellular localization, inhibition of anti-apoptosis protein, and in vivo antitumor activities of the functionalized nanocarriers were evaluated in this study.

2. RESULTS AND DISCUSSION 2.1. Synthesis of PB. The 1H nuclear magnetic resonance (NMR) spectrum of the synthesized betaines is provided in Figure S1A. The 1H NMR data (δ ppm, in D2O) are as follows: 1.98 (m, 2H), 2.16 (m, 2H), 2.95 (t, J = 8 Hz, 2H), 3.08 (m, 6H), 3.35 (m, 4H), 3.42 (t, J = 8 Hz, 2H), 5.75 (d, J =


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12.0 Hz, 1H), 6.12−6.24 (m, 2H). PB conjugates were obtained through the Michael addition reaction between PEI and betaines. The unconjugated betaines were removed via dialysis. The 1H NMR spectrum of PB is provided in Figure S1B. The graft ratio (mole percentage of betaines/total amino groups in PEI) of the resulting conjugates was around 85% in the light of the 1H NMR spectra. In this study, the Michael addition reaction readily occurred and the graft ratio could be readily controlled, thereby ensuring the repeatability of the facile approach. 2.2. Characterization of the Functionalized SWCNT 2.2.1. Fourier Transform Infrared (FTIR) Spectra. Figure S2 shows the FTIR spectra of raw SWCNT, O-SWCNT, SP, and SPB. O-SWCNT presented an absorption band at 1620 cm−1, which was owing to the carbonyl stretching of the carboxylic acid group. After the grafting of PEI and PB, new absorption bands appeared at 1630 cm−1 and 1578 cm−1. These corresponded with the amide carbonyl vibration and the N–H bending vibration, respectively.24 The peaks at 2924 cm−1 and 2854 cm−1 were attributed to the C–H stretching vibrations from PEI and PB. This condition indicated the consumption of carboxyl acid groups in the carboxyl-terminated SWCNT because of the formation of the amide structure after the grafting of PEI and PB. 2.2.2. Ultraviolet–Visible (UV–Vis) Spectra. The UV–Vis spectra of the functionalized SWCNT are shown in Figure S3. Compared with that of raw SWCNT, the absorbance bands of the functionalized SWCNT (i.e., O-SWCNT, SP, and SPB) appeared at 250 nm. This result indicated that SP and SPB were partly connected to O-SWCNT through a covalent interaction. 2.2.3. X-ray Diffraction (XRD) Analysis. The structure of the functionalized SWCNT was characterized using an XRD technique. Figure S4 shows the XRD patterns of the functionalized SWCNT.25 Two peaks appeared, i.e., at 26° and 43°, which were assigned to the (002), (110) diffraction plane of SWCNT.5 These two diffraction peaks were due to the characteristic structure of SWCNT. The XRD patterns of O-SWCNT, SP, and SPB were highly similar to that of raw SWCNT, indicating that the modified SWCNT still presented the similar wall structure as raw SWCNT. The functionalization process exhibited no interference on the general structure of SWCNT. 2.2.4. Thermogravimetric Analysis (TGA). The number of functional groups in the modified SWCNT was analyzed via TGA, as shown in Figure S5. The weight loss of the modified conjugates was directly correlated with an increase in mass around the CNTs that were introduced at each step when calculated from the thermogravimetric curves at 400 °C.26 The mass attributed to functionalities



increased from 18.6% in O-SWCNT to 25.8% in SP. The SPB curves showed a 34.7% mass loss. The difference in weight loss at 400 °C among O-SWCNT, SP, and SPB indicated that PEI and PB grafting amounts were approximately 7.2% and 16.1%, respectively. 2.2.5. Morphology, Size distribution, and Zeta potential. As shown in Figure 1A, the surface morphology of the functionalized SWCNT was investigated via transmission electron microscopy (TEM). Untreated raw SWCNTs adhered to one another with a large number of black clumps, thereby forming an uneven distribution of tube bundle with a length reaching up to dozens of micrometers. After acid treatment, O-SWCNT appeared to be smooth without impurities, indicating the complete removal of metal particles and amorphous carbon. The purified SWCNTs were relatively short and well separated, and thus, formed only small bundles. After PB coating, copolymer chains were observed on the sidewalls of SWCNT. SPB was well dispersed via ultrasonication. As shown in Figure 1B, O-SWCNT was readily dispersed in an aqueous phase due to the carboxyl groups in the structure. After covalent binding with PEI or PB, SP and SPB exhibited decent dispersibility due to the amino groups on the surface. As shown in Figure 1C, O-SWCNT, SP, and SPB presented a narrow particle size distribution. The particle size of O-SWCNT was 710 nm, and the zeta potential (−50 mV) was negative because of the formation of carboxyl groups (Figure 1D). After PB modification, particle size decreased to 250 nm and the zeta potential changed to a positive value of 25 mV due to the amidogen of the PB chain, suggesting that the functionalized SWCNT could be a suitable nanocarrier for the co-delivery of drugs and siRNA.27 2.3. In Vitro Cytotoxicity of the Functionalized SWCNT. Before conducting the transfection experiments, the cytotoxicity of the modified SWCNT gene vectors should be evaluated. First, the cell viability of functional groups, such as PB and PEI, in the concentration range of 3.125–100 μg/mL was evaluated in adenocarcinomic human alveolar basal epithelial (A549) cells via MTT assay, as shown in Figure S6. High cytotoxicity was found when PEI concentration was over 12.5 μg/mL, whereas PB exhibited substantially lower cytotoxicity. This result concurs with the assumption that the conjugation of betaines to PEI can reduce cation density on the polymer surface, attenuating damage to the cell membrane. The uniform modification and distribution of betaines allow minimal protein adsorption onto the PEI surface and contribute to the excellent biocompatibility and increased cell viability of nanomaterials.28


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Second, the cytotoxicity of the functionalized SWCNT on A549 and human embryonic kidney (293T) cells was evaluated via WST-1 assay. As shown in Figure 2, SPB exhibited attenuated cytotoxicity on both cell lines compared with raw SWCNT and SP, with cells maintaining more than 80% viability after 24 h and 48 h of treatment with SPB at a high concentration of 50 μg mL−1. As a result, cell viability data strongly indicated that the cytotoxicity of SPB on A549 and 293T cells was negligible. 2.4. Agarose Gel Electrophoresis Assay. The formulations of SPB–siRNA and SP–siRNA complexes with different N/P ratios (amino group of PB/PEI versus phosphate group of siRNA phosphate backbone) were assessed via agarose gel retardation assay as previously described.29 The siRNA binding ability of SP or SPB is shown in Figures 3A and 3B, respectively. When the N/P ratio reached 25, no signal of siRNA was observed in the loading sample of SPB, suggesting that negatively charged siRNA was fully neutralized and Goldview™ was unable to intercalate. Meanwhile, the ratio for siRNA neutralization was >30 for SP. This result suggests that SPB exhibits the excellent binding ability of siRNA and satisfactory capacity to form stable complexes with siRNA. Heparin is one of anionic polysaccharides that can compete with siRNA binding on SPB, thereby causing destabilization of the SPB–siRNA complex. Figure 3C shows that the degree of siRNA degradation by ribonuclease (RNase A) depends on heparin concentration. Heparin can completely replace free siRNA from SPB–siRNA when its concentration reached 0.80 mg/mL. In addition, the ability of SPB to protect siRNA against degradation was realized via incubation with RNase A at different time points. As shown in Figures 3D and 3E, naked siRNA was completely degraded when incubated with RNase A for 10 min, whereas SPB prolonged siRNA stability in RNase A at an N/P ratio of 25:1 for >6 h, as demonstrated by the intact fluorescence in the wells. Consequently, SPB demonstrated an excellent ability to protect siRNA against degradation. 2.5. Characterization of DOX–SPB. The DOX-loaded functionalized SWCNT formed stable suspensions in water and was lighter in color (i.e., red-brown) than its carriers. The formation of DOX– SPB was monitored via UV–vis absorption spectroscopy. As shown in Figure S7, DOX exhibited two main absorption peaks (i.e., at 490 nm and 233 nm), whereas O-SWCNT presented an absorption peak only at 233 nm. The absorption peaks of DOX–SWCNT and DOX–SPB were enhanced slightly at 490 nm, indicating the adsorption of DOX onto the surface of the nanocarriers. 2.6. Drug Loading Capacity and Encapsulation Efficiency. Drug loading capacity is one of the



important indexes for a drug delivery system. As a widely used anticancer drug for chemotherapy treatment, DOX was used as a model drug to study the loading capacity of SPB.30 Our results showed that DOX loading capacity onto SPB was 70.8%, which is similar to that onto O-SWCNT (73.8%). The powerful drug-loading capacities of the two carriers indicated that the positively charged molecules were easily adsorbed onto the carriers with negetive zeta potentials, suggesting that electrostatic and π– π stacking interactions play important roles with respect to drug loading. DOX can attach to the surface of CNTs via π–π interactions due to their inherent aromatic structure. In addition, the drug molecules contain pH-responsive amine groups, which make their binding to CNTs pH-dependent.30 Meanwhile, the encapsulation efficacies of the functionalized SWCNT were at 92% for DOX–SWCNT and 94% for DOX–SPB, indicating that nearly all the DOX was loaded onto the SWCNT derivatives. 2.7. Drug Release Studies. Controlled and site-specific drug release is the major objective of cancer therapy. When drug-loaded nanocarriers are internalized by cells, the antitumor effect will only occur when the nanocarriers encounter an acidic endosome/lysosome, which enables complex destabilization and drug release into the cytoplasm. Therefore, the release rate of DOX from the functionalized SWCNT was evaluated in acidic (pH = 5.0) and neutral (pH = 7.4) phosphate-buffered saline (PBS) solutions at 37℃ (Figure S8).30 The release rates of DOX from O-SWCNT and SPB were faster in pH 5.0 PBS than in pH 7.4 PBS. Approximately 15% and 30% DOX were released from O-SWCNT and SPB in pH 5.0 PBS solution at 48 h, respectively. By comparison, only approximately 6% and 15% of DOX were released from O-SWCNT and SPB in pH 7.4 PBS solution even at 72 h. In the pH 5.0 environment, DOX became positively charged, increasing electrostatic repulsion between DOX molecules. Meanwhile, PB was further protonated with increasing electrostatic repulsion among PEI branches, which promoted rapid DOX release from SPB in an acidic environment. In addition, the release profiles of DOX from O-SWCNT and SPB in 0.5% Tween 80 PBS were faster than those in PBS. In PBS (pH 5.0) containing 0.5% Tween 80, DOX release from SPB could reach up to 50% within 72 h. Therefore, DOX–SPB can be protonated to release more drugs from the nanocarrier in acidic lysosomal/endosomal vacuoles. The pH-triggered drug release from SPB in the low pH environments of lysosomes/or endosomes/or cancerous tissues in general, has provided a built-in mechanism for selective drug release. 2.8. Cellular Uptake of Various Peptides. To enhance the endocytic ability of nanocarriers, cell-penetrating and cell-targeting peptides were studied and selected for nanocarrier modification. The


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internalization of classical peptides, including TAT, RGD, and BR2, in different cell types (A549, HeLa, and 293T) was investigated via flow analysis. As shown in Figure S9, TAT peptide penetrated cancerous and normal cells without selection, reaching more than 80% of the cellular uptake rate among all cell types. This result could be attributed to the highly penetrating property of TAT through the cellular membrane.31 The cellular uptake of RGD was remarkably higher in HeLa and 293T cells than in A549 cells because integrin αvβ3 is highly expressed on the surface of HeLa and 293T cells.32 The BR2 peptide was transported more efficiently into cancer cells (HeLa and A549) than into normal cells (293T). More than 80% of BR2 was transported into HeLa and A549 cells, whereas only 35% was internalized into 293T cells.32 The cellular uptake of peptides (TAT, RGD, and BR2) was also visualized using a fluorescence microscope.33 As shown in Figure S10, BR2 was easily transported into cancer cells (A549 and HeLa) within 6 h and distributed throughout all the cancer cells. However, BR2 was hardly internalized into normal cells (293T) under the same conditions. By contrast, RGD demonstrated high cellular uptakes in both HeLa and 293T cells, indicating its translocation ability across the cell membranes of normal cells. Fluorescent signal from TAT-treated cells was also distinctly detected in cancer and normal cells. These results evidently showed that BR2 peptide exhibits higher endocytic efficiency and specificity on cancerous cells than on noncancerous cells. 2.9. In Vitro Transfection. FAM-labeled siRNA was used as an indicator to investigate the transfection efficiencies of free siRNA, Lipofectamine 2000, SP, SPB, and SPBB in A549 cells. After 6 h of transfection, the intracellular fluorescence intensities and the cell percentage with internalized FAM-labeled siRNA (Figure 4A and 4B) were evaluated via flow cytometry. The order of intracellular fluorescence intensity of FAM–siRNA was as follows: SPBB > SPB > Lipofectamine 2000 > SP. Compared with the untreated cells, the percentage of siRNA positive cells of SP was 74.57% and that of SPB was 90.27%. Furthermore, SPBB presented the highest fluorescence intensity compared with SPB (p

Enhanced Lysosomal Escape of pH-Responsive Polyethylenimine

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