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gene co-delivery systems
Journal of Industrial and Engineering Chemistry 75 (2019) 148–157
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Phenylboronic acid-conjugated cationic methylcellulose for hepatocellular carcinoma-targeted drug/gene co-delivery systems Ju Hyeon Jeona,1, Jae Hong Parka,1, Tae-il Kima,b,* a Department of Biosystems & Biomaterials Science and Engineering, College of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea b Research Institute of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 January 2019 Received in revised form 28 February 2019 Accepted 8 March 2019 Available online 16 March 2019
Phenylboronic acid-conjugated cationic methylcellulose (MCPEI-PBA) was synthesized and characterized for hepatocellular carcinoma-targeted drug/gene co-delivery systems. MCPEI-PBA was obtained by reductive amination of polyethylenimine 2k to oxidized methylcellulose and conjugation of PBA. MCPEIPBA could form nano-aggregates via B–N coordinate bonding and hydrophobic interaction and also form positively charged and nano-sized polyplexes. Cytotoxicity of MCPEI-PBA was low and MCPEI-PBA showed higher transfection efficiency than PEI25k in hepatocellular carcinoma. Galactose pretreatment experiment suggested its specific interaction with asialoglycoprotein receptors. MCPEI-PBA could encapsulate doxorubicin (Dox) efficiently. Finally, MCPEI-PBA/Dox/pJDK-apoptin complexes showed high anticancer activity due to the combinatorial effect of Dox and expressed apoptin. © 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Keywords: Drug/gene co-delivery Methylcellulose Phenylboronic acid Nano-aggregates Hepatocellular carcinoma-targeting
Introduction Drug and gene co-delivery systems have been reported to induce improved therapeutic efficacy via synergistic effect of drug and therapeutic gene in comparison with single delivery [1]. Especially, it has advantages for cancer therapy because of heterogeneity of cancer cells and complexity of cancer cell growth and development [2,3]. Anthracycline derivative anticancer agent, doxorubicin (Dox) shows anticancer activity by inhibiting topoisomerase II and forming free radicals in cells [4]. It is a hydrophobic molecule, which requires proper encapsulating delivery carriers for solvation and administration. Methylcellulose (MC) is one of the amphiphilic cellulose ether derivatives containing methylated hydroxyl groups of cellulose [5]. From its classic applications for food additives or cosmetics, applications of MC were extended to hydrogel and drug delivery systems [6–8] due to its biocompatibility and sol–gel transition property [9]. Recently, polyethylenimine-grafted MC (MCPEI) has
* Corresponding author at: Department of Biosystems & Biomaterials Science and Engineering, College of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea. E-mail address: [email protected] (T.-i. Kim). 1 These authors contributed equally to this work.
been reported as an efficient gene delivery system, possessing serum compatibility and endosome buffering ability [10]. In this work, MCPEI was employed as a backbone polymer for drug/gene co-delivery carrier systems. Then, phenylboronic acid (PBA) was conjugated to MCPEI, synthesizing MCPEI-PBA. PBA has been utilized for hydrogel, cancer-targeting drug or gene delivery carriers, and probes [11,12] because of its forming ability of reversible boronate ester bond with cis-1,2 or cis-1, 3 diol of sugar molecules [13,14], especially sialic acids of glycoproteins and glycolipids overexpressed in cancer cells. In addition, PBA also can interact with amine group via B–N coordinate bond formation [15]. Therefore, it was hypothesized that MCPEI-PBA can form nanoaggregates encapsulating hydrophobic drug molecules by B–N coordinate bond formation between PBA and PEI and that cationic MCPEI-PBA/Dox nano-aggregates can further form complexes with anionic pDNA, finally constructing drug/gene co-delivery carrier. As a therapeutic gene, apoptin-encoding pDNA, pJDK-apoptin was used [16]. Apoptin is known as an antitumor agent inducing tumor cell-selective apoptosis [17]. Although several gene delivery systems have been developed for apoptin gene delivery [16,18], its therapeutic application has limitations such as lack of efficient delivery systems [19,20]. Therefore, we tried to show the capability of MCPEI-PBA for efficient apoptin gene delivery systems in this work. Here, MCPEI-PBA was synthesized and its characterizations for drug and gene co-delivery carrier systems were reported for cancer therapy. Moreover, its unique targeting ability for hepatocellular carcinoma via asialoglycoprotein receptor (ASGPR) interaction was
https://doi.org/10.1016/j.jiec.2019.03.016 1226-086X/© 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
J.H. Jeon et al. / Journal of Industrial and Engineering Chemistry 75 (2019) 148–157
identified and analyzed. To the best of our knowledge, this is the first report regarding the development of PBA-conjugated drug/ gene co-delivery carrier with ASGPR-mediated hepatocellular carcinoma-targeting ability. Experimental Materials Methylcellulose (15 cP, 2% in H2O), poly(ethylenimine) (PEI, molecular weight 2k and 25 kDa), agarose, ethylenediaminetetraacetic acid (EDTA), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and pyrene were purchased from SigmaAldrich (St. Louis, MO). Sodium periodate, sodium tetrahydroborate, and ethylacetate was purchased from Junsei (Tokyo, Japan). 4(Bromomethy)phenylboronic acid (4-BMPBA), 3-aminophenylboronic acid (3-APBA), 4-carboxyphenylboronic acid (4-CPBA), D(+)-galactose, and DMEM + GlutaMax-I medium were purchased from Thermo Fisher Scientific TM(Waltham, MA). YOYO-1 iodide, fetal bovine serum (FBS), 0.25% Trypsin-EDTA, Dulbecco’s phosphate buffered saline (DPBS), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Invitrogen (Carlsbad, CA). Minimum Essential Medium with Earle’s Balanced Salts (MEM/ EBSS) was purchased from GE Healthcare Life Sciences (South Logan, UT). Luciferase assay system and reporter lysis buffer were TM purchased from Promega (Madison, WI). BCA protein assay kit was purchased from PIERCE (Rockford, IL). Doxorubicin was purchased from Bio-techne (Minneapolis, MN). All other chemicals were purchased and used without any further purification. Synthesis of MCPEI-PBA First, backbone polymer, MCPEI2k was synthesized according to the previous report [10]. Briefly, methyl cellulose was oxidized by sodium periodate. After 24 h of oxidation reaction, the reaction mixture was dialyzed against ultrapure water with dialysis membrane (MWCO = 6–8 k). Then, PEI2k (5 molar to glucose units of oxidized MC, OXMC) water solution was added to OXMC solution for the conjugation of PEI2k to OXMC. After 24 h of reaction, the reaction mixture was reduced by sodium tetrahydroborate (water) for another 24 h. The product, MCPEI2k was obtained after dialysis followed by lyophilization. In order to synthesize MCPEI-PBA, 4-BMPBA (3, 5, and 10-fold molar excess to PEI units of MCPEI2k, respectively) was reacted with MCPEI2k in MeOH solution at 50 C for 24 h. Unreacted 4BMPBA was extracted with ethylacetate. After extraction, the reaction mixture was purified by dialysis against 0.2 M NaCl solution and continuously water for 1 day each with dialysis membrane (MWCO = 6–8k). The final product, MCPEI-PBA was obtained by lyophilization. The each step of polymer synthesis was confirmed by 1H NMR (D2O, 400 MHz JEOL JNM-LA400). Molecular weight measurements by gel permeation chromatography (GPC) The molecular weights of polymers were determined by GPC (YL-9100, Young Lin Instrument, Korea) with Ultrahydrogel 250 column (Waters, Milford, MA) with 1% formic acid as an eluent. Polyethyleneglycols with various molecular weights were used as standards. The concentration of the samples and the flow rate were set to 10 mg/mL and 0.6 mL/min, respectively. Critical aggregation concentration (CAC) measurements In order to characterize CAC of MCPEI-PBA, pyrene absorbance method was used as previously reported [21]. 20 mL of pyrene
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ethanol solutions (0.1 mM) were added to vials and dried under vacuum overnight in dark condition. After formation of pyrene film, 1 mL of MCPEI-PBA water solutions with various concentrations were added to the vials, giving the final pyrene concentration of 6 107 M. The MCPEI-PBA/pyrene solutions were sonicated for 10 min and incubated with gentle mixing at room temperature for 24 h. Then, absorbance of pyrene was measured by UV/Vis spectrometer (Optizen POP BIO, Mecasys, Korea) using quartz cuvettes in the range of 200–400 nm. The total absorbance by sum of four absorbance points (242, 272, 320, and 336 nm) was plotted against MCPEI-PBA concentrations. CAC of MCPEI-PBA sample was set to the center of the sigmoid. Encapsulation of doxorubicin (Dox) in MCPEI-PBA nanoparticles DoxHCl solution (2 mg Dox, DMSO) was mixed with 2 equivalent moles of triethylamine. After 1 day, Dox solution was added to MCPEI-PBA solution (20 mg, DMSO) for 1.5 h. MCPEI-PBA/ Dox solution was added dropwise to water. After 1 day of stirring, the mixture solution was dialyzed against water with dialysis membrane (MWCO = 6-8k) for 1 day and the final MCPEI-PBA/Dox was obtained after lyophilization. Evaluation of Dox loading in MCPEI-PBA nanoparticles MCPEI-PBA/Dox was dissolved in DMSO and the fluorescence of Dox was measured by a microplate reader at excitation wavelength of 480 nm and emission wavelength of 590 nm. Loaded Dox amount was determined according to the previously prepared calibration curve of Dox solution (DMSO). Drug loading content (DLC) and drug loading efficiency (DLE) were calculated as following formula. DLC ð% Þ ¼
Weight of Loaded DOX 100 Weight of MCPEI PBA
DLE ð% Þ ¼
Weight of Loaded DOX 100 Weight of total DOX f or loading
Average particle size and zeta-potential value measurements Average particle sizes of MCPEI-PBA were measured by a Zetasizer Nano ZS (Malvern Instruments, UK) with He-Ne laser beam (633 nm) at 25 C. MCPEI-PBA water solutions with various concentrations were used for measurements. MCPEI2k water solutions were also used for comparison. In the case of polyplexes, 1 mL of MCPEI-PBA/pDNA polyplex solutions were prepared in ultrapure water at various weight ratios. After 30 min of incubation, average particle sizes and Zetapotential values were measured by a Zeta-sizer. Furthermore, MCPEI-PBA/Dox/pDNA water solution was prepared by mixing of MCPEI-PBA/Dox solution (50 mg/mL) and pDNA (1 mg) at a weight ratio of 25. Average particle sizes and Zetapotential values of MCPEI-PBA/Dox and MCPEI-PBA/Dox/pDNA were measured by a Zeta-sizer. Transmission electron microscopy (TEM) The morphology of MCPEI-PBA nanoparticles were observed by Energy-filtering transmission electron microscopy (EF-TEM, LIBRA 120, Carl Zeiss, Germany). 500 mg/mL of MCPEI-PBA water solutions were prepared and deposited on TEM copper grid plates 4 times. The samples were then stained with filtered uranium acetate for 10 s. After absorption of residual solutions, the images were visualized with an accelerating voltage of 120 kV.
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Plasmid DNA purification Plasmid DNAs (pDNAs), pCN-Luci encoding luciferase, pEGFPC2 encoding GFP, and pJDK-apoptin encoding apoptin were amplified with Escherichia Coli DH5a, respectively and purified with Nucleobond Xtra Midi kit (Macherey-Nagel, Germany). Agarose gel electrophoresis Agarose gel electrophoresis was performed to assess pDNA condensation ability of MCPEI-PBA. Agarose gel (0.7%, w/v) containing ethidium bromide (0.5 mg/mL) was prepared in TrisAcetate-EDTA (TAE) buffer. The MCPEI-PBA polyplexes were prepared in Hepes buffer (pH 7.4) at various weight ratios (polymer/pDNA) for 30 min of incubation at room temperature. After loading of samples, the electrophoresis was run for 12 min at 100 V (Mupid-2plus, Takara Bio Inc., Japan). The locations of pDNA bands were visualized by UV illuminator (Gel Doc XRS + gel documentation system, Bio-Rad, Hercules, CA). Cell culture Human cervical adenocarcinoma cells (HeLa), mouse myoblast cells (C2C12), and human hepatocellular carcinoma cells (HepG2) were maintained in a 5% CO2 incubator at 37 C in DMEM + GlutaMax-I medium, DMEM medium, and MEM/EBSS medium, respectively, which were supplemented with 10% FBS and 1% penicillin/streptomycin. MTT assay MTT assay was performed to examine the cytotoxicity of polymers. Cells were seeded in a 96-well plate at a density of 1 104 cells/well, respectively. Having achieved 70–80% confluence after 24 h, the cells were exposed to 100 mL polymer solutions (serum-free medium) with various concentrations for 4 h. Subsequently, the media was changed with fresh medium (10% FBS). After 24 h, the cells were treated with 25 mL of MTT stock solution (2 mg/mL in DPBS) and incubated for 2 h at 37 C. After removing each medium carefully, 150 mL of DMSO was added to each well to dissolve the formazan crystal formed by proliferating cells. The absorbance was measured at 570 nm using a microplate reader. Results were presented as relative cell viabilities (RCV, percentage values relative to value of untreated control cells). In vitro transfection experiments in serum or without serum condition The transfection efficiencies of polymers were examined by measuring luciferase reporter transgene expression in non-serum or serum conditions. Cells were seeded in a 24-well plate at a density of 5 104 cells/well and were grown to reach 70–80% confluence. Before transfection, the media were exchanged with serum-free medium for the assay in non-serum condition and with serum-containing medium (10% FBS) for the assay in serum condition, respectively. Then, the cells were treated with polyplex solutions (0.5 mg pDNA) with various weight ratios. The weight ratio of PEI25k polyplex was 1.0. In the case of PBA or galactose inhibition in HepG2 cells, 4-CPBA solutions (10 mM 1% MeOH/ medium, v/v) or galactose solutions (100 mM medium) were pretreated for 30 min. After 4 h of incubation, the transfection media were exchanged with fresh medium (10% FBS) and the cells were incubated for further 2 days. Subsequently, media were removed and the cells were rinsed with DPBS and shaken for 30 min at room temperature in Reporter Lysis Buffer. The collected cell lysates were centrifuged and the supernatants were used for luciferase
activity measurements with a microplate reader. A protein TM quantification assay was performed using a BCA Protein Assay Reagent Kit to measure total amount of cellular proteins. The final results were presented in terms of RLU/mg cellular protein. Green fluorescence protein (GFP) expression The galactose inhibition effect to transfection efficiencies of polymers were also examined by observing GFP transgene expression. HepG2 cells were seeded in a 6-well plate at a density of 2 105 cells/well and were grown to reach 70–80% confluence. Before transfection, the media were exchanged with serum-free medium. Then, the cells were treated with polyplex solutions (4 mg pEGFP-C2, PEI25k: weight ratio = 1.0, MCPEI2k, MCPEI-PBA: weight raio = 25). In the case of galactose inhibition, galactose solutions (100 mM medium) were pre-treated for 30 min. After 4 h of incubation, the transfection media were exchanged with fresh medium (10% FBS) and the cells were incubated for further 2 days. Then, expressed GFP of the cells was observed by fluorescence microscopy (iRis Digital Cell Imaging System, Logos biosystems, Korea). Flow cytometry Flow cytometry was performed to investigate the cellular uptake of MCPEI-PBA polyplexes. HepG2 cells were seeded at a density of 2 105 cells/well in a 6-well plate and grown to reach 70–80% confluence prior to transfection. Before transfection, medium of each well was exchanged for fresh serum-free medium. pDNA was labeled with YOYO-1 iodide (1 molecule of the dye per 50 base pairs of the nucleotide). The cells were treated with the polyplex solutions (1 mg pDNA, serum-free medium) at a weight ratio of 25 for 4 h. In the case of galactose inhibition, galactose solutions (100 mM medium) were pre-treated for 30 min. Then, medium was aspirated off from the wells and the cells were washed two times with ice-cold DPBS. After trypsinization, the cells were suspended in 1 mL DPBS. The cellular uptake of fluorescence-labeled polyplexes was measured by BD Accuri C6 flow cytometer (Becton Dickinson, San Jose, CA) at a minimum of 1 104 cells gated per sample. Analysis was performed by using BD Accuri C6 software. Anticancer activity of MCPEI-PBA/Dox/pJDK-apoptin polyplexes Cells were seeded in a 24-well plate at a density of 5 104 cells/ well. Having achieved 70–80% confluence after 24 h, the cells were exposed to Dox solution, pJDK-apoptin solution, PEI25k/pJDKapoptin polyplexes solution (weight ratio of 1.0), MCPEI-PBA/Dox solution, MCPEI-PBA/pJDK-apoptin polyplexes solution (weight ratio of 25), and MCPEI-PBA/Dox/pJDK-apoptin polyplexes solution (weight ratio of 25) for 4 h. Each solution contains 1 mg pDNA or 2.4 mg Dox in serum-free medium. Subsequently, the media was changed with fresh medium (10% FBS). After 48 h of incubation, MTT assay was performed in an identical manner with above procedures. Results and discussions Synthesis and characterization of MCPEI-PBA In order to synthesize MCPEI-PBA, backbone polymer, MCPEI2k was synthesized by periodate oxidation of MC followed by reductive amination of PEI2k to oxidized MC, according to the previous study [10]. Then, 4-BMPBA was conjugated to amines of MCPEI2k via nucleophilic substitution of amine to bromide of 4BMPBA. Fig. S1 shows the synthesis scheme of MCPEI-PBA.
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Fig. 1. Molecular structures and 1H NMR spectra of MC (A), OXMC (B), MCPEI2k (C), and MCPEI-PBA (D).
The synthesis of MCPEI-PBA was confirmed by 1H NMR (Fig. 1). H-2(2-OH) protons of MC (Fig. 1A, 3.3–3.4 ppm (a)) disappeared after oxidation (Fig. 1B) due to the conversion of hydroxyl group to aldehyde and H-2(2-OMe) protons of MC (Fig. 1A, 3.1–3.2 ppm (b)) remained unchanged. After conjugation of PEI2k to OXMC via reductive amination, PEI proton peaks (2.5–3.0 ppm) were observed in Fig. 1C. It was calculated by 1H NMR analysis that the degree of PEI conjugation was 10.1%, which means that one molecule of PEI2k was conjugated to every 9.87 glucose units of MC. In Fig. 1D, phenyl proton peaks of PBA (7.0–8.0 ppm (a), (b)) were detected after 4-BMPBA conjugation. Comparing the proton peaks of PBA (a and b) and PEI2k, it was identified that 1.8, 3.6, and 9.0 molecules of PBA were conjugated to one molecule of PEI2k with 3, 5, and 10 times excess of molar feed ratio, respectively. In this work, only MCPEI-PBA3.6 was used for further experiments because MCPEI-PBA1.8 was found to have high critical aggregation concentration and MCPEI-PBA9.0 showed low water solubility. Molecular weights of the polymers were measured by GPC. Mw of MCPEI2k was found to be 29.1 kDa (PDI = 1.64). However, MCPEI-PBA showed the apparently decreased Mw (13.8 kDa, PDI = 1.58), even though it was expected to have higher Mw than MCPEI2k due to the conjugation of PBA moieties. This underestimation of MCPEI-PBA molecular weight in GPC analysis may be explained by the possibility that MCPEI-PBA can form more condensed conformation or nano-aggregates via hydrophobic interaction between PBAs or B–N interaction between boronic acid of PBA and amine of PEI. Therefore, we examined that possibility by average particle size measurement and EF-TEM imaging.
Fig. 2. Concentration-dependent Z-average sizes of MCPEI-PBA nano-aggregates (A) and TEM images of MCPEI-PBA nano-aggregates at 500 mg/mL (B, C). Scale bars represent 200 nm (B) and 50 nm (C), respectively.
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Characterization of MCPEI-PBA nano-aggregates In Fig. 2, it was found that MCPEI-PBA itself could form nanoaggregates in aqueous solution by Zeta-sizer and TEM analysis. Zaverage size of MCPEI-PBA nano-aggregates was decreased with increasing concentration (Fig. 2A). Nano-aggregates of MCPEI-PBA
Fig. 3. Agarose gel electrophoresis result of MCPEI2k and MCPEI-PBA polyplexes (A). Numbers represent weight ratios (polymer/pDNA) of the polyplexes. Z-average sizes and Zeta-potential values of MCPEI2k polyplexes (B) and MCPEI-PBA polyplexes (C).
with about 760 nm Z-average size at 50 mg/mL was condensed to about 280 nm at 500 mg/mL with the concentration increase. It is thought that polymer molecules/unit volume increases with increasing polymer concentration, which would induce the multiplied hydrophobic interactions and B–N interactions between polymer molecules, finally leading to the formation of more compact and stable particles. The morphology of MCPEI-PBA nanoaggregates was also observed by EF-TEM (Fig. 2B and C). It showed the formation of spherical nano-aggregates with about 50 nm
Fig. 4. MTT assay results of MCPEI-PBA in HeLa (A), C2C12 (B), and HepG2 (C) cells.
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diameter at 500 mg/mL. It is thought that nano-aggregates observed by TEM in dry condition would be more condensed and appear to have smaller sizes than same nano-aggregates analyzed by Zeta-sizer for hydrodynamic size measurement in aqueous solution. In contrast with MCPEI-PBA result, particles of MCPEI2k without PBA moieties were not detected by Zeta-sizer, as expected. Then, molecular interactions of PBAs within MCPEI-PBA was investigated by fluorescence quenching method. PBA is known to emit fluorescence (lex: 302 nm, lem: 310–480 nm), which is reduced by its bond formation with diols or amines, via photoinduced electron transfer [22,23]. Therefore, MC, MCPEI2k, or PEI2k solution with various concentration was mixed with 50 mM of 3-APBA solution and each fluorescence change was observed. As shown in Fig. S2A, the fluorescence of 3-APBA displayed rapid decrease with increasing MCPEI2k concentration. However, the fluorescence of 3-APBA was not changed in spite of increasing MC concentration (Fig. S2B), which means that PBA would not interact with MC, probably due to the lack of diols in MC chain. In the case of PEI2k, similar fluorescence quenching with MCPEI-PBA result was also observed. This result suggests that PBA moiety in MCPEI-PBA would interact amine of PEI2k moiety via intramolecular or intermolecular B–N coordinate bonding, finally leading to the formation of MCPEI-PBA nano-aggregates. In addition, based on the fact that MCPEI-PBA with higher PBA conjugation degree was not soluble in aqueous solution, hydrophobic interaction of PBA is also considered as another major factor for nano-aggregates formation of MCPEI-PBA. Critical aggregation concentration (CAC) of MCPEI-PBA was measured as 7.43 102 mg/mL by pyrene absorbance method, which means that MCPEI-PBA can encapsulate hydrophobic small molecules inside of the nano-aggregates. Characterization of MCPEI-PBA polyplexes pDNA condensing ability of MCPEI-PBA was examined by agarose gel electrophoresis. As shown in Fig. 3, MCPEI2k could retard pDNA from a weight ratio of 0.5. In contrast with MCPEI2k, MCPEI-PBA was found to retard pDNA from a weight ratio of 1. This reduced pDNA condensing ability of MCPEI-PBA is probably due to
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the decreased free amine moiety in MCPEI-PBA by PBA conjugation [24] and to the steric hindrance of PBA ring structure to electrostatic interaction with pDNA [25]. Z-average size and Zeta-potential value of polyplexes were measured by Zeta-sizer. Zeta-potential value of MCPEI2k polyplex was negative potential value of 33.1 mV at a weight ratio of 0.25 and rapidly switched to positive potential values at higher weight ratios over 1 (Fig. 3B). Average size of MCPEI2k polyplex was about 1 mm at a weight ratio of 0.25 and abruptly decreased to 200– 250 nm with increasing polyplex weight ratios over 2, which means the formation of stable polyplexes. In contrast with MCPEI2k, MCPEI-PBA was found to form nano-sized polyplex (127.7 nm) at a low weight ratio of 0.25, which was gradually increased to 200–250 nm at higher weight ratios over 5 (Fig. 3C). As already confirmed, this may be because MCPEI-PBA itself could form nano-aggregates without polyplex formation and bulky MCPEI-PBA nano-aggregates could be more condensed by complexation with pDNA. Contrary to MCPEI2k, Zeta-potential values of MCPEI-PBA was switched to positive values from a weight ratio of 5 by less chargeable amine moiety of MCPEI-PBA than that of MCPEI2k. Also, it is thought that cationic MCPEI-PBA could bind to negatively charged polyplex at weight ratios ranging from 0.25 to 5, increasing polyplex size unlike MCPEI2k. These results demonstrate that MCPEI-PBA could form positively charged and nano-sized polyplex with pDNA which have suitable values for efficient adsorption onto cellular membrane and cellular uptake [26]. In vitro cell experiments of MCPEI-PBA Cytotoxicity of MCPEI-PBA was assessed by MTT assay in HeLa, C2C12, and HepG2 cells. As shown in Fig. 4, PEI25k displayed drastic decrease of cell viability with increasing concentration in all cells (IC50, HeLa: 9.2 mg/mL, C2C12: 3.3 mg/mL, HepG2: 24.3 mg/ mL), which indicates its significant cytotoxicity. Backbone polymer, MCPEI2k also showed considerable cytotoxicity (IC50, HeLa: 75.3 mg/mL, C2C12: 28.7 mg/mL, HepG2: 84.7 mg/mL), although it was relatively low in comparison with that of PEI25k. However, viabilities of MCPEI-PBA-treated cells were found to be similar to or much higher than that of MCPEI2k-treated cells in all cells (IC50,
Fig. 5. Transfection results of MCPEI-PBA in HeLa (A, C), C2C12 (B, E), and HepG2 (C, F) cells. Serum-free condition: A–C, serum-containing condition: D– F.
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HeLa: >100 mg/mL, C2C12: 71.4 mg/mL, HepG2: 73.2 mg/mL), demonstrating its low cytotoxicity. It may be induced by decreased charge density of MCPEI-PBA due to PBA conjugation. Transfection efficiency of MCPEI-PBA was evaluated by luciferase transgene expression assay in HeLa, C2C12, and HepG2 cells. Fig. 5A–C shows transfection experiment results in the absence of serum. Although the transfection efficiency of MCPEI-PBA was gradually increased with elevating weight ratios ranging from 5 to 25, it was found to be similar to or lower than that of MCPEI2k in HeLa and C2C12 cells. This result means that PBA conjugation to MCPEI2k could not affect the transfection efficiency in those cells. The transfection efficiency of MCPEI2k and MCPEI-PBA was 2.7 and 5.6 times lower in HeLa cells and 7.5 and 7.9 times lower in C2C12 cells than that of PEI25k at a weight ratio of 25 respectively. However, interestingly, MCPEI-PBA showed 1.4 times higher transfection efficiency than MCPEI2k and 4.2 times higher than PEI25k in HepG2 cell at a same weight ratio. In the presence of serum (Fig. 5D–F), although all the transfection efficiency of the polymers were decreased probably due to non-specific interactions with serum proteins, MCPEI2k and MCPEI-PBA exhibited less decreased transfection efficiency than PEI25k, suggesting their serum stability due to the charge shielding effect of hydroxy and methoxy functional groups in MC chains [10]. Especially, MCPEIPBA displayed 5.6 times higher transfection efficiency than PEI25k in HepG2 cells at a weight ratio of 25. This improved transfection efficiency of MCPEI-PBA in HepG2 cells suggests the possibility of its specific interaction with hepatocellular carcinoma. Hepatocellular carcinoma-targeting of MCPEI-PBA Human hepatocellular carcinoma, HepG2 cells are known to overexpress sialyl-Lewis X (sLex) antigen [27] or asialoglycoprotein receptors (ASGPRs) [28] on cellular membrane. Many literatures have reported the targeted drug or gene delivery to cancer cells by utilizing specific interaction of PBA moieties of the delivery carriers with diol of sLex [29,30]. Also, galactosylated drug or gene delivery carriers have been developed for Hepatocellular carcinoma-targeting via strong binding of galactose to ASGPRs [31,32]. In order to examine the specific interaction of MCPEI-PBA with sLex or ASGPRs, free PBA or galactose was pretreated in the transfection experiment for competition assay in HepG2 cells. As shown in Fig. 6A, transfection efficiencies of PEI25k (122.7% of PBAfree condition value) and MCPEI2k (88.6%) were not decreased significantly in PBA pretreatment condition, due to the lack of PBA moieties which can recognize sLex on HepG2 cell surfaces. Interestingly, MCPEI-PBA (107.7%) also did not show the decreased transfection efficiency even in the presence of PBA. It may be explained that almost PBA moieties of MCPEI-PBA would be consumed for intramolecular or intermolecular B–N coordinate bonding of amine of PEI2k. In the case of galactose pretreatment, all the transfection efficiencies of the polymers were generally decreased, probably due to the nonspecific interaction of galactose with high concentration (Fig. 6B). However, the transfection efficiency of MCPEI-PBA in galactose condition was dramatically decreased to 6.7% value of the efficiency in galactose-free condition in comparison with other polymers (PEI25k: 38.6%, MCPEI2k: 26.7% values), probably due to decreased interaction with ASGPRs by galactose binding. These results means that the transfection mechanism of MCPEI-PBA in hepatocellular carcinoma would be mediated by interaction with ASGPRs, not with sLex. In addition, the transfection experiments using non-hepatocellular carcinoma cells, HeLa and C2C12 cells, were performed in galactose-pretreatment condition. As shown in Fig. S3, MCPEI-PBA showed similarly or even less decreased transfection efficiency (HeLa: 5.1% of normal condition value, C2C12: 8.9%) in comparison with PEI25k (HeLa: 5.3%, C2C12: 3.7%) and MCPEI2k (HeLa: 3.6%,
Fig. 6. Transfection results of MCPEI-PBA with PBA pretreatment (A) and galactose pretreatment (B) in HepG2 cells.
C2C12: 4.2%) in the galactose-pretreatment condition. These results displayed that the transfection efficiencies of all tested polymers were generally decreased probably due to the nonspecific interaction of galactose, in non-hepatocellular carcinoma cells lacking ASGPRs, contrary to HepG2 cell result. The size change of the polyplexes in galactose condition was analyzed to examine the effect of galactose to the polyplex stability (Fig. S4). Similarly enlarged MCPEI-PBA polyplexes (203.2 → 293.2 nm) to MCPEI2k polyplexes (236.3 → 318.4 nm) suggested that the interaction of galactose with MCPEI-PBA polyplex would be similar to that of MCPEI2k polyplex and not specific for PBA moieties, meaning that galactose with even high concentration would not induce abrupt swelling or disruption of MCPEI-PBA polyplexes, which may lead to the significant decrease of the transfection efficiency. Interaction of MCPEI-PBA with ASGPRs was further investigated by GFP expression comparison in HepG2 cells (Fig. 7A). It was observed by fluorescence microscopy that the cells treated by PEI25k and MCPEI2k polyplexes (GFP pDNA) showed no significant GFP expression decrease in galactose pretreatment condition, meaning their low interaction with ASGPRs. However, GFP
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Fig. 7. GFP transgene expression results of polyplexes with galactose pretreatment (A) and cellular uptake efficiency of polyplexes with galactose pretreatment (B) in HepG2 cells.
expression of MCPEI-PBA polyplexes was considerably reduced in galactose pretreatment condition. In addition, the effect of galactose pretreatment to cellular uptake of the polyplexes (YOYO-1 iodide-labeled pDNA) was also analyzed by FACS. As shown in Fig. 7B, the cellular uptake efficiency of PEI25k and MCPEI2k polyplexes was slightly changed in galactose pretreatment condition. MCPEI-PBA polyplexes displayed about 10% decrease cellular uptake efficiency (65.3%) in galactose pretreatment condition, comparing with non-treatment condition (74.7%). These results suggest that pretreated galactose could bind to ASGPRs on HepG2 cell surfaces, inhibiting interaction of MCPEIPBA with ASGPRs and decreasing cellular uptake and transgene expression. The mechanism of interaction of MCPEI-PBA and ASGPRs was not fully understood. However, it was reported that ASGPRs also can recognize other sugar molecules such as glucose derivatives, except galactose or N-acetyl galactosamine [33]. Considering this report, one of possible explanation about ASGPRs-targeting of MCPEI-PBA may be the synergistic effect of interaction of ASGPRs with methyl glucose unit of MCPEI-PBA and hydrogen bonding and hydrophobic interaction with tryptophan of ASGPRs [28] or p–p interaction of phenyl ring of PBA with tyrosine residues of ASGPRs [34]. Characterization of MCPEI-PBA/Dox Hydrophobic anticancer agent, Doxorubicin (Dox) was encapsulated in MCPEI-PBA nano-aggregates by dialysis method. When the mixing ratio (Dox/MCPEI-PBA, w/w) was 1/10, the drug loading efficiency of was measured to be about 100% and drug loading content was 8.8%, which means that MCPEI-PBA nano-aggregates could encapsulate Dox very efficiently. Average sizes and Zeta-potentials of MCPEI-PBA/Dox nanoaggregates and MCPEI-PBA/Dox/pDNA complexes were examined. Interestingly, average size of MCPEI-PBA/Dox nano-aggregates was measured to be 287.3 79.6 nm, which was significantly decreased in comparison with that of MCPEI-PBA nano-aggregates (about
760 nm) at same concentration probably due to the condensation by hydrophobic interaction with Dox molecules. In addition, MCPEIPBA/Dox/pDNA complexes showed 259.3 24.4 nm, which suggests the formation of more compact and homogeneous nano-particles by electrostatic interactionwith negatively charged pDNA. In the case of Zeta-potential measurements, MCPEI-PBA/Dox nano-aggregates and MCPEI-PBA/Dox/pDNA complexes displayed 44.9 4.3 mV and 49.3 2.1 mV, respectively. Zeta-potential value of MCPEIPBA/Dox/pDNA complexes was almost similar with that of MCPEIPBA/pDNA polyplexes, demonstrating that MCPEI-PBA/Dox nanoaggregates can form MCPEI-PBA/Dox/pDNA complexes with pDNA by covering the surfaces and these nano-particles can adsorb readily onto negatively charged cellular membrane. Release behavior of Dox from MCPEI-PBA/Dox nano-aggregates was monitored at pH 5.5 and 7.4, which mimic late endosome/ lysosome and physiological extracellular environment (Fig. S5). The release rate of Dox was fast until 12 h, showing somewhat initial burst. The release of Dox occurred sustainably and was up to 30.5% at pH 5.5 and 22.5% at pH 7.4 after 48 h of incubation. This result means that Dox could be released faster at pH 5.5 than pH 7.4 because protonation of Dox could increase its solubility and protonated PEI could be swelled via charge repulsion. Low release rate of Dox means that Dox can interact strongly with MCPEI-PBA molecules, leading to the inhibition of its release. It may be interpreted by several following inferences. Firstly, Dox molecules also can be encapsulated in the interior of PEI [35]. Secondly, average size of MCPEI-PBA nano-aggregates was dramatically decreased by encapsulation of Dox, which suggests this spatial shrinkage may disturb the diffusion of Dox. Thirdly, PBA moieties may interrupt the release of Dox by hydrogen bonding, hydrophobic interaction or p–p stacking, or B–N boding between PBA and Dox [36]. Although the release of Dox was slow, it may minimize the loss of Dox from the nano-aggregates via spontaneous release during delivery procedure to the target cells. Fig. S6 shows the schematic illustration for MCPEI-PBA/Dox/ pDNA nano-aggregates. According to the above results, it is
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Conclusion PBA-conjugated cationic methylcellulose derivative (MCPEIPBA) was synthesized for drug/gene co-delivery systems. MCPEIPBA could form nano-aggregates by self-assembly via hydrophobic interaction and B–N bonding, which can encapsulate hydrophobic Dox molecules efficiently and further condense pDNA to construct nano-sized complexes with pDNA. MCPEI-PBA showed high transfection efficiency especially in hepatocellular carcinoma and it may be deduced that MC and PBA can specifically interact with ASGPRs on cell membrane. Finally, MCPEI-PBA/Dox/pDNA (apoptin) complexes exhibited efficient anticancer activity due to the co-delivery of Dox and apoptin gene by nano-particles. These results demonstrate the potential of MCPEI-PBA as a targeted codelivery carrier of drug/gene for hepatocellular carcinoma. Acknowledgments
Fig. 8. Anticancer activity result of MCPEI-PBA/Dox/pJDK-apoptin complexes in HepG2 cells.
expected that Dox molecules would be encapsulated inside of the hydrophobic core and PEI chains and pDNA would be complexed in outer region of the nano-aggregates, covered by cationic MCPEIPBA chains. Cancer cell killing activity of MCPEI-PBA/Dox nano-aggregates and MCPEI-PBA/Dox/pDNA complexes Cancer cell killing effect of MCPEI-PBA/Dox nano-aggregates and MCPEI-PBA/Dox/pDNA complexes were investigated by MTT assay in HepG2 cells in order to identify the combinatorial effect for co-delivery of Dox and pDNA. As shown in Fig. 8, only pDNA could not induce any cytotoxic effect to HepG2 cells because of very low delivery efficiency by the absence of the carrier. PEI25k polyplexes showed 77.6% cell viability, meaning the low level of apoptin transgene expression. On the other hand, cell viability of MCPEI-PBA polyplexes-treated cell was decreased to 54.9%, which showed superior gene delivery efficiency to PEI25k polyplexes as already shown in luciferase assay. In the case of Dox treatment, Free Dox could reduce the cell viability to 16.9%. MCPEI-PBA/Dox nano-aggregates exhibited 39.4% cell viability. Finally, MCPEI-PBA/Dox/pDNA was found to reduce cell viability to 30.5%. These results demonstrated that co-delivery of Dox and pDNA by MCPEI-PBA showed enhanced anti-cancer activity due to the combinatorial therapeutic effect of cytotoxicity of Dox and apoptosis by expressed apoptin, in comparison with other singular delivery of Dox or pDNA. Although cancer cell killing effect of free Dox was very high, several studies reported that free Dox delivery could induce serious heart damage [37] and multi-drug resistance (MDR) of cancer cells could reduce the therapeutic effect by exporting internalized Dox [38]. This MDR of HepG2 cells was also observed in Fig. S7, which maintained the cell viability similarly (~40%) even at elevated concentration (2–10 mg/mL). However, MCPEI-PBA/Dox nanoaggregates could kill HepG2 cells gradually in proportion to its concentration and finally showed higher anticancer activity over 5 mg/mL than free Dox, demonstrating these nano-aggregates could overcome MDR of the cells, similarly with other nanocarriers [39].
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Phenylboronic acid-conjugated cationic methylcellulose for hepatocellular carcinoma-targeted drug/gene co-delivery systems Ju Hyeon Jeona,1, Jae Hong Parka,1, Tae-il Kima,b,* a Department of Biosystems & Biomaterials Science and Engineering, College of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea b Research Institute of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 January 2019 Received in revised form 28 February 2019 Accepted 8 March 2019 Available online 16 March 2019
Phenylboronic acid-conjugated cationic methylcellulose (MCPEI-PBA) was synthesized and characterized for hepatocellular carcinoma-targeted drug/gene co-delivery systems. MCPEI-PBA was obtained by reductive amination of polyethylenimine 2k to oxidized methylcellulose and conjugation of PBA. MCPEIPBA could form nano-aggregates via B–N coordinate bonding and hydrophobic interaction and also form positively charged and nano-sized polyplexes. Cytotoxicity of MCPEI-PBA was low and MCPEI-PBA showed higher transfection efficiency than PEI25k in hepatocellular carcinoma. Galactose pretreatment experiment suggested its specific interaction with asialoglycoprotein receptors. MCPEI-PBA could encapsulate doxorubicin (Dox) efficiently. Finally, MCPEI-PBA/Dox/pJDK-apoptin complexes showed high anticancer activity due to the combinatorial effect of Dox and expressed apoptin. © 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Keywords: Drug/gene co-delivery Methylcellulose Phenylboronic acid Nano-aggregates Hepatocellular carcinoma-targeting
Introduction Drug and gene co-delivery systems have been reported to induce improved therapeutic efficacy via synergistic effect of drug and therapeutic gene in comparison with single delivery [1]. Especially, it has advantages for cancer therapy because of heterogeneity of cancer cells and complexity of cancer cell growth and development [2,3]. Anthracycline derivative anticancer agent, doxorubicin (Dox) shows anticancer activity by inhibiting topoisomerase II and forming free radicals in cells [4]. It is a hydrophobic molecule, which requires proper encapsulating delivery carriers for solvation and administration. Methylcellulose (MC) is one of the amphiphilic cellulose ether derivatives containing methylated hydroxyl groups of cellulose [5]. From its classic applications for food additives or cosmetics, applications of MC were extended to hydrogel and drug delivery systems [6–8] due to its biocompatibility and sol–gel transition property [9]. Recently, polyethylenimine-grafted MC (MCPEI) has
* Corresponding author at: Department of Biosystems & Biomaterials Science and Engineering, College of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea. E-mail address: [email protected] (T.-i. Kim). 1 These authors contributed equally to this work.
been reported as an efficient gene delivery system, possessing serum compatibility and endosome buffering ability [10]. In this work, MCPEI was employed as a backbone polymer for drug/gene co-delivery carrier systems. Then, phenylboronic acid (PBA) was conjugated to MCPEI, synthesizing MCPEI-PBA. PBA has been utilized for hydrogel, cancer-targeting drug or gene delivery carriers, and probes [11,12] because of its forming ability of reversible boronate ester bond with cis-1,2 or cis-1, 3 diol of sugar molecules [13,14], especially sialic acids of glycoproteins and glycolipids overexpressed in cancer cells. In addition, PBA also can interact with amine group via B–N coordinate bond formation [15]. Therefore, it was hypothesized that MCPEI-PBA can form nanoaggregates encapsulating hydrophobic drug molecules by B–N coordinate bond formation between PBA and PEI and that cationic MCPEI-PBA/Dox nano-aggregates can further form complexes with anionic pDNA, finally constructing drug/gene co-delivery carrier. As a therapeutic gene, apoptin-encoding pDNA, pJDK-apoptin was used [16]. Apoptin is known as an antitumor agent inducing tumor cell-selective apoptosis [17]. Although several gene delivery systems have been developed for apoptin gene delivery [16,18], its therapeutic application has limitations such as lack of efficient delivery systems [19,20]. Therefore, we tried to show the capability of MCPEI-PBA for efficient apoptin gene delivery systems in this work. Here, MCPEI-PBA was synthesized and its characterizations for drug and gene co-delivery carrier systems were reported for cancer therapy. Moreover, its unique targeting ability for hepatocellular carcinoma via asialoglycoprotein receptor (ASGPR) interaction was
https://doi.org/10.1016/j.jiec.2019.03.016 1226-086X/© 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
J.H. Jeon et al. / Journal of Industrial and Engineering Chemistry 75 (2019) 148–157
identified and analyzed. To the best of our knowledge, this is the first report regarding the development of PBA-conjugated drug/ gene co-delivery carrier with ASGPR-mediated hepatocellular carcinoma-targeting ability. Experimental Materials Methylcellulose (15 cP, 2% in H2O), poly(ethylenimine) (PEI, molecular weight 2k and 25 kDa), agarose, ethylenediaminetetraacetic acid (EDTA), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and pyrene were purchased from SigmaAldrich (St. Louis, MO). Sodium periodate, sodium tetrahydroborate, and ethylacetate was purchased from Junsei (Tokyo, Japan). 4(Bromomethy)phenylboronic acid (4-BMPBA), 3-aminophenylboronic acid (3-APBA), 4-carboxyphenylboronic acid (4-CPBA), D(+)-galactose, and DMEM + GlutaMax-I medium were purchased from Thermo Fisher Scientific TM(Waltham, MA). YOYO-1 iodide, fetal bovine serum (FBS), 0.25% Trypsin-EDTA, Dulbecco’s phosphate buffered saline (DPBS), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Invitrogen (Carlsbad, CA). Minimum Essential Medium with Earle’s Balanced Salts (MEM/ EBSS) was purchased from GE Healthcare Life Sciences (South Logan, UT). Luciferase assay system and reporter lysis buffer were TM purchased from Promega (Madison, WI). BCA protein assay kit was purchased from PIERCE (Rockford, IL). Doxorubicin was purchased from Bio-techne (Minneapolis, MN). All other chemicals were purchased and used without any further purification. Synthesis of MCPEI-PBA First, backbone polymer, MCPEI2k was synthesized according to the previous report [10]. Briefly, methyl cellulose was oxidized by sodium periodate. After 24 h of oxidation reaction, the reaction mixture was dialyzed against ultrapure water with dialysis membrane (MWCO = 6–8 k). Then, PEI2k (5 molar to glucose units of oxidized MC, OXMC) water solution was added to OXMC solution for the conjugation of PEI2k to OXMC. After 24 h of reaction, the reaction mixture was reduced by sodium tetrahydroborate (water) for another 24 h. The product, MCPEI2k was obtained after dialysis followed by lyophilization. In order to synthesize MCPEI-PBA, 4-BMPBA (3, 5, and 10-fold molar excess to PEI units of MCPEI2k, respectively) was reacted with MCPEI2k in MeOH solution at 50 C for 24 h. Unreacted 4BMPBA was extracted with ethylacetate. After extraction, the reaction mixture was purified by dialysis against 0.2 M NaCl solution and continuously water for 1 day each with dialysis membrane (MWCO = 6–8k). The final product, MCPEI-PBA was obtained by lyophilization. The each step of polymer synthesis was confirmed by 1H NMR (D2O, 400 MHz JEOL JNM-LA400). Molecular weight measurements by gel permeation chromatography (GPC) The molecular weights of polymers were determined by GPC (YL-9100, Young Lin Instrument, Korea) with Ultrahydrogel 250 column (Waters, Milford, MA) with 1% formic acid as an eluent. Polyethyleneglycols with various molecular weights were used as standards. The concentration of the samples and the flow rate were set to 10 mg/mL and 0.6 mL/min, respectively. Critical aggregation concentration (CAC) measurements In order to characterize CAC of MCPEI-PBA, pyrene absorbance method was used as previously reported [21]. 20 mL of pyrene
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ethanol solutions (0.1 mM) were added to vials and dried under vacuum overnight in dark condition. After formation of pyrene film, 1 mL of MCPEI-PBA water solutions with various concentrations were added to the vials, giving the final pyrene concentration of 6 107 M. The MCPEI-PBA/pyrene solutions were sonicated for 10 min and incubated with gentle mixing at room temperature for 24 h. Then, absorbance of pyrene was measured by UV/Vis spectrometer (Optizen POP BIO, Mecasys, Korea) using quartz cuvettes in the range of 200–400 nm. The total absorbance by sum of four absorbance points (242, 272, 320, and 336 nm) was plotted against MCPEI-PBA concentrations. CAC of MCPEI-PBA sample was set to the center of the sigmoid. Encapsulation of doxorubicin (Dox) in MCPEI-PBA nanoparticles DoxHCl solution (2 mg Dox, DMSO) was mixed with 2 equivalent moles of triethylamine. After 1 day, Dox solution was added to MCPEI-PBA solution (20 mg, DMSO) for 1.5 h. MCPEI-PBA/ Dox solution was added dropwise to water. After 1 day of stirring, the mixture solution was dialyzed against water with dialysis membrane (MWCO = 6-8k) for 1 day and the final MCPEI-PBA/Dox was obtained after lyophilization. Evaluation of Dox loading in MCPEI-PBA nanoparticles MCPEI-PBA/Dox was dissolved in DMSO and the fluorescence of Dox was measured by a microplate reader at excitation wavelength of 480 nm and emission wavelength of 590 nm. Loaded Dox amount was determined according to the previously prepared calibration curve of Dox solution (DMSO). Drug loading content (DLC) and drug loading efficiency (DLE) were calculated as following formula. DLC ð% Þ ¼
Weight of Loaded DOX 100 Weight of MCPEI PBA
DLE ð% Þ ¼
Weight of Loaded DOX 100 Weight of total DOX f or loading
Average particle size and zeta-potential value measurements Average particle sizes of MCPEI-PBA were measured by a Zetasizer Nano ZS (Malvern Instruments, UK) with He-Ne laser beam (633 nm) at 25 C. MCPEI-PBA water solutions with various concentrations were used for measurements. MCPEI2k water solutions were also used for comparison. In the case of polyplexes, 1 mL of MCPEI-PBA/pDNA polyplex solutions were prepared in ultrapure water at various weight ratios. After 30 min of incubation, average particle sizes and Zetapotential values were measured by a Zeta-sizer. Furthermore, MCPEI-PBA/Dox/pDNA water solution was prepared by mixing of MCPEI-PBA/Dox solution (50 mg/mL) and pDNA (1 mg) at a weight ratio of 25. Average particle sizes and Zetapotential values of MCPEI-PBA/Dox and MCPEI-PBA/Dox/pDNA were measured by a Zeta-sizer. Transmission electron microscopy (TEM) The morphology of MCPEI-PBA nanoparticles were observed by Energy-filtering transmission electron microscopy (EF-TEM, LIBRA 120, Carl Zeiss, Germany). 500 mg/mL of MCPEI-PBA water solutions were prepared and deposited on TEM copper grid plates 4 times. The samples were then stained with filtered uranium acetate for 10 s. After absorption of residual solutions, the images were visualized with an accelerating voltage of 120 kV.
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Plasmid DNA purification Plasmid DNAs (pDNAs), pCN-Luci encoding luciferase, pEGFPC2 encoding GFP, and pJDK-apoptin encoding apoptin were amplified with Escherichia Coli DH5a, respectively and purified with Nucleobond Xtra Midi kit (Macherey-Nagel, Germany). Agarose gel electrophoresis Agarose gel electrophoresis was performed to assess pDNA condensation ability of MCPEI-PBA. Agarose gel (0.7%, w/v) containing ethidium bromide (0.5 mg/mL) was prepared in TrisAcetate-EDTA (TAE) buffer. The MCPEI-PBA polyplexes were prepared in Hepes buffer (pH 7.4) at various weight ratios (polymer/pDNA) for 30 min of incubation at room temperature. After loading of samples, the electrophoresis was run for 12 min at 100 V (Mupid-2plus, Takara Bio Inc., Japan). The locations of pDNA bands were visualized by UV illuminator (Gel Doc XRS + gel documentation system, Bio-Rad, Hercules, CA). Cell culture Human cervical adenocarcinoma cells (HeLa), mouse myoblast cells (C2C12), and human hepatocellular carcinoma cells (HepG2) were maintained in a 5% CO2 incubator at 37 C in DMEM + GlutaMax-I medium, DMEM medium, and MEM/EBSS medium, respectively, which were supplemented with 10% FBS and 1% penicillin/streptomycin. MTT assay MTT assay was performed to examine the cytotoxicity of polymers. Cells were seeded in a 96-well plate at a density of 1 104 cells/well, respectively. Having achieved 70–80% confluence after 24 h, the cells were exposed to 100 mL polymer solutions (serum-free medium) with various concentrations for 4 h. Subsequently, the media was changed with fresh medium (10% FBS). After 24 h, the cells were treated with 25 mL of MTT stock solution (2 mg/mL in DPBS) and incubated for 2 h at 37 C. After removing each medium carefully, 150 mL of DMSO was added to each well to dissolve the formazan crystal formed by proliferating cells. The absorbance was measured at 570 nm using a microplate reader. Results were presented as relative cell viabilities (RCV, percentage values relative to value of untreated control cells). In vitro transfection experiments in serum or without serum condition The transfection efficiencies of polymers were examined by measuring luciferase reporter transgene expression in non-serum or serum conditions. Cells were seeded in a 24-well plate at a density of 5 104 cells/well and were grown to reach 70–80% confluence. Before transfection, the media were exchanged with serum-free medium for the assay in non-serum condition and with serum-containing medium (10% FBS) for the assay in serum condition, respectively. Then, the cells were treated with polyplex solutions (0.5 mg pDNA) with various weight ratios. The weight ratio of PEI25k polyplex was 1.0. In the case of PBA or galactose inhibition in HepG2 cells, 4-CPBA solutions (10 mM 1% MeOH/ medium, v/v) or galactose solutions (100 mM medium) were pretreated for 30 min. After 4 h of incubation, the transfection media were exchanged with fresh medium (10% FBS) and the cells were incubated for further 2 days. Subsequently, media were removed and the cells were rinsed with DPBS and shaken for 30 min at room temperature in Reporter Lysis Buffer. The collected cell lysates were centrifuged and the supernatants were used for luciferase
activity measurements with a microplate reader. A protein TM quantification assay was performed using a BCA Protein Assay Reagent Kit to measure total amount of cellular proteins. The final results were presented in terms of RLU/mg cellular protein. Green fluorescence protein (GFP) expression The galactose inhibition effect to transfection efficiencies of polymers were also examined by observing GFP transgene expression. HepG2 cells were seeded in a 6-well plate at a density of 2 105 cells/well and were grown to reach 70–80% confluence. Before transfection, the media were exchanged with serum-free medium. Then, the cells were treated with polyplex solutions (4 mg pEGFP-C2, PEI25k: weight ratio = 1.0, MCPEI2k, MCPEI-PBA: weight raio = 25). In the case of galactose inhibition, galactose solutions (100 mM medium) were pre-treated for 30 min. After 4 h of incubation, the transfection media were exchanged with fresh medium (10% FBS) and the cells were incubated for further 2 days. Then, expressed GFP of the cells was observed by fluorescence microscopy (iRis Digital Cell Imaging System, Logos biosystems, Korea). Flow cytometry Flow cytometry was performed to investigate the cellular uptake of MCPEI-PBA polyplexes. HepG2 cells were seeded at a density of 2 105 cells/well in a 6-well plate and grown to reach 70–80% confluence prior to transfection. Before transfection, medium of each well was exchanged for fresh serum-free medium. pDNA was labeled with YOYO-1 iodide (1 molecule of the dye per 50 base pairs of the nucleotide). The cells were treated with the polyplex solutions (1 mg pDNA, serum-free medium) at a weight ratio of 25 for 4 h. In the case of galactose inhibition, galactose solutions (100 mM medium) were pre-treated for 30 min. Then, medium was aspirated off from the wells and the cells were washed two times with ice-cold DPBS. After trypsinization, the cells were suspended in 1 mL DPBS. The cellular uptake of fluorescence-labeled polyplexes was measured by BD Accuri C6 flow cytometer (Becton Dickinson, San Jose, CA) at a minimum of 1 104 cells gated per sample. Analysis was performed by using BD Accuri C6 software. Anticancer activity of MCPEI-PBA/Dox/pJDK-apoptin polyplexes Cells were seeded in a 24-well plate at a density of 5 104 cells/ well. Having achieved 70–80% confluence after 24 h, the cells were exposed to Dox solution, pJDK-apoptin solution, PEI25k/pJDKapoptin polyplexes solution (weight ratio of 1.0), MCPEI-PBA/Dox solution, MCPEI-PBA/pJDK-apoptin polyplexes solution (weight ratio of 25), and MCPEI-PBA/Dox/pJDK-apoptin polyplexes solution (weight ratio of 25) for 4 h. Each solution contains 1 mg pDNA or 2.4 mg Dox in serum-free medium. Subsequently, the media was changed with fresh medium (10% FBS). After 48 h of incubation, MTT assay was performed in an identical manner with above procedures. Results and discussions Synthesis and characterization of MCPEI-PBA In order to synthesize MCPEI-PBA, backbone polymer, MCPEI2k was synthesized by periodate oxidation of MC followed by reductive amination of PEI2k to oxidized MC, according to the previous study [10]. Then, 4-BMPBA was conjugated to amines of MCPEI2k via nucleophilic substitution of amine to bromide of 4BMPBA. Fig. S1 shows the synthesis scheme of MCPEI-PBA.
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Fig. 1. Molecular structures and 1H NMR spectra of MC (A), OXMC (B), MCPEI2k (C), and MCPEI-PBA (D).
The synthesis of MCPEI-PBA was confirmed by 1H NMR (Fig. 1). H-2(2-OH) protons of MC (Fig. 1A, 3.3–3.4 ppm (a)) disappeared after oxidation (Fig. 1B) due to the conversion of hydroxyl group to aldehyde and H-2(2-OMe) protons of MC (Fig. 1A, 3.1–3.2 ppm (b)) remained unchanged. After conjugation of PEI2k to OXMC via reductive amination, PEI proton peaks (2.5–3.0 ppm) were observed in Fig. 1C. It was calculated by 1H NMR analysis that the degree of PEI conjugation was 10.1%, which means that one molecule of PEI2k was conjugated to every 9.87 glucose units of MC. In Fig. 1D, phenyl proton peaks of PBA (7.0–8.0 ppm (a), (b)) were detected after 4-BMPBA conjugation. Comparing the proton peaks of PBA (a and b) and PEI2k, it was identified that 1.8, 3.6, and 9.0 molecules of PBA were conjugated to one molecule of PEI2k with 3, 5, and 10 times excess of molar feed ratio, respectively. In this work, only MCPEI-PBA3.6 was used for further experiments because MCPEI-PBA1.8 was found to have high critical aggregation concentration and MCPEI-PBA9.0 showed low water solubility. Molecular weights of the polymers were measured by GPC. Mw of MCPEI2k was found to be 29.1 kDa (PDI = 1.64). However, MCPEI-PBA showed the apparently decreased Mw (13.8 kDa, PDI = 1.58), even though it was expected to have higher Mw than MCPEI2k due to the conjugation of PBA moieties. This underestimation of MCPEI-PBA molecular weight in GPC analysis may be explained by the possibility that MCPEI-PBA can form more condensed conformation or nano-aggregates via hydrophobic interaction between PBAs or B–N interaction between boronic acid of PBA and amine of PEI. Therefore, we examined that possibility by average particle size measurement and EF-TEM imaging.
Fig. 2. Concentration-dependent Z-average sizes of MCPEI-PBA nano-aggregates (A) and TEM images of MCPEI-PBA nano-aggregates at 500 mg/mL (B, C). Scale bars represent 200 nm (B) and 50 nm (C), respectively.
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Characterization of MCPEI-PBA nano-aggregates In Fig. 2, it was found that MCPEI-PBA itself could form nanoaggregates in aqueous solution by Zeta-sizer and TEM analysis. Zaverage size of MCPEI-PBA nano-aggregates was decreased with increasing concentration (Fig. 2A). Nano-aggregates of MCPEI-PBA
Fig. 3. Agarose gel electrophoresis result of MCPEI2k and MCPEI-PBA polyplexes (A). Numbers represent weight ratios (polymer/pDNA) of the polyplexes. Z-average sizes and Zeta-potential values of MCPEI2k polyplexes (B) and MCPEI-PBA polyplexes (C).
with about 760 nm Z-average size at 50 mg/mL was condensed to about 280 nm at 500 mg/mL with the concentration increase. It is thought that polymer molecules/unit volume increases with increasing polymer concentration, which would induce the multiplied hydrophobic interactions and B–N interactions between polymer molecules, finally leading to the formation of more compact and stable particles. The morphology of MCPEI-PBA nanoaggregates was also observed by EF-TEM (Fig. 2B and C). It showed the formation of spherical nano-aggregates with about 50 nm
Fig. 4. MTT assay results of MCPEI-PBA in HeLa (A), C2C12 (B), and HepG2 (C) cells.
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diameter at 500 mg/mL. It is thought that nano-aggregates observed by TEM in dry condition would be more condensed and appear to have smaller sizes than same nano-aggregates analyzed by Zeta-sizer for hydrodynamic size measurement in aqueous solution. In contrast with MCPEI-PBA result, particles of MCPEI2k without PBA moieties were not detected by Zeta-sizer, as expected. Then, molecular interactions of PBAs within MCPEI-PBA was investigated by fluorescence quenching method. PBA is known to emit fluorescence (lex: 302 nm, lem: 310–480 nm), which is reduced by its bond formation with diols or amines, via photoinduced electron transfer [22,23]. Therefore, MC, MCPEI2k, or PEI2k solution with various concentration was mixed with 50 mM of 3-APBA solution and each fluorescence change was observed. As shown in Fig. S2A, the fluorescence of 3-APBA displayed rapid decrease with increasing MCPEI2k concentration. However, the fluorescence of 3-APBA was not changed in spite of increasing MC concentration (Fig. S2B), which means that PBA would not interact with MC, probably due to the lack of diols in MC chain. In the case of PEI2k, similar fluorescence quenching with MCPEI-PBA result was also observed. This result suggests that PBA moiety in MCPEI-PBA would interact amine of PEI2k moiety via intramolecular or intermolecular B–N coordinate bonding, finally leading to the formation of MCPEI-PBA nano-aggregates. In addition, based on the fact that MCPEI-PBA with higher PBA conjugation degree was not soluble in aqueous solution, hydrophobic interaction of PBA is also considered as another major factor for nano-aggregates formation of MCPEI-PBA. Critical aggregation concentration (CAC) of MCPEI-PBA was measured as 7.43 102 mg/mL by pyrene absorbance method, which means that MCPEI-PBA can encapsulate hydrophobic small molecules inside of the nano-aggregates. Characterization of MCPEI-PBA polyplexes pDNA condensing ability of MCPEI-PBA was examined by agarose gel electrophoresis. As shown in Fig. 3, MCPEI2k could retard pDNA from a weight ratio of 0.5. In contrast with MCPEI2k, MCPEI-PBA was found to retard pDNA from a weight ratio of 1. This reduced pDNA condensing ability of MCPEI-PBA is probably due to
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the decreased free amine moiety in MCPEI-PBA by PBA conjugation [24] and to the steric hindrance of PBA ring structure to electrostatic interaction with pDNA [25]. Z-average size and Zeta-potential value of polyplexes were measured by Zeta-sizer. Zeta-potential value of MCPEI2k polyplex was negative potential value of 33.1 mV at a weight ratio of 0.25 and rapidly switched to positive potential values at higher weight ratios over 1 (Fig. 3B). Average size of MCPEI2k polyplex was about 1 mm at a weight ratio of 0.25 and abruptly decreased to 200– 250 nm with increasing polyplex weight ratios over 2, which means the formation of stable polyplexes. In contrast with MCPEI2k, MCPEI-PBA was found to form nano-sized polyplex (127.7 nm) at a low weight ratio of 0.25, which was gradually increased to 200–250 nm at higher weight ratios over 5 (Fig. 3C). As already confirmed, this may be because MCPEI-PBA itself could form nano-aggregates without polyplex formation and bulky MCPEI-PBA nano-aggregates could be more condensed by complexation with pDNA. Contrary to MCPEI2k, Zeta-potential values of MCPEI-PBA was switched to positive values from a weight ratio of 5 by less chargeable amine moiety of MCPEI-PBA than that of MCPEI2k. Also, it is thought that cationic MCPEI-PBA could bind to negatively charged polyplex at weight ratios ranging from 0.25 to 5, increasing polyplex size unlike MCPEI2k. These results demonstrate that MCPEI-PBA could form positively charged and nano-sized polyplex with pDNA which have suitable values for efficient adsorption onto cellular membrane and cellular uptake [26]. In vitro cell experiments of MCPEI-PBA Cytotoxicity of MCPEI-PBA was assessed by MTT assay in HeLa, C2C12, and HepG2 cells. As shown in Fig. 4, PEI25k displayed drastic decrease of cell viability with increasing concentration in all cells (IC50, HeLa: 9.2 mg/mL, C2C12: 3.3 mg/mL, HepG2: 24.3 mg/ mL), which indicates its significant cytotoxicity. Backbone polymer, MCPEI2k also showed considerable cytotoxicity (IC50, HeLa: 75.3 mg/mL, C2C12: 28.7 mg/mL, HepG2: 84.7 mg/mL), although it was relatively low in comparison with that of PEI25k. However, viabilities of MCPEI-PBA-treated cells were found to be similar to or much higher than that of MCPEI2k-treated cells in all cells (IC50,
Fig. 5. Transfection results of MCPEI-PBA in HeLa (A, C), C2C12 (B, E), and HepG2 (C, F) cells. Serum-free condition: A–C, serum-containing condition: D– F.
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HeLa: >100 mg/mL, C2C12: 71.4 mg/mL, HepG2: 73.2 mg/mL), demonstrating its low cytotoxicity. It may be induced by decreased charge density of MCPEI-PBA due to PBA conjugation. Transfection efficiency of MCPEI-PBA was evaluated by luciferase transgene expression assay in HeLa, C2C12, and HepG2 cells. Fig. 5A–C shows transfection experiment results in the absence of serum. Although the transfection efficiency of MCPEI-PBA was gradually increased with elevating weight ratios ranging from 5 to 25, it was found to be similar to or lower than that of MCPEI2k in HeLa and C2C12 cells. This result means that PBA conjugation to MCPEI2k could not affect the transfection efficiency in those cells. The transfection efficiency of MCPEI2k and MCPEI-PBA was 2.7 and 5.6 times lower in HeLa cells and 7.5 and 7.9 times lower in C2C12 cells than that of PEI25k at a weight ratio of 25 respectively. However, interestingly, MCPEI-PBA showed 1.4 times higher transfection efficiency than MCPEI2k and 4.2 times higher than PEI25k in HepG2 cell at a same weight ratio. In the presence of serum (Fig. 5D–F), although all the transfection efficiency of the polymers were decreased probably due to non-specific interactions with serum proteins, MCPEI2k and MCPEI-PBA exhibited less decreased transfection efficiency than PEI25k, suggesting their serum stability due to the charge shielding effect of hydroxy and methoxy functional groups in MC chains [10]. Especially, MCPEIPBA displayed 5.6 times higher transfection efficiency than PEI25k in HepG2 cells at a weight ratio of 25. This improved transfection efficiency of MCPEI-PBA in HepG2 cells suggests the possibility of its specific interaction with hepatocellular carcinoma. Hepatocellular carcinoma-targeting of MCPEI-PBA Human hepatocellular carcinoma, HepG2 cells are known to overexpress sialyl-Lewis X (sLex) antigen [27] or asialoglycoprotein receptors (ASGPRs) [28] on cellular membrane. Many literatures have reported the targeted drug or gene delivery to cancer cells by utilizing specific interaction of PBA moieties of the delivery carriers with diol of sLex [29,30]. Also, galactosylated drug or gene delivery carriers have been developed for Hepatocellular carcinoma-targeting via strong binding of galactose to ASGPRs [31,32]. In order to examine the specific interaction of MCPEI-PBA with sLex or ASGPRs, free PBA or galactose was pretreated in the transfection experiment for competition assay in HepG2 cells. As shown in Fig. 6A, transfection efficiencies of PEI25k (122.7% of PBAfree condition value) and MCPEI2k (88.6%) were not decreased significantly in PBA pretreatment condition, due to the lack of PBA moieties which can recognize sLex on HepG2 cell surfaces. Interestingly, MCPEI-PBA (107.7%) also did not show the decreased transfection efficiency even in the presence of PBA. It may be explained that almost PBA moieties of MCPEI-PBA would be consumed for intramolecular or intermolecular B–N coordinate bonding of amine of PEI2k. In the case of galactose pretreatment, all the transfection efficiencies of the polymers were generally decreased, probably due to the nonspecific interaction of galactose with high concentration (Fig. 6B). However, the transfection efficiency of MCPEI-PBA in galactose condition was dramatically decreased to 6.7% value of the efficiency in galactose-free condition in comparison with other polymers (PEI25k: 38.6%, MCPEI2k: 26.7% values), probably due to decreased interaction with ASGPRs by galactose binding. These results means that the transfection mechanism of MCPEI-PBA in hepatocellular carcinoma would be mediated by interaction with ASGPRs, not with sLex. In addition, the transfection experiments using non-hepatocellular carcinoma cells, HeLa and C2C12 cells, were performed in galactose-pretreatment condition. As shown in Fig. S3, MCPEI-PBA showed similarly or even less decreased transfection efficiency (HeLa: 5.1% of normal condition value, C2C12: 8.9%) in comparison with PEI25k (HeLa: 5.3%, C2C12: 3.7%) and MCPEI2k (HeLa: 3.6%,
Fig. 6. Transfection results of MCPEI-PBA with PBA pretreatment (A) and galactose pretreatment (B) in HepG2 cells.
C2C12: 4.2%) in the galactose-pretreatment condition. These results displayed that the transfection efficiencies of all tested polymers were generally decreased probably due to the nonspecific interaction of galactose, in non-hepatocellular carcinoma cells lacking ASGPRs, contrary to HepG2 cell result. The size change of the polyplexes in galactose condition was analyzed to examine the effect of galactose to the polyplex stability (Fig. S4). Similarly enlarged MCPEI-PBA polyplexes (203.2 → 293.2 nm) to MCPEI2k polyplexes (236.3 → 318.4 nm) suggested that the interaction of galactose with MCPEI-PBA polyplex would be similar to that of MCPEI2k polyplex and not specific for PBA moieties, meaning that galactose with even high concentration would not induce abrupt swelling or disruption of MCPEI-PBA polyplexes, which may lead to the significant decrease of the transfection efficiency. Interaction of MCPEI-PBA with ASGPRs was further investigated by GFP expression comparison in HepG2 cells (Fig. 7A). It was observed by fluorescence microscopy that the cells treated by PEI25k and MCPEI2k polyplexes (GFP pDNA) showed no significant GFP expression decrease in galactose pretreatment condition, meaning their low interaction with ASGPRs. However, GFP
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Fig. 7. GFP transgene expression results of polyplexes with galactose pretreatment (A) and cellular uptake efficiency of polyplexes with galactose pretreatment (B) in HepG2 cells.
expression of MCPEI-PBA polyplexes was considerably reduced in galactose pretreatment condition. In addition, the effect of galactose pretreatment to cellular uptake of the polyplexes (YOYO-1 iodide-labeled pDNA) was also analyzed by FACS. As shown in Fig. 7B, the cellular uptake efficiency of PEI25k and MCPEI2k polyplexes was slightly changed in galactose pretreatment condition. MCPEI-PBA polyplexes displayed about 10% decrease cellular uptake efficiency (65.3%) in galactose pretreatment condition, comparing with non-treatment condition (74.7%). These results suggest that pretreated galactose could bind to ASGPRs on HepG2 cell surfaces, inhibiting interaction of MCPEIPBA with ASGPRs and decreasing cellular uptake and transgene expression. The mechanism of interaction of MCPEI-PBA and ASGPRs was not fully understood. However, it was reported that ASGPRs also can recognize other sugar molecules such as glucose derivatives, except galactose or N-acetyl galactosamine [33]. Considering this report, one of possible explanation about ASGPRs-targeting of MCPEI-PBA may be the synergistic effect of interaction of ASGPRs with methyl glucose unit of MCPEI-PBA and hydrogen bonding and hydrophobic interaction with tryptophan of ASGPRs [28] or p–p interaction of phenyl ring of PBA with tyrosine residues of ASGPRs [34]. Characterization of MCPEI-PBA/Dox Hydrophobic anticancer agent, Doxorubicin (Dox) was encapsulated in MCPEI-PBA nano-aggregates by dialysis method. When the mixing ratio (Dox/MCPEI-PBA, w/w) was 1/10, the drug loading efficiency of was measured to be about 100% and drug loading content was 8.8%, which means that MCPEI-PBA nano-aggregates could encapsulate Dox very efficiently. Average sizes and Zeta-potentials of MCPEI-PBA/Dox nanoaggregates and MCPEI-PBA/Dox/pDNA complexes were examined. Interestingly, average size of MCPEI-PBA/Dox nano-aggregates was measured to be 287.3 79.6 nm, which was significantly decreased in comparison with that of MCPEI-PBA nano-aggregates (about
760 nm) at same concentration probably due to the condensation by hydrophobic interaction with Dox molecules. In addition, MCPEIPBA/Dox/pDNA complexes showed 259.3 24.4 nm, which suggests the formation of more compact and homogeneous nano-particles by electrostatic interactionwith negatively charged pDNA. In the case of Zeta-potential measurements, MCPEI-PBA/Dox nano-aggregates and MCPEI-PBA/Dox/pDNA complexes displayed 44.9 4.3 mV and 49.3 2.1 mV, respectively. Zeta-potential value of MCPEIPBA/Dox/pDNA complexes was almost similar with that of MCPEIPBA/pDNA polyplexes, demonstrating that MCPEI-PBA/Dox nanoaggregates can form MCPEI-PBA/Dox/pDNA complexes with pDNA by covering the surfaces and these nano-particles can adsorb readily onto negatively charged cellular membrane. Release behavior of Dox from MCPEI-PBA/Dox nano-aggregates was monitored at pH 5.5 and 7.4, which mimic late endosome/ lysosome and physiological extracellular environment (Fig. S5). The release rate of Dox was fast until 12 h, showing somewhat initial burst. The release of Dox occurred sustainably and was up to 30.5% at pH 5.5 and 22.5% at pH 7.4 after 48 h of incubation. This result means that Dox could be released faster at pH 5.5 than pH 7.4 because protonation of Dox could increase its solubility and protonated PEI could be swelled via charge repulsion. Low release rate of Dox means that Dox can interact strongly with MCPEI-PBA molecules, leading to the inhibition of its release. It may be interpreted by several following inferences. Firstly, Dox molecules also can be encapsulated in the interior of PEI [35]. Secondly, average size of MCPEI-PBA nano-aggregates was dramatically decreased by encapsulation of Dox, which suggests this spatial shrinkage may disturb the diffusion of Dox. Thirdly, PBA moieties may interrupt the release of Dox by hydrogen bonding, hydrophobic interaction or p–p stacking, or B–N boding between PBA and Dox [36]. Although the release of Dox was slow, it may minimize the loss of Dox from the nano-aggregates via spontaneous release during delivery procedure to the target cells. Fig. S6 shows the schematic illustration for MCPEI-PBA/Dox/ pDNA nano-aggregates. According to the above results, it is
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Conclusion PBA-conjugated cationic methylcellulose derivative (MCPEIPBA) was synthesized for drug/gene co-delivery systems. MCPEIPBA could form nano-aggregates by self-assembly via hydrophobic interaction and B–N bonding, which can encapsulate hydrophobic Dox molecules efficiently and further condense pDNA to construct nano-sized complexes with pDNA. MCPEI-PBA showed high transfection efficiency especially in hepatocellular carcinoma and it may be deduced that MC and PBA can specifically interact with ASGPRs on cell membrane. Finally, MCPEI-PBA/Dox/pDNA (apoptin) complexes exhibited efficient anticancer activity due to the co-delivery of Dox and apoptin gene by nano-particles. These results demonstrate the potential of MCPEI-PBA as a targeted codelivery carrier of drug/gene for hepatocellular carcinoma. Acknowledgments
Fig. 8. Anticancer activity result of MCPEI-PBA/Dox/pJDK-apoptin complexes in HepG2 cells.
expected that Dox molecules would be encapsulated inside of the hydrophobic core and PEI chains and pDNA would be complexed in outer region of the nano-aggregates, covered by cationic MCPEIPBA chains. Cancer cell killing activity of MCPEI-PBA/Dox nano-aggregates and MCPEI-PBA/Dox/pDNA complexes Cancer cell killing effect of MCPEI-PBA/Dox nano-aggregates and MCPEI-PBA/Dox/pDNA complexes were investigated by MTT assay in HepG2 cells in order to identify the combinatorial effect for co-delivery of Dox and pDNA. As shown in Fig. 8, only pDNA could not induce any cytotoxic effect to HepG2 cells because of very low delivery efficiency by the absence of the carrier. PEI25k polyplexes showed 77.6% cell viability, meaning the low level of apoptin transgene expression. On the other hand, cell viability of MCPEI-PBA polyplexes-treated cell was decreased to 54.9%, which showed superior gene delivery efficiency to PEI25k polyplexes as already shown in luciferase assay. In the case of Dox treatment, Free Dox could reduce the cell viability to 16.9%. MCPEI-PBA/Dox nano-aggregates exhibited 39.4% cell viability. Finally, MCPEI-PBA/Dox/pDNA was found to reduce cell viability to 30.5%. These results demonstrated that co-delivery of Dox and pDNA by MCPEI-PBA showed enhanced anti-cancer activity due to the combinatorial therapeutic effect of cytotoxicity of Dox and apoptosis by expressed apoptin, in comparison with other singular delivery of Dox or pDNA. Although cancer cell killing effect of free Dox was very high, several studies reported that free Dox delivery could induce serious heart damage [37] and multi-drug resistance (MDR) of cancer cells could reduce the therapeutic effect by exporting internalized Dox [38]. This MDR of HepG2 cells was also observed in Fig. S7, which maintained the cell viability similarly (~40%) even at elevated concentration (2–10 mg/mL). However, MCPEI-PBA/Dox nanoaggregates could kill HepG2 cells gradually in proportion to its concentration and finally showed higher anticancer activity over 5 mg/mL than free Dox, demonstrating these nano-aggregates could overcome MDR of the cells, similarly with other nanocarriers [39].
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