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Synthesis of Polysaccharide-Block-Polypeptide Copolymer for Potential Co-Delivery of Drug and Plasmid DNAa Qianqian Li, Wenya Liu, Jian Dai,* Chao Zhang A pH-sensitive, biodegradable, and biocompatible polysaccharide-block-polypeptide Copolymer derivative {Ac-Dex-b-PAsp(DET)} is synthetized from acetal-modified dextran (Ac-Dex) and diethylenetriamine (DET) grafted poly(L-aspartic acid) {PAsp(DET)} by using click and aminolysis reaction. The copolymer can self-assemble into cationic nanopaticles for potential co-delivery of plasmid DNA (pEGFP-N3) and anticancer drug (doxorubicin, DOX), by using water/oil/water (w/o/w) emulsion method. Gel retardation assay reveals that pDNA can be effectively complexed into cationic nanoparticles at N/P ratio ¼ 12. In vitro drug release behavior of DOX-NPs and DOX/pDNA-NPs is achieved by using fluorescence spectra and UV– Vis spectra and confocal laser scanning microscopy (CLSM). And, pEGFP-N3-NPs at N/P ratio ¼ 42 presents the considerable potential in cell transfection. Cell viability assay shows that nanoparticles exhibit low cell cytotoxicity. These results suggest that the copolymer has excellent performance and potential for the co-delivery of gene and drugs.

1. Introduction In recent years, much research has focused on designing nanocarriers for drug delivery and tumor therapy, due to passive tumor targeting and active targeting bioconjugation, compared to chemotherapy.[1,2] Moreover, the great progress has been achieved during the whole process, including biomaterials design for nanocarriers, controlled release of encapsulated drug in vitro, drug delivery, and tumor treatment in vivo.[3,4] For example, the spherical nanoparticles coated with a shell that incorporates Q. Li, W. Liu, Dr. J. Dai, Prof. C. Zhang School of Engineering, Sun Yat-sen University, Guangzhou 510006, P. R. China E-mail: [email protected] a Supporting Information is available from the Wiley Online Library or from the author.

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hyaluronic acid (HA) was developed for ATP-triggered anticancer drug delivery, in which ATP was used as a molecular trigger for controlled release of anticancer drugs, both in vitro and in vivo;[5] and, self-peptides designed from human CD47 reported as a ‘‘marker of self’’ delayed macrophage-mediated clearance of nanoparticles, which promotes persistent circulation that enhances dye and drug delivery to tumors.[6] However, although a large number of nanocarriers have been demonstrated for drug delivery and tumor therapy, representing a promising candidate for the clinical application, there remains a need for an efficient method that can solve the problem of drug-resistance during the period of tumor chemotherapy. Recently, numerous studies have established that combining several treatment methods can achieve promising results of reducing drugresistance and the dose of injections and achieving the synergistic therapeutic effect.[7,8] For example, cationic

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DOI: 10.1002/mabi.201400454

Synthesis of Polysaccharide-Block-Polypeptide Copolymer for Potential Co-Delivery of Drug and Plasmid DNA www.mbs-journal.de

core-shell nanoparticles for co-delivery of drugs and DNA were prepared using cationic amphiphilic copolymer, poly {(N-methyldietheneaminesebacate)-co-[(cholesteryloxocarbonylamido ethyl) methyl bis(ethylene) ammonium bromide] sebacate} (P(MDS-co-CES)). And the synergistic effect of the co-delivery of paclitaxel with an interleukin12-encoded plasmid using these nanoparticles was demonstrated, suppressing cancer growth more efficiently than the delivery of either paclitaxel or the plasmid in a 4T1 mouse breast cancer model.[9] Diblock copolymers (PEI-PCL) of poly(e-caprolactone) (PCL) and linear poly(ethylene imine) (PEI) were synthesized and assembled to form biodegradable nanocarriers for co-delivery of BCL-2 siRNA and doxorubicin(DOX), yielding synergistic effect of RNA interference and chemotherapy in cancer.[10] Meanwhile, co-delivery of doxorubicin and BCL-2 siRNA by mesoporous silica nanoparticles achieved the favorable efficacy of chemotherapy in multidrug-resistant cancer cells.[11] So far, combining the emerging gene therapy with the traditional chemotherapy together has caused a wide attention in cancer treatment. On the one hand, since tumor cells get more sensitive to the chemotherapy drugs owing to the effects of some certain gene, the amount of the dosages and toxic effects to the normal cells are greatly reduced; on the other hand, the anticancer drugs can improve the expression of the gene and activate of the death receptors, resulting in the cells quickly apoptosis with the effects of therapeutic genes. Those factors, coupled with the in-depth realization of tumors, result in the rapidly development of the co-delivery system.[12–15] However, due to intracellular drug availability relating with efficiency for killing cancer cells, little evidence is available to study nanocarriers of microenvironment-related drug release for co-delivery of drugs with DNA. Thus, the demand for material innovations is increasing for efficient tumor therapy. Nowadays, stimuli-responsive nanocarriers for control release of drugs have been attracting extensive attention,[16–20] especially related with cell microenvironment such as reducibility, or low pH value.[18] For example, the highly packed interlayer-crosslinked micelle with reduction and pH dual sensitivity were reasonably constructed for in vivo highly efficient drug delivery and microenvironment-sensitive rapid release of drugs, revealing the great potential for achieving an optimal therapeutic effect of the transported drugs in tumor treatment.[21] As such, acid-triggered rapid release of drugs can be achieved inside tumor tissue (pH below 6.8) or lysosomal compartments (pH about 5.0) of cancer cells by using micelles of copolymers bearing pH-sensitive blocks, such as poly(L-histidine),[22– 24] poly(b-amino ester)[25] and 2-(diisopropylamino) ethylamine-grafted poly(L-aspartic acid)[21] or biomaterials bearing pH-dependent hydrolysis groups, such as acetal group[26] and cyclic carbonate.[27]

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In the present study, we prepared pH-dependent hydrolysis nanoparticle for co-delivery of anticancer drug and plasmid DNA, based on a novel block copolymer [AcDex-b-PAsp(DET)] synthesized from acetal-modified dextran (Ac-Dex) and diethylenetriamine (DET) grafted poly(Laspartic acid) [PAsp(DET)] via click and aminolysis reactions (shown in Scheme 1). The amphiphilic block copolymer was characterized by proton magnetic resonance (1H NMR), gel permeation chromatograph (GPC), and FT-IR spectrometer (FTIR). Blank nanoparticles (NPs) and anticancer drug doxorubicin/plasmid DNA co-encapsulated nanoparticles (DOX/pDNA-NPs) both were prepared by w/o/w method. The size and potential of the nanoparticles were characterized by dynamic light scattering and transmission electron microscope; DOX release from the pH-sensitive polymeric nanoparticles was qualitatively assessed using fluorescence spectra and quantitatively using UV–Vis spectra at pH 7.4 and pH 5.0. Moreover, the toxicity of carriers was evaluated via MTT assay on BEL-7402 cells. Intracellular DOX release from pH-sensitive cationic nanoparticles and migration into nuclei was observed by confocal laser scanning microscopy (CLSM). Transfection efficiency of pDNA-complexed nanoparticles (pDNA-NPs) and PEI25k/pDNA complexes was determined by flow cytometry, and fluorescent images of EGFP expression were obtained by an inverted microscope in BEL-7402 cells transfected with pDNA-encapsulated nanoparticles at N/P ratio ¼ 42 and PEI25k/pDNA complexes at N/P ¼ 10. These results indicate that the copolymer has considerable potential for the co-delivery of gene and drugs.

2. Experimental Section All experimental details are provided in the Supporting Information.

3. Results and Discussion 3.1. Characterization of the Ac-Dex-b-PAsp(DET) Block Copolymer Herein, we present the synthesis of polysaccharide-blockpolypeptide copolymer, in which dextran block is linked linearly to poly(L-aspartate) block by using click reaction, and finally obtain the amphiphilic block copolymer, AcDex-b-PAsp(DET) as shown in Scheme 1. 3.1.1. Preparation of Acetal-Modified Alkyne-Dextran (AcDex) Using theory of Schiff bases, a terminal alkyne group was modified to dextran (Dex) and achieved alkyne-dextran (alkyne-Dex).[28] Then, the alkyne-Dex was reacted with 2-

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Scheme 1. Schematic illustration of the synthesis of the block copolymer Ac-Dex-b-PAsp(DET).

methoxypropene to prepare acetal-modified alkyne-dextran (Ac-Dex) by using the catalyst Pyridinium toluene-4sulphonate. Acetals were chosen to modify dextran because of their tunable pH-dependent hydrolysis rates.[29] In a result, the Ac-Dex became pH-sensitive and insoluble in water. The polymer characterization was investigated via 1 H NMR and FT-IR. Although the terminal alkyne group was not detected obviously by 1H NMR of Dex, alkyne-Dex and Ac-Dex in the DMSO-d6 (Figure S1, Supporting Information), the peaks of reducing-end-group (d6.7 ppm, d6.3 ppm) completely disappeared demonstrating that the reaction successfully occurred. Compared with alkyne-Dex, the new strong peak at d1.26 ppm exhibited the existence of the methyl-functional group. As shown in FT-IR (Figure S2, Supporting Information), methyl-functional group absorption at 1 378 cm1, and isopropyl groups absorption at 846 cm1 were distinctly observed in the spectrum of AcDex. Meanwhile, OH(3) and OH(4) of Dex were shifted from 4.68–4.79 ppm to a single peak at 5.4 ppm, and OH(2) of Dex was shifted from 4.5 to 5.1 ppm (Figure S1, Supporting Information), it is probably resulted from hydrogen bond between hydroxyl and methoxy groups. According to the integration ratio of the special proton (H1) of the Dex at 4.63 ppm with methanol at 3.09 ppm and acetone at 1.88 ppm from acid hydrolysis of Ac-Dex (Figure S3, Supporting Information), the molecular weight of the AcDex was theoretically calculated as 7 341 g  mol1 by 1H NMR integration ratio.

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3.1.2. Synthesis of Ac-Dex-b-PAsp(DET) In the present study, the block copolymer Ac-Dex-bPAsp(DET) was synthesized via click reaction between Ac-Dex and PBLA-N3 with the catalyst system of CuBr and PMDETA and aminolysis reactions with DET. PBLA-N3 was synthesized from the ring-opening polymerization of eBenzyloxycarbonyl L-aspartic acid NCA (BLA-NCA) using 1azido-3-aminopropane as an initiator. Based on the integration ratio of d3.06 ppm (2H, CH2N3)/d7.26 ppm (85H,–CH– of benzene ring) in 1H NMR spectrum, the Mn of PBLA-N3 was theoretically calculated as 3 602 g  mol1. Comparing with PBLA-N3, the azide vibrational peak at 2 098 cm1 was not observed in the FT-IR spectrum of the block copolymer Ac-Dex-b-PAsp(DET) (Figure S4, Supporting Information). It revealed that the click synthesis was well completed. Meanwhile, the completely disappearance of the absorption signal at 1 738 cm1, which was assigned to ester of PBLA-N3, indicating that the aminolysis reaction occurred.[30] On the other hand, since 1H NMR peak of CH2 of DET and proton peak of H2O overlapped together in Figure 1, the aminolysis rate could not have been determined accurately and meanwhile the integration ratio between CH2 of DET and CH3 of Ac-Dex could not be analyzed because of this overlap. However, the block copolymer Ac-Dex-bPAsp(DET) can be used to prepare the nanoparticles with the core of Ac-Dex and the shell of PAsp(DET) via self-assembly in pH7.4 PBS. The 1H NMR spectrum of the nanoparticles in

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Figure 1. 1H NMR spectrum of the block copolymer Ac-Dex-b-PAsp(DET) in d6-DMSO.

D2O (Figure S5, Supporting Information) exhibited the peaks at 2.65, 2.74, and 2.92 ppm, which were assigned to the protons peaks of methylene groups in the DET, indicating the successful aminolysis reaction of Ac-Dexb-PAsp(DET). The results were supported by the 1H NMR spectrogram of the block copolymer Ac-Dex-b-PAsp(DET) in Figure S6 (Supporting Information); the completely disappearance of signal at 7.22 ppm attributable to benzene ring protons, and the methylene protons in the PBLA backbone were shifted from 2.60 and 2.80 ppm to 3.15 and 2.69 ppm, corresponding to the article reported.[30] GPC measurements provided further evidence for the successful synthesis of the block copolymer Ac-Dex-b-PAsp(DET). As shown in Figure 2, Ac-Dex, and PBLA-N3 and Ac-Dex-bPAsp(DET), all showed an unimodal molecular weight distribution in the GPC chromatograms, indicating that the completely aminolysis reaction occurred. Moreover, the molecular weight of the block copolymer Ac-Dex-b-PAsp(DET) exhibited an apparent increase, compared to Ac-Dex

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and PBLA-N3. It can be shown that Ac-Dex-b-PAsp(DET) (10 858 g  mol1 by 1H NMR and 35 800 g  mol1 by GPC measurement) was successfully synthesized. The

Figure 2. GPC analysis of the Ac-Dex, PBLA-N3 and Ac-Dex-bPAsp(DET).

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molecular weight determined by GPC is higher than that calculated based on the 1H NMR spectra in the Supporting Information. An underlying reason might be that, in the given solvent, the PEG standard for GPC calibration did not well match the structurally much more complexed polymers in the molecular morphology, leading to standard deviations in measurements. However, trend of the molecular weight variation of the three polymers (AcDex, PBLA-N3 and Ac-Dex-b-PAsp(DET)) are consistent in the two measurement methods. 3.2. Preparation and Characterization of Cationic Nanoparticles Since the particles with over 500 nm or low 10 nm size will be eliminated by the cells of the reticuloendothelial system (RES), particle size is one of the most critical factors for drug delivery system to access target cells. In the present study, all the four nanoparticles, such as blank nanoparticles (NPs), DOX-loaded nanoparticles (DOX-NPs), pDNA complexed nanoparticles (pDNA-NPs), and DOX/pDNA co-encapsulated nanoparticles (DOX/pDNA-NPs), were made from the amphiphilic block copolymer, Ac-Dex-b-PAsp(DET) by using a double emulsion water/oil/water (w/o/w) method, and were designed to study the ability of complexing pDNA, nanoparticle size, surface zeta potential, and shape with different N/P ratio. In the present study, we fabricated pH-sensitive cationic nanoparticles for drug/pDNA co-delivery using the amphiphilic block copolymer, Ac-Dex-b-PAsp(DET). Therefore, the ability of loading drug and complexing pDNA would be determined by using Agarose gel electrophoresis, drug loading efficiency, in vitro drug controlled release, etc. It is well known that cationic polymers complex pDNA upon electrostatic neutralization, through which the negative

charge of pDNA is partially or completely neutralized and consequently results in the retardation or complete loss of oriented migration of pDNA in the electric field.[31] Agarose gel electrophoresis was conducted to confirm the pDNA complexation ability of the amphiphilic block copolymer, Ac-Dex-b-PAsp(DET) in comparison with naked pDNA. As shown in Figure 3, at N/P ¼ 12, Ac-Dex-b-PAsp(DET)/pDNA was completely retained in the sampling whole, indicating the complexation of full DNA chains into the nanoparticles.[32] Since all the nanoparticles are prepared using a double emulsion water/oil/water (w/o/w) method, pDNA is encapsulated into the core of the nanoparticle by electrostatic interaction. At N/P ¼ 12, the zeta potential of the nanoparticles is near zero and the size is about 700 nm. It means that at N/P < 12, the zeta potential of the nanoparticles is negative value. As a result, it suggested that encapsulating pDNA into cationic nanoparticle by using a double emulsion method is different from that of simply complexing pNDA by water-soluble polycation, as shown in the reference.[33] In addition, Ac-Dex-b-PAsp(DET)/pDNA complexes with size and zeta potential of different N/P ratio were evaluated by dynamic light scattering (DLS). In Figure 3, the curve of the nanoparticle size climbed up first and then tended to a certain value at around 250 nm, with the increasing of the N/P ratios. It presumably resulted from the electrostatic neutralization of the positively charged copolymer and negatively charged pDNA. And, the stability of the complexes may be destroyed and began to aggregate when the charge of the complexes was close to neutral, according to our previous report.[31] Meanwhile, the zeta potential of the complexes increased significantly from 26.3 to 19 mv as the N/P ratios increased in Figure 3. Although the higher positive charge makes the NPs easier for cell internalization through the electrostatic interaction, it also resulted in the higher

Figure 3. Gel retardation assay (left) of pDNA-NPs and the size and zeta potential (right) of pDNA-NPs at different N/P ratios. 2: N/P ¼ 1; 3: N/ P ¼ 12; 4: N/P ¼ 24; 5: N/P ¼ 48; 6: N/P ¼ 60; 7: N/P ¼ 72.

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cytotoxicity. Hence, in the present study, we chose N/P ¼ 42 as the optimistic condition with around 250 nm size and 10 mv zeta potential for co-delivery of drug and gene. On the other hand, since the vehicle stability is essential to in vivo drug delivery for effective therapy, the stability of blank nanoparticles (NPs) were evaluated by DLS, as shown in Figure S7 (Supporting Information), the particle size slightly fluctuated around 200 nm, and the zeta potential ranged from 20 to 29 mv within 120 h, indicating the NPs could be stable for a long time. It exhibited considerably stable performance for the drug/gene delivery system. As showed in Table S1 (Supporting Information), the result of dynamic light scattering (DLS) presented the size and zeta potential of NPs were 110.0  16.0 nm and 29.4  0.7 mV, the size and zeta potential of DOX-NPs were 134  39 nm and 22.2  2.2 mV, the size and zeta potential of pDNA-NPs were 271.1  11.1 nm and 10.23  1.87 mV, and the size and zeta potential of DOX/pDNA-NPs were 308  52 nm and 7.9  2.7 mV. There is a slightly increasing in the size of DOX/pDNA-NPs. It was probably due to the pDNA increased the volume of the hydrophobic core. Meanwhile, the particle morphology was determined in terms of transmission electron microscope (TEM). As shown in Figure S8 (Supporting Information), all the nanoparticles were spherical, uniform, and dispersed. The size of the blank nanoparticles (NPs), DOX-loaded nanoparticles (DOX-NPs), and DOX and pDNA co-encapsulated nanoparticles (DOX/ pDNA-NPs) were measured respectively as 50, 60, and 100 nm from TEM micrograms. It was figured out that the sizes of all the nanoparticles observed by TEM were much lower than that obtained from DLS, presumably due to the fact that TEM observes particles in dry state while DLS determines hydrodynamic size of particles in solution. Moreover, although Ac-Dex exhibited hydrophobic performance, there still existed some hydroxyl residues, leading to the nanoparticles swelling in the aqueous

solution. Hence, it probably suggests that the nanoparticles would shrink under the vacuum environment and become smaller.

3.3. Drug Release of DOX-NPs and DOX/pDNA-NPs Comparing to the hydrophobic DOX encapsulated in the particles, the free DOX dissolving in the solution presents much higher fluorescence intensity.[21] Therefore, DOX release from pH-sensitive cationic nanoparticles was assessed using two different assays. Firstly, the fluorescence spectra were used for the qualitative analysis of the drug release at pH 7.4 and 5.0. As shown in Figure 4, the fluorescence intensity of the DOX-NPs or DOX/pDNA-NPs exhibited four or five times higher intensity at pH 5.0 than that at pH 7.4, indicating the excellent pH-sensitive properties. However, DOX/pDNA-NPs exhibited slightly lower intensity comparing to the DOX-NPs at pH 5.0; this is probably due to the electrostatic interaction between pDNA with negative charge and PAsp(DET) with positive charge of Ac-Dex-b-PAsp(DET), which slightly hampered DOX release from the DOX/pDNA-NPs. To further study the quantitative drug release of pHsensitive cationic nanoparticles, at a certain time intervals, the release medium was sampled and the same volume of the fresh buffer solution was added, then the amount of the DOX release was obtained by measuring the absorb intensity at 480 nm using a UV spectrophotometer. The encapsulation efficiency and loading level of DOX-NPs was determined to be 36.7 and 5.2%, and the DOX/pDNA-NPs was determined to be 31.86 and 5.3%, indicating pDNA complexed was not a significant influence on the loading level or the encapsulation efficiency. As expected, in Figure 4, the drug release at pH 5.0 exhibited much faster than pH 7.4, which was in good agreement with the

Figure 4. In vitro drug release behavior of DOX-NPs and DOX/pDNA-NPs by using DOX fluorescence (left) and DOX UV-Vis (right) at pH 7.4 and 5.0 (mean  standard deviation, n ¼ 3).

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qualitative analysis. The DOX release was slower at pH 7.4, and the totally amount of the drug release was only 12% in 30 h, showing that the drugs can be encapsulated well in the NPs, which is benefit for the NPs circulation in vivo, as well as reducing in vivo cytotoxicity. At pH5.0, there is an initial burst of drug release about 26% at first 5 h, and then keeping in a sustainable manner for further 25 h. It is probably pHhydrolysis of Ac-Dex at pH 5.0, leading to drugs diffused rapidly through the swelling nanoparticles. The DOX/ pDNA-NPs has a similar behavior in line with that of DOXNPs. However, the Ac-Dex presently synthesized was a slow-hydrolyzing polymer variant by designing the reaction time (1 h) of converting the hydroxyl groups of the native dextran to pendant acetals with the 80% extent of coverage of hydroxyls as well as the type of acetals (ratio of cyclic acetals/acyclic acetals ¼ 3.46) that had been formed by using 1H NMR (Figure S3, Supporting Information).[26] Meanwhile, another polycation block, PAsp(DET) involves primary amine and secondary amine pendants, which can exhibit the ability of complexing pDNA and proton buffering function in intracellular endosome environment.[34] It indicated that DOX release profile in the nanoparticles was considerably complex with only 50% release efficiency within 50 h. Meanwhile, it implied that

the DOX-NPs and DOX/pDNA-NPs could pH-sensitively release the encapsulated cargo (DOX) after entering cancer cells via endocytosis and being entrapped inside lysosomes, which will be confirmed in cell study as well.

3.4. Intracellular DOX Release and Migration into Nuclei As nanoparticles are eventually entrapped inside lysosomes after endocytosis, a rapid lysosomal release of drugs is crucial for obtaining ideal therapeutic effects. Therefore, the intracellular release of DOX from DOX-NPs in human hepatoma BEL-7402 cells was investigated. It is well known that, as a nuclei-targeted drug, free DOX enters nuclei quickly after cell uptake, whereas DOX transported by nonsensitive micelles accumulates very slowly in nuclei.[21] Therefore, free DOX and DOX-loaded non-sensitive micelle based on the diblock copolymer of poly(ethylene glycol) and poly(e-caprolactone), PEG2k-PCL3k, were employed as positive and negative controls, respectively. As shown in Figure 5, DOX-NPs designed as pH-sensitivity controlled release system did not exhibit the rapid release of loaded DOX inside lysosomes after endocytosis, comparing to free

Figure 5. Intracellular DOX release and migration into nuclei observed by confocal laser scanning microscopy (CLSM). BEL-7402 cells were incubated (37 8C) for 6 h at a DOX-equivalent dosage of 10 mg per dish. DOX loading contents: 5.2% in DOX-NPs and 5.1% in PEG2k-PCL3k micelles. Nuclei were stained with Hoechst 33342 (blue).

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Figure 6. Fluorescent images of EGFP expression obtained by an inverted microscope and transfection efficiency determined by flow cytometry of pEGFP-NPs (A, B, C) at N/P ¼ 42 and pEGFP/PEI25KD (D, E, F) as control group at N/P ¼ 10. Transfection was performed at a dose of 1 mg of DNA (mean  SE, n ¼ 3) in BEL-7402 cells; Incubation time: 48 h; scale bar is 50 mm.

DOX rapidly accumulated into nuclei of BEL-7402 cells. Meanwhile, it differed also with DOX release of DOX-loaded non-sensitive micelle which was not detected in the nuclei of BEL-7402 cells after cell incubation for 6 h. The Ac-Dex presently synthesized was a slow-hydrolyzing polymer variant, indicating that pH-hydrolysis of the nanoparticles designed was controlled by acetal hydrolysis rate of pendant ratio of cyclic acetals (slow hydrolysis)/acyclic acetals (rapid hydrolysis).[29] So, DOX release rate from pHhydrolysis of nanoparticles is moderate between that of free DOX and that of DOX-loaded non-sensitive micelle, in good agreement with DOX release in vitro.

3.5. Gene Transfection To evaluate the effects of pH-sensitive cationic nanoparticles on transfection, pEGFP-N3 plasmid was employed in vitro. The transfection efficiency was evaluated in BEL7402 cells line using flow cytometry in order to correlate the actual cell populations transfected and the levels of protein produced from BEL-7402 cells at N/P ratio ¼ 42, in comparison to the PEI 25 KDa control at N/P ratio ¼ 10. In Figure 6, on the whole, the transfection efficiency with 4.2% of pDNA-NPs at N/P ratio ¼ 42 is quantitatively similar to that with 5.5% of pDNA/PEI25KD at N/P ratio ¼ 10. Meanwhile, the inverted fluorescence microscope can be directly observed by enhanced green fluorescence protein in cells.

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The result presents the successful potential in cell transfection ability of pDNA-NPs designed.

3.6. The Cytotoxicity of NPs In the present study, the cytotoxicity of blank nanoparticle (NPs) was evaluated in the terms of MTT in BEl-7402 cells line. The untreated cells were used as negative control, and the viability of cells was assayed at 570 nm using a microplate reader after 24 h of treatment. As shown in Figure S9 (Supporting Information), the viability of BEl7402 cells was not affected with the increasing of the concentration of the NPs from 0.1 to 250 mg.ml1 and maintained over 87% cell viability, indicating that nanoparticles did not exhibit substantial cytotoxicity to the cells.

4. Conclusion In conclusion, we synthesized an amphiphilic copolymer Ac-Dex-b-PAsp(DET) and designed pH-sensitive cationic nanoparticles for potential co-delivery of drugs and gene by using w/o/w double emulsion method. The pH-sensitive cationic nanoparticles were stable in neutral pH environment by using DLS characterization, while inside the cells, the hydrophobic fragment was expected to hydrolyze in the acidic lysosome environment, and erupted to release the

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anticancer drugs and pDNA. DOX release rate from pHhydrolysis of nanoparticles is moderate between that of free DOX and that of DOX-loaded non-sensitive micelle, and the transfection efficiency with 4.2% of pDNA-NPs at N/P ratio ¼ 42 presents the considerable potential in cell transfection ability of pDNA-NPs designed. Additionally, the nanoparticles did not result in significantly cytotoxicity to the cells through the MTT assay, these promising data exhibited that Ac-Dex-b-PAsp(DET) is an ideal candidate for the co-delivery of gene and drugs. However, considering the aim is the synergistic manner of gene and drugs, the further experiments are necessary to elucidate this point. At present, we are studying pH-sensitive cationic nanoparticles decorated with c(RGDyC)-peptide for targeted drug/pDNA co-delivery to integrin-rich tumors. Our results mean that pH-sensitive cationic nanoparticles designed exhibit attractive potential of drugs/pDNA co-delivery for synergetic therapeutic efficacy.

Acknowledgements: The authors thank the financial support of the National Natural Science Foundation of China (No. 21104097) and the Fundamental Research Funds for the Central Universities of China (No. 12lgpy01).

Received: October 17, 2014; Revised: February 10, 2015; Published online: March 11, 2015; DOI: 10.1002/mabi.201400454 Keywords: co-delivery; drugs; plasmid DNA; polypeptide; polysaccharide

[1] T. J. Wickham, Nat. Med. 2003, 9, 135. [2] R. Haag, Angew. Chem. Int. Ed. 2004, 43, 278. [3] J. W. Yoo, D. J. Irvine, D. E. Discher, S. Mitragotri, Nat. Rev. Drug Discov. 2011, 10, 521. [4] R. A. Petros, J. M. DeSimone, Nat. Rev. Drug Discov. 2010, 9, 615. [5] R. Mo, T. Jiang, R. DiSanto, W. Tai, Z. Gu, Nat. Commun. 2014, DOI:10.1038/ncomms4364 [6] P. L. Rodriguez, T. Harada, D. A. Christian, D. A. Pantano, R. K. Tsai, D. E. Discher, Science. 2013, 339, 971. [7] M. Khan, Z. Y. Ong, N. Wiradharm, A. B. E. Attia, Y.-Y. Yang, Adv. Healthcare Mater. 2012, 1, 373.

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[8] M. Creixell, N. A. Peppas, Nano Today 2012, 7, 367. [9] Y. Wang, S. Gao, W. Ye, H. S. Yoon, Y.-Y. Yang, Nat. Mater. 2006, 5, 791. [10] N. Cao, D. Cheng, S. Zou, H. Ai, J. Gao, X. Shuai, Biomaterials 2011, 32, 2222. [11] A. M. Chen, M. Zhang, D. Wei, D. Stueber, O. Taratula, T. Minko, H. He, Small 2009, 5, 2673. [12] S. Liu, Y. Guo, R. Huang, J. Li, S. Huang, Y. Kuang, L. Han, C. Jiang, Biomaterials 2012, 31, 4907. [13] C. W. Beh, W. Y. Seow, Y. Wang, Y. Zhang, Z. Y. Ong, P. L. Rachel Ee, Y.-Y. Yang, Biomacromolecules, 2009, 10, 41. [14] H. Fan, Q. D. Hu, F. J. Xu, W. Q. Liang, G. P. Tang, W. T. Yang, Biomaterials 2012, 33, 1428. [15] Q. Hu, W. Li, X. Hu, Q. Hu, J. Shen, X. Jin, J. Zhou, G. Tang, P. K. Chu, Biomaterials 2012, 33, 6580. [16] Y. Li, K. Xiao, W. Zhu, W. Deng, K. S. Lam. Adv. Drug Deliver. Rev. 2014, 66, 58. [17] M. Simona, N. Julien, C. Patrick, Nat. Mater. 2013, 12, 991. [18] Z. Ge, S. Liu. Chem. Soc. Rev. 2013, 42, 7289. [19] E. Fleige, M. A. Quadir, R. Haag, Adv. Drug Deliver. Rev. 2012, 64, 866. [20] M. Motornov, Y. Roiter, I. Tokarev, S. Minko, Prog. Polym. Sci. 2010, 35, 174. [21] J. Dai, S. Lin, D. Cheng, S. Zou, X. Shuai, Angew. Chem. Int. Ed. 2011, 50, 9404. [22] E. S. Lee, K. Na, Y. H. Bae. J. Control. Release 2005, 103, 405. [23] E. S. Lee, K. T. Oh, D. Kim, Y. S. Youn, Y. H. Bae. J. Control. Release 2007, 123, 19. [24] H. Yin, E. S. Lee, D. Kim, K. H. Lee, K. T. Oh, Y. H. Bae, J. Control. Release 2008, 126, 130. [25] S. Tang, Q. Yin, Z. Zhang, W. Gu, L. Chen, H. Yu, Y. Huang, X. Chen, M. Xu, Y. Li, Biomaterials 2014, 35, 6047. [26] E. M. Bachelder, T. T. Beaudette, K. E. Broaders, J. Dashe, J. M. J. Frechet, J. Am. Chem. Soc. 2008, 130, 10494. [27] W. Chen, F. Meng, F. Li, S. J. Ji, Z. Zhong. Biomacromolecules 2009, 10, 1727. [28] C. Schatz, S. Louguet, J. F. L. Meins, S. Lecommandoux, Angew. Chem. 2009, 121, 2610. [29] K. E. Broaders, J. A. Cohen, T. T. Beaudette, E. M. Bachelder, J. M. J. Frechet, Proc. Natl. Acad. Sci. USA. 2009, 106, 5497. [30] M. Nakanishi, J. S. Park, W. D. Jang, M. Oba, K. Kataoka, React. Funct. Polym. 2007, 67, 1361. [31] J. Dai, S. Y. Zou, Y. Y. Pei, D. Cheng, H. Ai, X. T. Shuai, Biomaterials 2011, 32, 1694. [32] D. A. Wang, A. S. Narang, M. Kotb, A. O. Gaber, D. D. Miller, S. W. Kim, R. I. Mahato, Biomacromolecules 2002, 3, 1197. [33] J. A. Cohen, T. T. Beaudette, J. L. Cohen, K. E. Broaders, E. M. Bachelder, J. M. J. Frechet, Adv. Mater. 2010, 22, 3593. [34] H. Uchida, K. Miyata, M. Oba, T. Ishii, T. Suma, K. Itaka, N. Nishiyama, K. Kataoka, J. Am. Chem. Soc. 2011, 133, 15524.

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Synthesis of polysaccharide-block-polypeptide copolymer for potential co-delivery of drug and plasmid DNA.

A pH-sensitive, biodegradable, and biocompatible polysaccharide-block-polypeptide Copolymer derivative {Ac-Dex-b-PAsp(DET)} is synthetized from acetal...
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