Codelivery of doxorubicin and p53 by biodegradable micellar carriers based on chitosan derivatives

Abstract

In this work, novel biodegradable cationic micelles were prepared based on poly-(N-ε-carbobenzyloxy-L-lysine) (PZLL) and chitosan (CS) by click reaction, and applied for co-delivery of doxorubicin (DOX) and p53 plasmid. The structure of the copolymer was characterized by ¹H NMR and FTIR. The loading amount of DOX in the micelles was 12.8%. Fluorescence spectra confirmed that DOX interacted via π–π stacking with micelles when DOX was encapsulated into the micelles. In particular, its complexation with plasmid DNA was investigated using agarose gel electrophoresis, flow cytometry, zeta potential, and particle size analyses as well as transmission electron microscopy observations. The results showed that the copolymers have a strong pDNA condensation ability and provide protection of pDNA against deoxyribonuclease I degradation. CS-g-PZLL/DOX/p53 nanoparticles showed good gene transfection efficiency in vitro. Fluorescence images and flow cytometry tests revealed that p53 and DOX could be efficiently transported into Hela tumor cells simultaneously, and the optimum N/P ratio for p53 transfection was 20/1. For co-delivery analysis, the obtained CS-g-PZLL/DOX/p53 complexes showed a better inhibitory effect on Hela tumor cells than DOX or p53 used singly.

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1. Introduction

To date, cancer treatment still faces serious challenges. Chemotherapy is one of the reliable choices for the treatment of many cancers. However, treatment with chemotherapy has been limited because of the emergence of multi-drug resistance (MDR), which is commonly associated with cancer cell over expression of drug transporter proteins.¹ A novel approach to address cancer drug resistance is to take advantage of the co-delivery of anticancer drugs and nucleic acid using multi-functionalized nanocarriers. Co-delivery of anticancer drugs and nucleic acid to the tumor site could efficiently control the drug transporter proteins. This promising approach may sidestep MDR and lead to an improved therapeutic effect.

For the co-delivery of drugs and genes while maintaining their biological functions, there has been an increasing interest in the development of multifunctional polymeric carriers using polymers,^2–4 liposomes^5,6 dendrimers,^7,8 silica,^9,10 quantum dot¹¹ based nanoparticles and so on. As one of the most promising nanocarrier systems, self-assembled cationic polymeric micelles have been widely utilized as drug and gene co-delivery systems. Cationic micelles are very effective nano-carriers for the co-delivery of genes and drugs into various cancer cell lines. Shi et al. prepared a series of cationic micelles based on triblock copolymers (MPEG–PCL-g-PEI) to deliver doxorubicin and the gene Msurvivin T34A. Their results showed that DOX and the gene were successfully co-delivered to the MCF-7 and CT26 cells. Introduction of T34A in combination with doxorubicin could greatly reduce systemic toxicity as well as improve the anti-tumor efficiency.¹² Lee et al. designed an amphiphilic copolymer poly{(N-methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido ethyl) methyl bis ammonium bromide] sebacate} [P(MDS-co-CES)] to deliver human TRAIL and paclitaxel simultaneously. They found that the co-delivery nanoparticulate system induced synergistic anti-cancer activities with relatively low toxicity in non-cancerous cells.¹³ However, these kinds of cationic polymers are still associated with problems of biodegradability, biocompatibility and cytotoxicity, which need to be overcome for in vivo application.

In this work, novel cationic micelles based on a polysaccharide and polypeptide were prepared by click chemistry, and applied for co-delivery of doxorubicin (DOX) and p53 plasmid. Poly-(N-ε-carbobenzyloxy-L-lysine) (PZLL) is a hydrophobic polypeptide derivate. Due to its good biocompatibility and biodegradability, PZLL has been widely used as the hydrophobic inner cores of micelles.^14–16 DNA can be bound tightly to the surfaces of the micelles because of the amino groups in the chitosan chains and PZLL branch chains. Their proton buffering capability, DNA condensation ability, protection of pDNA against deoxyribonuclease I degradation, in vitro cytotoxicity and gene transfection efficiency into Hela cells were investigated via acid–base titration, agarose gel electrophoresis, MTT, flow cytometry assay and fluorescence microscopy. Their drug loading capacity and in vitro release behavior were studied using DOX as a model drug. The co-delivery of an anti-cancer DOX and functional gene (p53 plasmid) into Hela cells was also investigated and discussed in this study.

2. Experimental section

2.1. Materials

N-ε-Carbobenzyloxy-L-lysine, doxorubicin hydrochloride, azido propylamine phthalic anhydride, hydrazine monohydrate, N-bromosuccinimide (NBS), triphenylphosphine (TPP), copper sulfate, 1-methyl-2-pyrrolidinone (NMP) and triphosgene were purchased from Aladdin Chemical Reagent Co., Ltd. (China). Chitosan (M_w = 10 kDa) was purchased from Haidebei Marine Bioengineering Co. Ltd. (China). Propargylamine, sodium azide (NaN₃, 99%) and sodium ascorbate (99%) were purchased from Alfa Aesar. The Dulbecco’s modified Eagle medium (DMEM), trypsin–ethylenediaminetetraacetic acid (trypsin–EDTA), and fetal bovine serum (FBS) were purchased from Gibco-BRL (Canada). Polyethylenimine (PEI, 25 kDa) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (U.S.A.). Deoxyribonuclease I (DNaseI) was purchased from Feibo Life Sciences (China). The plasmid p53 was obtained from Invitrogen. All other reagents were analytical grade and were used as received.

2.2. Preparation of CS-g-PZLL copolymers

CS-g-PZLL was prepared by click reaction of α-alkyne-poly-(N-ε-carbobenzyloxy-L-lysine) (α-alkyne-PZLL) and azide focal point chitosan (6-N₃–CS), as shown in Scheme 1. First, α-alkyne-PZLL (Scheme 1A) was synthesized following a procedure reported by Lin.¹⁷

Scheme 1 Synthesis of (A) α-alkyne-PZLL, (B) 6-N₃–CS, (C) CS-g-PZLL. Reaction conditions: (a) phthalic anhydride, DMF, 120 °C, 8 h; (b) N-bromosuccinimide, triphenylphosphine, NMP, 80 °C, 2 h; (c) sodium azide, NMP, 80 °C, 4 h; (d) hydrazine monohydrate, water, 100 °C, 10 h; (e) CuSO₄·5H₂O/sodium ascorbate, DMSO/water, 40 °C, 24 h.

In brief, 5.0 g of N-ε-carbobenzyloxy-L-lysine (17.8 mmol) was reacted with 3 g of triphosgene (10.1 mmol) using tetrahydrofuran (THF) as solvent. The reaction time was 1 h and reaction temperature was 50 °C. After reaction, the solvent was removed in vacuo, and the obtained residue was first dissolved in ethyl acetate, then washed with cold 5% NaHCO₃ solution. The ethyl acetate layer was collected and dried using anhydrous Na₂SO₄. The ethyl acetate was removed in vacuo and a ε-carbobenzyloxy-L-lysine N-carboxyanhydride (Lys (Z)-NCA) white solid with a yield of 77% was obtained. After that, 1.0 g of Lys (Z)-NCA was reacted with 0.01 g of propargylamine in anhydrous dimethylformamide (DMF), and the reaction time was 3 days. After reaction, methanol was added into the solution and a white powder deposit was obtained. The chemical structure was confirmed by ¹H NMR and FTIR. ¹H NMR (400 MHz, D₂O): δ 7.36–7.41 (5H, m, –Ph), 5.08 (s, 2H, –OCH₂Ph), 3.95 (s, 2H, –CCH₂NH–), 2.89 (d, 2H, –NHCH₂CH₂), 1.7 (t, 2H, –CHCH₂CH₂–), 1.24 (m, 2H, –CHCH₂CH₂–). IR (KBr, cm⁻¹): 3299, 3053, 2970, 1645, 1544, 1258, 1165, 1013, 539.

Second, 6-N₃–CS (Scheme 1B) was synthesized by a similar method as that reported by Deng et al.¹⁸ In brief, chitosan and phthalic anhydride were dissolved in DMF and reacted for 8 h at 120 °C to obtain N-phthaloyl-chitosan (2). (2) was then reacted with N-bromosuccinimide to obtain 6-bromide-6-deoxy-N-phthaloyl-chitosan (3), and after that, (3) was reacted with sodium azide in N-methylpyrrolidone for 8 h at 80 °C. After reaction, the solution was filtered and precipitated with ethanol, and the precipitate was collected and washed using acetone three times, then dried under vacuum to get 6-azido-6-deoxy-N-phthaloyl-chitosan (4) with a yield of 73%. ¹H NMR (400 MHz, D₂O): δ 3.30–3.90 (m, D-glucosamine unit, H-3, H-4, H-5, H-6, H-60), 3.34–3.78 (2H, –CONHCH₂–), 2.90 (protons next to amines). IR (KBr, cm⁻¹): 3442, 2930, 2114, 1667, 1382, 1071, 1013, 657.

Finally, CS-g-PZLL was synthesized as shown in Scheme 1C. α-Alkyne-PZLL (3 mmol), 6-N₃–CS (1 mmol) and the catalyst (CuSO₄·5H₂O/sodium ascorbate, 0.5 mmol/1 mmol) were dissolved in 20.0 mL of DMSO/water (5 : 1, v/v) mixture solution and reacted at 50 °C for 24 h. The mixture was then precipitated with ethanol and purified by dialysis in water for 2 days. After dialysis and lyophilization, the CS-g-PZLL was collected as a brown powder with a yield of 87%. ¹H NMR (400 MHz, D₂O): δ 7.36–7.41 (5H, m, –Ph), 5.08 (s, 2H, –OCH₂Ph), δ 3.30–3.90 (m, D-glucosamine unit, H-3, H-4, H-5, H-6, H-60), 3.34–3.78 (2H, –CONHCH₂–), 2.90 (protons next to amines) 1.7 (t, 2H, –CHCH₂CH₂–), 1.24 (m, 2H, –CHCH₂CH₂–). IR (KBr, cm⁻¹): 3299, 3053, 2930, 1645, 1382, 1258, 1165, 1013, 539.

2.3. DOX loading and in vitro release

DOX was loaded using a dialysis method reported by Lin et al.⁹ Briefly, 10 mg of CS-g-PZLL and 5.0 mg of DOX hydrochloride were first dissolved using 5.0 mL of DMSO as solvent, and for neutralization of HCl, a drop of triethylamine was then added to the solution. The complete dissolved solution was then transferred to a dialysis bag (MWCO 3000) and subjected to dialysis against distilled water for 48 h. After that, the dialysis solution was filtered through a 0.45 μm filter and then lyophilized. To investigate the interactions between CS-g-PZLL and DOX, the obtained CS-g-PZLL/DOX complex was analysed using obtained fluorescence spectra. The excitation wavelength was 330 nm and the fluorescence emission spectra were recorded in the range from 400 to 700 nm. To determine the loading amount of DOX, the obtained DOX/CS-g-PZLL was dissolved in DMSO and analyzed using UV-vis spectrophotometry (TU-1900, China) at 480 nm. It was found that the loading amount of DOX was 12.8%. The loading amount of DOX was calculated according to the following equation:

LC = M₁/M₀ × 100%

where M₀ is the weight of micelles, and M₁ is the weight of the loaded DOX.

The release of DOX from CS-g-PZLL was assayed at 37 °C in PBS buffer of pH 5.8 (to simulate the pH of a tumor) and 7.4 (to simulate the pH of blood plasma). A predetermined amount of the DOX/CS-g-PZLL complexes in 5.0 mL of PBS (pH 5.0 and 7.4) was sealed in a dialysis bag (MWCO = 3 kDa), and then the dialysis bag was submerged in 20 mL of the corresponding buffer. At predetermined time intervals, 2.0 mL of aqueous solution was taken out for drug concentration measurement and replaced by an equal volume of fresh PBS. The released DOX in the incubation buffer was analyzed using UV-vis spectrophotometry (TU-1900, China) at 480 nm. All measurements were performed in triplicate.

2.4. Plasmid binding

2.4.1. Formation. For the plasmid binding to the CS-g-PZLL, p53 solution was added to the CS-g-PZLL or DOX loaded CS-g-PZLL solutions at various N/P ratios, then mixed by gentle agitation for 5 s and incubated at 37 °C for 30 min before use.

2.4.2. Gel electrophoresis. An agarose gel retardation assay was carried out to determine the DNA condensation ability of the CS-g-PZLL micellar nanoparticles for p53. CS-g-PZLL/p53 complexes were prepared at various N/P ratios (CS-g-PZLL to p53: 5, 10 and 20). The complexes were mixed with appropriate amounts of loading buffer and incubated for 30 min at room temperature, then loaded onto a 1.0% agarose gel containing GeneGreen (0.1 mg mL⁻¹, Sigma) and electrophoresed with tris–acetate buffer for 30 min at 100 V. The location of the DNA in the gel was analyzed using a UV transilluminator and a digital imaging system (Fisher Scientific, PA, USA).

DNase I was added to the CS-g-PZLL/p53 complexes (N/P 5, 10 and 20) to examine the protective ability of CS-g-PZLL against DNase degradation. DNase I and the complexes were incubated at 37 °C for 30 min, after that, EDTA (4.0 mL, 250 mM) and sodium dodecyl sulfate (SDS) solution (4.0 mL, 10%, w/v) were added and the mixture was incubated at room temperature for another 1 h. The samples were then electrophoresed on the 1.0% agarose gel to examine the integrity of the DNA.

2.4.3. Size and morphology. The particle size and surface charge of the complexes were determined using a Zeta Potential Analyzer instrument (ZetaPALS, Brookhaven Instruments Corporation, USA). The morphology of the complex was observed using a JEM-2010HR high-resolution transmission electron microscope instrument.

2.4.4. In vitro transfection. For transfection, Hela cells were plated in 24-well plates at 1 × 10⁴ cells per well. Prior to transfection, the cells were washed with PBS buffer first, then medium in each well was replaced with serum-free media for 12 h. After that, cells were replenished with 10% fetal bovine serum media containing CS-g-PZLL/p53 complexes at different N/P ratios (containing 2.0 μg of p53 in each N/P ratio). After 48 h of transfection at 37 °C, the cells were observed using an Olympus IX71 fluorescence microscope (Melville, NY, U.S.A.). The transfected cells were washed once with PBS, detached with 0.25% trypsin and collected, and transfection efficiency was analyzed via flow cytometry quantitatively by scoring the percentage of cells expressing GFP (FACS Aria flow cytometer Germany).

2.5. In vitro cytotoxicity assay

An in vitro cytotoxicity assay of CS-g-PZLL and DOX loaded CS-g-PZLL nanoparticles was performed against Hela cells using the MTT assay. Five multiple holes were set for every sample. Briefly, Hela cells were cultured on 96-well plates at a density of 1 × 10⁴ cells per well and incubated in a humidified atmosphere of 5% CO₂ at 37 °C for 12 h. After that, the growth medium was replaced with 100 μL of complete DMEM containing an indicated amount of sample and the cells were further incubated for 24 h. Then 10 μL of MTT (0.5 mg mL⁻¹) in PBS solution was added to each well, and the cells were incubated for another 4 h to form formazan crystals. Finally, the medium was removed and 100 μL of DMSO was added to each well. The optical density values of the samples were measured at 490 nm using a MRX-Microplate Reader (Thermo, USA). Cells treated with the same amount of PBS were used as a control. The relative cell viability was calculated as follow:

Cell viability (%) = [A₄₉₀ (sample)/A₄₉₀ (control)] × 100

where A₄₉₀ (sample) and A₄₉₀ (control) were obtained in the presence and absence of sample, respectively.

3. Result and discussion

3.1. Synthesis and characterization of CS-g-PZLL

Fig. 1 shows the ¹H NMR spectra of the CS-g-PZLL, 6-N₃–CS, and PZLL. As seen in the spectrum of the CS-g-PZLL, the new peaks at 5.0, and 7.0–7.30 ppm showed the presence of the PZLL compared to the spectrum of the 6-N₃–CS. Moreover, the peak at 8.1 ppm in the spectrum of the CS-g-PZLL indicated the presence of the triazole proton, which could be attributed to the formation of 6-N₃–CS and PZLL by the click reaction.

Fig. 1 ¹H NMR spectra of CS-g-PZLL, N₃–CS and PZLL.

Fig. 2 shows the FTIR spectra of the CS-g-PZLL, N₃–CS and PZLL. As seen in the FTIR spectrum of N₃–CS, the characteristic vibration band for the azide group at 2110 cm⁻¹ demonstrated that azide groups were successfully incorporated in N₃–CS. A new peak at 1450 cm⁻¹ appeared in the samples of CS-g-PZLL, which is attributed to the 1,2,3-triazole structure formed during click modification.¹⁷ The FTIR data indicated that the PZLL dendrimer was successfully grafted onto the chitosan chains via click chemistry.

Fig. 2 FTIR spectra of CS-g-PZLL, N₃–CS and PZLL.

3.2. DOX-loaded micelles and in vitro drug release

The in vitro release of drugs from the micelles was investigated at pH 7.4 and 5.0 to imitate the pH of blood and a tumor site. DOX was chosen as the model drug, and DOX was encapsulated into the hydrophobic core of the micelles. Fig. 3A shows the interaction of DOX and micelles by fluorescence spectra. It was found that the fluorescence of the complex was nearly completely quenched when DOX was encapsulated into the micelles, confirming the π–π stacking interaction between DOX and the phenyl groups of the PZLL chains.^19,20 Moreover, the π–π stacking interaction helps to improve the high loading amount of DOX. It was found that the loading amount of DOX in the micelles was 12.8%. As shown in Fig. 3B, the release profiles showed the sustained release behaviors of DOX at pH 7.4 and pH 5.0, where 35.7% and 37.8% of the loaded DOX was released at pH 7.4 and pH 5.0 in the initial 12 h, and the accumulated release reached 61.4% and 75.4% after 96 h, respectively (Fig. 3B). It is interesting to find that the release rate and released amount of DOX at pH 5.0 were a little higher than those at pH 7.4, which revealed a promoted drug release behavior at the tumor site. DOX was released from micelles mainly by free diffusion. The better solubility of DOX at pH 5.0 than at pH 7.4 promoted the rapid release behavior. Moreover, the pH-responsive property of the copolymers leads to the higher release rate at lower pH. Since a tumor shows a lower extracellular pH, this observation demonstrated that the CS-g-PZLL micelle might show potential applications as a tumor-targeting drug delivery platform.

Fig. 3 (A) The fluorescence spectra of the micelles, DOX solution and complex (λ_excitation = 330 nm). (B) In vitro release profiles of the loaded DOX from the CS-g-PZLL nanoparticles in phosphate buffer (pH 7.4 or 5.8) at 37 °C.

3.2.1. Gel electrophoresis. The formation of the CS-g-PZLL/p53 complexes was examined using an agarose gel electrophoresis assay. As shown in Fig. 4A, the migration of the pDNA was completely retarded when the MSN-x-G3/pDNA weight ratio exceeded 5. These results indicated that CS-g-PZLL has a strong binding ability for p53. Fig. 4B shows the protective effect of CS-g-PZLL against p53 degradation by DNase I. It was found that the naked p53 was completely digested, while CS-g-PZLL/p53 at all N/P ratios (5, 10 and 20) exhibited distinct protective effects against DNase I. These results indicated that the micelles formed by the CS-g-PZLL copolymer could be used as a co-delivery system for loading hydrophobic drugs and genes. Moreover, the co-delivery system could protect genes against DNase simultaneously.

Fig. 4 (A) Agarose gel electrophoresis retardation assay of CS-g-PZLL/p53 complexes at different N/P ratios. (B) Protection and release assay of p53. p53 was released by adding 10% SDS to CS-g-PZLL/p53 complexes at different N/P ratios.

3.2.2. Size and morphology. The mean particle sizes and zeta potential of the CS-g-PZLL/DOX/p53 complexes were investigated using a Zeta Potential Analyzer and TEM, as shown in Fig. 5. It was found that the zeta potential increased with the N/P ratio of 2–80, and the range was between 0.5 and 12.5 mV. By contrast, the size of the complexes tended to decrease with the increase of the N/P ratio, and remained in the size range from 400 to 150 nm. The TEM photos show that the CS-g-PZLL/DOX/p53 complexes (Fig. 5B) have a spherical shape and compact structure. The complexes had an average diameter of about 100 nm, which was consistent with the results measured using the zeta potential test. It is reported that the size range of pDNA containing complexes from 50 to 400 nm is suitable for cellular endocytosis.^21,22 In this study, the test results indicate that CS-g-PZLL/DOX/p53 complexes could meet the requirement of efficient gene delivery.

Fig. 5 (A) Particle sizes and zeta potentials of CS-g-PZLL/DOX/p53 complexes formed at various N/P ratios. (B) Typical particle size distribution and TEM image of the CS-g-PZLL/DOX/p53 complex formed at an N/P ratio of 20.

3.2.3. Co-delivery and cell viability. To investigate the co-delivery ability of CS-g-PZLL, Hela cells were studied by fluorescence microscopy using the plasmid eGFP-N1-53 and DOX as a model gene and hydrophobic drug. The representative fluorescence images are shown in Fig. 6. The green and red fluorescence were both observed in the Hela cells at different N/P ratios, the green fluorescence was from eGFP and the red fluorescence was from DOX (Fig. 6A). The results suggested that both p53 and DOX can be delivered into Hela cells by CS-g-PZLL at different N/P ratios. The intensity of the red fluorescence at different N/P ratios did not show apparent differences. It is suggested that DOX could be efficiently transported into the cells at all N/P ratios. On the other hand, the intensity of the green fluorescence from the eGFP was stronger at the N/P ratio of 20 than the others, indicating that the N/P ratio of 20 was the optimum N/P ratio for p53 transfection. This result was consistent with the results of the flow cytometry, as shown in Fig. 5B. The highest transfection ratio was 31.2% while the N/P ratio was 20. The percentage of transfected cells at the N/P ratio of 40 was slightly less than that at the N/P ratio of 20. This result may be caused by the slightly reduced tolerance of Hela cells under the high CS-g-PZLL concentration (Fig. 6C).

Fig. 6 (A) Fluorescence field images of Hela cells transfected with CS-g-PZLL/p53/DOX complexes formed at various N/P ratios (1 N/P = 10, 2 N/P = 20, 3 N/P = 40). (B) Quantitative determination of transfected Hela cells by flow cytometry (C) Hela cell viability after treatment with CS-g-PZLL, CS-g-PZLL/p53 and CS-g-PZLL/p53/DOX.

The co-delivery of drugs and genes has become the primary strategy in cancer and other disease therapy in recent years, because this technique could promote synergistic actions, improve target selectivity and deter the development of drug resistance. For the co-delivery of antitumor drugs and genes while maintaining their chemo-physical properties and biological functions, a multifunctional carrier in which gene and drug could be loaded simultaneously is necessary. In previous studies, cationic polymeric micelles have been widely used as drug and gene co-delivery carriers. Zhu et al.²² prepared biodegradable cationic micelles based on the self-assembly of PDMAEMA–PCL–PDMAEMA triblock copolymers as siRNA and paclitaxel co-delivery carriers. The results demonstrated that combinatorial delivery of VEGF siRNA and paclitaxel showed an efficient knockdown of VEGF expression. Zheng et al.²³ prepared polypeptide cationic micelles based on poly(ethylene glycol)-b-poly(L-lysine)-b-poly(L-leucine) (PEG–PLL–PLLeu) triblock copolymers as docetaxel (DTX) and siRNA-Bcl-2 co-delivery vectors. The results showed that the co-delivery of DTX and siRNA-Bcl-2 (siRNA that suppresses the expression of the anti-apoptotic Bcl-2 gene) significantly inhibited tumor growth as compared to the individual siRNA or DTX treatment.

To confirm the cell inhibition effect of the complexes containing both DOX and p53, we evaluated their cytotoxic effects using a MTT assay. The results showed that CS-g-PZLL was non-toxic at the concentration used in this assay, while the samples containing p53 showed an obvious cytotoxicity, where the cell viability was decreased to 70, 58 and 55% at the N/P ratios of 10, 20 and 40 (Fig. 6C). A further inhibitory effect was found in the co-delivery group, where the cell viability was decreased to 63, 41 and 44%, respectively. The better effect may be attributed to the released DOX being able to damage DNA; meanwhile, p53 could instigate mRNA transcription to down-regulate protein expression. The result suggested that the co-delivery induces synergistic actions and leads to an effective method for tumor therapy.

4. Conclusion

For the co-delivery of an anti-tumor drug and gene to tumor cells, a new cationic micelle consisting of poly-(N-ε-carbobenzyloxy-L-lysine) (PZLL) and chitosan has been synthesized, and used to co-deliver DOX and p53 for cancer therapy. CS-g-PZLL/DOX/p53 nanoparticles showed good gene transfection efficiency in vitro, and could deliver p53 and DOX simultaneously to Hela tumor cells. Through co-delivery analysis, the obtained CS-g-PZLL/DOX/p53 complexes were shown to have a better inhibitory effect on Hela tumor cells than p53 used singly. Such a cationic micelle delivery system may be used as a potential multifunctional vector for future cancer therapy applications.

Acknowledgements

This work was financially supported by National Natural Science Foundation of China (81402563), Traditional Chinese Medicine Bureau Foundation of Guangdong Province (20141158), Doctoral Research Program of Guangdong Medical College (XB1303 and XB1387) and Special Foundation for Young Innovation Scientists of Department of Education of Guangdong Province (4CX14119G and 4CX14118g).

References

1. X.-B. Xiong and A. Lavasanifar, ACS Nano, 2011, 5, 5202–5213.
2. Y. Wang, S. Gao, W.-H. Ye, H. S. Yoon and Y.-Y. Yang, Nat. Mater., 2006, 5, 791–796.
3. Q.-D. Hu, H. Fan, Y. Ping, W.-Q. Liang, G.-P. Tang and J. Li, Chem. Commun., 2011, 47, 5572–5574.
4. M. Ma, Z. Yuan, X. Chen, F. Li and R. Zhuo, Acta Biomater., 2012, 8, 599–607.
5. M. S. Suh, G. Shim, H. Y. Lee, S.-E. Han, Y.-H. Yu, Y. Choi, K. Kim, I. C. Kwon, K. Y. Weon and Y. B. Kim, J. Controlled Release, 2009, 140, 268–276.
6. Z. Xu, Z. Zhang, Y. Chen, L. Chen, L. Lin and Y. Li, Biomaterials, 2010, 31, 916–922.
7. S. Liu, Y. Guo, R. Huang, J. Li, S. Huang, Y. Kuang, L. Han and C. Jiang, Biomaterials, 2012, 33, 4907–4916.
8. T. L. Kaneshiro and Z.-R. Lu, Biomaterials, 2009, 30, 5660–5666.
9. J.-T. Lin, C. Wang, Y. Zhao and G.-H. Wang, Mater. Res. Express, 2014, 1, 035403.
10. M. Akin, R. Bongartz, J. G. Walter, D. O. Demirkol, F. Stahl, S. Timur and T. Scheper, J. Mater. Chem., 2012, 22, 11529–11536.
11. J.-M. Li, Y.-Y. Wang, M.-X. Zhao, C.-P. Tan, Y.-Q. Li, X.-Y. Le, L.-N. Ji and Z.-W. Mao, Biomaterials, 2012, 33, 2780–2790.
12. S. Shi, K. Shi, L. Tan, Y. Qu, G. Shen, B. Chu, S. Zhang, X. Su, X. Li and Y. Wei, Biomaterials, 2014, 35, 4536–4547.
13. A. L. Lee, Y. Wang, S. Pervaiz, W. Fan and Y. Y. Yang, Macromol. Biosci., 2011, 11, 296–307.
14. H.-Y. Wen, H.-Q. Dong, W.-j. Xie, Y.-Y. Li, K. Wang, G. M. Pauletti and D.-L. Shi, Chem. Commun., 2011, 47, 3550–3552.
15. J. Ding, J. Chen, D. Li, C. Xiao, J. Zhang, C. He, X. Zhuang and X. Chen, J. Mater. Chem. B, 2013, 1, 69–81.
16. C. Deng, X. Chen, H. Yu, J. Sun, T. Lu and X. Jing, Polymer, 2007, 48, 139–149.
17. J.-T. Lin, Y. Zou, C. Wang, Y.-C. Zhong, Y. Zhao, H.-E. Zhu, G.-H. Wang, L.-M. Zhang and X.-B. Zheng, Mater. Sci. Eng., C, 2014, 44, 430–439.
18. J. Deng, Y. Zhou, B. Xu, K. Mai, Y. Deng and L.-M. Zhang, Biomacromolecules, 2011, 12, 642–649.
19. M. Dong, L. Zong-Hua, Z. Qian-Qian, Z. Xiao-Yan, Z. Yi, S. Yun-Feng, L. Jian-Tao and X. Wei, Macromol. Rapid Commun., 2013, 34, 548–552.
20. D. Ma, J. Lin, Y. Chen, W. Xue and L. M. Zhang, Carbon, 2012, 50, 3001–3007.
21. M. Metzke, N. O’Connor, S. Maiti, E. Nelson and Z. Guan, Angew. Chem., Int. Ed., 2005, 44, 6529–6533.
22. C. Zhu, S. Jung, S. Luo, F. Meng, X. Zhu, T. G. Park and Z. Zhong, Biomaterials, 2010, 31, 2408–2416.
23. C. Zheng, M. Zheng, P. Gong, J. Deng, H. Yi, P. Zhang, Y. Zhang, P. Liu, Y. Ma and L. Cai, Biomaterials, 2013, 34, 3431–3438.

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Footnote

† These authors contributed equally to this work.

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