Water-Soluble Cationic Polyphosphazenes Grafted with Cyclic

Subscriber access provided by RMIT University Library

Article

Water-soluble Cationic Polyphosphazenes Grafted with Cyclic Polyamine and Imidazole as An Effective Gene Delivery Vector Chunying Ma, Xiao Zhang, Changguo Du, Baojing Zhao, Chunhua He, Chao Li, and Renzhong Qiao Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00048 • Publication Date (Web): 04 Mar 2016 Downloaded from http://pubs.acs.org on March 11, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Water-soluble Cationic Polyphosphazenes Grafted with Cyclic Polyamine and Imidazole as An Effective Gene Delivery Vector Chunying Ma†,§, Xiao Zhang†,§, Changguo Du†, Baojing Zhao†, Chunhua He†, Chao Li*,† and Renzhong Qiao*,†,‡ †

The State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical

Technology, Beijing, 100029, P. R. China ‡

State Key Laboratory of Natural and Biomimetic Drugs School of Pharmaceutical Sciences,

Peking University Health Science Center, Beijing, 100083, P. R. China

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 31

KEYWORDS: polyphosphazene; gene delivery; cyclic polyamine; Imidazole; non-viral carriers

ABSTRACT: Gene therapy holds immense potential as a future therapeutic strategy for the treatment of numerous genetic diseases which are incurable to date. Nevertheless, safe and efficient gene delivery remains the most challenging aspects of gene therapy. In this study, a series of polyphosphazenes (PPZ) bearing cyclic polyamine and imidazole groups were synthesized and investigated for gene delivery. Agarose gel electrophoresis assays showed that poly(Imidazole/1,4,7,10-tetraazyclodocane)phosphazene (Im-PPZ-cyclen) had good binding ability with plasmid DNA (pDNA), yielding positively charged particles with a size around 120140 nm from an ratio of 10:1 to 5:1 (Im-PPZ-cyclen/pDNA, w/w). The cytotoxicity of Im-PPZcyclen assayed by MTT was lower than that of PEI 25 kDa, and was similar with reported poly(di-2-dimethylaminoethylamine)phosphazene

(poly(di-DMAEA)phosphazene) in

some

degree. The maximum transfection efficiency of Im-PPZ-cyclen/pDNA complexes against 293 T cells at the ratio of 5:1 (Im-PPZ-cyclen/pDNA, w/w) is close to that of Lipofectamine 2000TM. The present work may provide a strategy for the design of new cationic polymers with reduced cytotoxicity and be applied to gene delivery as efficient non-viral vector.


2

Page 3 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Gene therapy has evolved as a promising therapeutic modality for the treatment of a wide range of diseases of both innate and acquired origin.1,2 The naked DNA is highly susceptible to nuclease degradation and displays poor cellular uptake, as well as low transfection efficiency.3,4 Therefore, the basic challenge for gene therapy is to design safe and effective carriers that assist efficient transfer of the therapeutic gene to targeted cells without degradation of the delivered gene. Viral carriers like recombinant retroviruses and adenoviruses have been widely studied for introducing therapeutic gene into cells.5 However, their applications have been restricted by some major problems such as immune and toxic response, random integration mediated by (retro)viruses, limits of plasmid size to be delivered and possible recombination with wild type viruses.6,7 Numerous non-viral delivery carriers including cationic polymers, liposomes, and organic or inorganic nanoparticles have been well developed and reported because of their low cost, flexibility in chemical design and safety.8-10 Recently, numerous researchers concentrate primarily on modifying the chemical composition and architecture of existed carriers by grafting functional ligands such as peptides11, lipids12,13, sugars14 or a combination15 to improve the efficiency and biocompatibility of vectors for gene delivery. Some non-viral vectors, such as polyethylenimine (PEI), chitosan, etc. are limited by their either low gene transfection efficiency or significant toxicity, and these limitations urge us to find new carries with low cytotoxicity and high gene transfection efficiency. Polyphosphazenes (PPZ) have been investigated for different biomedical and pharmaceutical applications because of their excellent hydrolytic degradability and non-toxic degradable products.16-20 A variety of


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 31

poly(organo)phosphazenes were prepared and evaluated for gene delivery after Hennink’s group firstly reported poly(2-dimethylaminoethylamine)phosphazene was able to transfect COS-7 cells in vitro and the toxicity was less compared to other used polymeric transfectants.21 Li’s group recently reported the synthesis and characteristics poly(imidazole/DMAEA)phosphazene consisting

of

a

biodegradable

PPZ

backbone

substituted

by

imidazole

and

2-

dimethylaminoethylamine groups, and found that poly(imidazole/DMAEA)phosphazene can condense DNA into complex nanoparticles and obviously enhance gene transfer activity with higher transfection efficiency and lower cytotoxicity compared with the control compound without imidazole moieties.22 It also should be noted that imidazole ring, which is one moiety of histidine with potent biocompatibility, could favor polymers/DNA complexes escaping from endosome by a “proton sponge” mechanism, possessing a buffering capacity in the endolysosomal pH range.23-25 Polyamine, which can act as a proton-sponge, is well studied materials for DNA delivery because of its buffering capability together with high binding capability towards DNA and relatively high transfection efficiency. Furthermore, our previous studies showed that a series of chitosan derivatives grafted by cyclic polyamine presented favorable transfection efficiency and acceptable cytotoxicity26, and further modification of phosphorylcholine improved the capability of the complexes to cross the cell membranes.27 Cyclic polyamine may have the potential to reduce charge associated cytotoxicity for gene delivery due to its centralized cyclic structures which can concentrate the positive charge into heterocyclic ring. These findings stimulated us to further investigate the cyclic polyamine-based PPZ polymers and their applications in gene delivery. Herein

we

reported

the

synthesis

and

characterization

of

biodegradable

poly(organo)phosphazenes bearing cyclic polyamine(cyclen, tacn and pip) and imidazole (Im)


4

Page 5 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


moieties, namely Im-PPZ-cyclen, Im-PPZ-tacn and Im-PPZ-pip (Scheme 1), and expected to induce DNA condensation which is necessary for gene delivery through charge interactions between polymer and the phosphate backbone of DNA. To further explore structure-activity relationship of DNA condensation induced by poly(organo)phosphazenes, control compounds including cyclen-PPZ-cyclen and Im-PPZ-Im were prepared and evaluated in comparison with the title compounds, as shown in Scheme 1. The abilities of these cationic polymers to bind and condense DNA into particles have been assessed using agarose gel electrophoresis, fluorescence quenching assay and circular dichroism (CD) experiments. Physical properties of polymer/DNA complexes have been characterized using atom force microscope (AFM) and dynamic light scattering (DLS). Their biological properties have been assessed by in vitro cytotoxicity and transfection activity. The results indicate that Im-PPZ-cyclen can interact with plasmid DNA to form around 120 nm particles, and protect DNA from enzymatic degradation. The further in vitro assays presented low cytotoxicity and favourable transfection activity of Im-PPZ-cyclen.

Scheme 1. Structures of poly(organo)phosphazene derivatives.

RESULTS


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 31

Polymer Synthesis and Characterization. Poly(dichlorophosphazene) (PDCP), as an essential intermediate for the synthesis of title compounds, was obtained by a simple and convenient one-pot synthesis in THF solution according to Carriedo’s method29, and the

31

P

NMR and FT-IR spectra are shown in SI Figure S1. The molecular weight of PDCP was 10 kDa, as determined by GPC analysis (Figure S2 and TableS1, SI). And molecular weight of Im-PPZpip, Im-PPZ-tacn and Im-PPZ-cyclen was 13 kDa, 17 kDa and 18 kDa, respectively, as also determined by GPC analysis. Cyclic polyamine including cyclen, tacn and pip is introduced to PDCP skeleton by a flexible linker; this process is realized by the replacing chlorine atom with NH2-terminal group, and followed by treatment with the excess of imidazole to achieve an attempted ratio of the two. Once the protected intermediates were obtained, tert-butoxycarbonyl (Boc-) groups were removed by trifluoroacetic acid (TFA) (90%, v/v) to give Im-PPZ-pip, Im-PPZ-tacn and ImPPZ-cyclen with cyclic polyamines, respectively. The route of synthesis and the NMR data are shown in SI Scheme S1 and Figures S3-S6, respectively. For instance,31P NMR spectrum of ImPPZ-cyclen showed signals at -2.40 ppm and -3.20 ppm attributable to –NP(cyclen)2– and – NP(imidazole/cyclen)– systems, respectively. A much lower signal at -10.2 ppm could be attributed to –NP(imidazole)2– group (SI Figure S6(C)). Because of high reactive polar phosphorus-chlorine bonds (P-Cl) in PDCP, chlorine atoms would be easily substituted by cyclen and Im moieties, and no signal that could be attributed to incomplete replacement product of chlorine atoms was observed in

31

PNMR spectrum (SI Figure S6(C)), indicating complete

chlorine replacement for Im-PPZ-cyclen. According to 1H NMR data, the degree of substitute of imidazole and cyclen on Im-PPZ-cyclen was 17% and 83%, respectively (SI Figure S5(A), and the degree of substitute of imidazole and tacn on Im-PPZ-tacn was 25% and 75%(SI Figure


6

Page 7 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


S4(A), respectively. And the degree of substitute of imidazole and pip on Im-PPZ-pip was 14% and 86% (SI Figure S3(A), respectively. To obtain Im-PPZ-Im or cyclen-PPZ-cyclen, excess of imidazole or Boc-cyclen was respectively coupled to PDCP following the same protocol as the synthesis of Im-PPZ-cyclen. The polymers of cyclen-PPZ-cyclen and Im-PPZ-Im were confirmed by NMR spectra (SI Figures S7 and S8). For Im-PPZ-Im, only two strong peaks occurred at 7.78 and 7.15 ppm in 1H NMR (SI Figure S8(A)) due to the introduction of imidazole and 31P NMR showed a singlet at 2.42 ppm (SI Figure S8(B)), which indicated complete chlorine replacement during substituent reaction. For cyclen-PPZ-cyclen, multiple peaks at 2.94-2.65 ppm confirmed the presence of cyclen in the polymer 1H NMR (SI Figure S7(A)), and

31

P NMR spectrum (SI Figure S7(B))

shows multiple peaks at 1.63 ppm due to the existence of much cyclen which may have an influence on splitting the peak. DNA Condensation. The ability of polymer to condense DNA plays a crucial role in efficient DNA transfection process. To investigate whether title compounds can induce DNA condensation, a series of polymer/pDNA complexes with different weight ratios were prepared and evaluated by gel retardation assay. As shown in Figure 1C, the electrophoretic mobility of DNA is retarded by the introduction of Im-PPZ-cyclen, and total DNA retardation is detected at and above the weight ratio of 10:1. The results indicate that Im-PPZ-cyclen has a good DNA binding ability, which could be attributed to the electrostatic interactions between positively charged cyclen and negatively charged DNA. In contrast, it is hard to monitor DNA retardation over the tested concentration range for Im-PPZ-pip and Im-PPZ-tacn (Figure 1A and 1B), which might be due to their weaker ability to capture the positive charge on the pip and tacn moieties, resulting in weak affinity to DNA. These results indicate that abundant positive amine groups in


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 31

cyclic polyamine are necessary for cationic polymers to capture the positive charge to interact with DNA through electrostatic interactions and result in DNA condensation.

Figure 1. Gel retardation assay by agarose gel electrophoresis in 40 mM pH 7.4 Tris-HCl buffer at 37°C for 12 h. Lane 1: DNA control; Lanes 2-7: poly(organo)phosphazene/pDNA (w/w) = 0.3, 1.0, 2.0, 3.0, 5.0, and 10.0, respectively. (A) Im-PPZ-pip; (B) Im-PPZ-tacn; (C) Im-PPZ-cyclen; (D) Im-PPZ-Im; (E) cyclen-PPZ-cyclen. Two control compounds Im-PPZ-Im and cyclen-PPZ-cyclen were prepared to verity potential valid groups for binding activity. Whether both cyclen and imidazole can neutralize negatively charges and effectively condense DNA that was evaluated by gel retarded assay. As shown in Figure 1D, migration of DNA in agarose gel was not retarded by the introduction of Im-PPZ-Im, indicating that only imidazole moieties on polymer backbone can not bind DNA to form large complexes in the absence of cyclen. In contrast, the extent of gel retarding at the same weight ratio remarkably enhanced compared with that of Im-PPZ-cyclen, which is attributed to the existence of more electropositive cyclen groups. Although imidazole ring with potent biocompatibility has successfully been grafted to many cationic polymers for regulating pH in the endolysosome, the introduction of polyamines as an important supplement played a key role in interaction with DNA.


8

Page 9 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Resistance to Nuclease. The structural integrity of the therapeutic gene is a prerequisite to ensure its desired function in vitro as well as in vivo.30 Thus, an efficient gene delivery vector must confer adequate protection to pDNA against nuclease degradation. In this study, the pDNA protection ability of polymers was evaluated using Dnase I as a model enzyme. As shown in SI Figure S9, after the naked pDNA (15 ng) treated with Dnase I (10 U·mL-1), the complete degradation of the pDNA occurred and the bands were not seen clearly (Lane 2). After the same amount of Im-PPZ-cyclen/pDNA and cyclen-PPZ-cyclen/pDNA complexes was treated with 1 U·mL-1 of Dnase I respectively, however, no detectable degradation of pDNA occurred (Lanes 4 and 6, SI Figure S9). The experimental results demonstrate that Im-PPZ-cyclen and cyclen-PPZcyclen can protect DNA from degradation of Dnase I. Interactions of Polymers with DNA. The binding ability of polymers to DNA was further studied by fluorescent spectroscopy with the use of ethidium bromide (EB) according to the literature.31-33 EB alone has weak fluorescence, but its emission intensity in the presence of DNA can be greatly enhanced because of its strong intercalation with the adjacent DNA base pairs. This enhanced fluorescence could be quenched, or at least partly quenched by the addition of a second molecule with higher DNA-binding ability. As shown in Figure 2A, Im-PPZ-Im showed a relatively weak and linear decrease of EB fluorescence intensity as a function of w/w ratios, which was according with the gel electrophoresis results that indicated weak interaction between Im-PPZ-Im and DNA. However, Im-PPZ-cyclen and cyclen-PPZ-cyclen showed a sharp decrease of EB fluorescence over the tested ratio range. The fluorescence quench results were also quite consistent with the following classical Sterne-Volmer equation:34 Fmax/Fx = 1 + Ksv (Q), where Fmax and Fx are as above-mentioned in Experimental section, Q is the mole concentration (mol/L) of polymers and Ksv is the quenching constant (namely binding constant)


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 31

which reflect the binding ability of polymers to DNA. For a more detail measurement, after linear fitting (Figure 2B), the binding constant Ksv values of the complexes formed from ImPPZ-Im, Im-PPZ-cyclen and cyclen-PPZ-cyclen were obtained as 0.12 × 104 M-1 (R2 = 0.9901), 0.68 × 105 M-1 (R2 = 0.9906) and 1.23 × 105 M-1(R2 = 0.9914), respectively, showing the DNA binding ability order of cyclen-PPZ-cyclen > Im-PPZ-cyclen > Im-PPZ-Im which is in agreement with gel electrophoresis assays.

Figure 2. (A) Fluorescence quenching of CT-DNA/EB system by addition of Im-PPZ-Im(■), Im-PPZ-cyclen (●) and cyclen-PPZ-cyclen (▼), respectively, in 10 mM pH 7.4 Tris-HCl buffer with 10 mM NaCl. (B) Linear fitting according the relative intensity (Fmax/F0) in different concentrations of Im-PPZ-Im(■), Im-PPZ-cyclen (●) and cyclen-PPZ-cyclen (▼). Diverse effects of poly(organo)phosphazenes on DNA were evaluated by CD experiment. The CD spectra of polymer/DNA complexes were determined at 25°C as depicted in Figure 3. Free DNA showed positive and negative ellipticities centered around 275 and 245 nm respectively, which revealed the B-type double-stranded structure for DNA (black line). Addition of Im-PPZIm does not induce CD signal change as shown in Figure 3 (red line), suggesting weak interaction between DNA and polymers. However, when DNA solution was mixed with Im-PPZcyclen, detectable changes were observed including significant decreasing both in positive and negative bands (blue line) compared to that of free DNA (black line). Though the shape of these bands was different from the typical ψ-phase, the decreased ellipticities in both sides suggested


10

Page 11 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


DNA helices could hardly remain a higher-order chiral structure. In addition, the addition of ImPPZ-cyclen does not induce the negative band shift in position, suggesting DNA remains in the B-type conformation.35,36 Cyclen-PPZ-cyclen (green line) shows similar CD behavior like that of Im-PPZ-cyclen under the same conditions.

Figure 3. CD spectra of poly(organo)phosphazene/DNA complexes (black, DNA only; red, ImPPZ-Im/DNA; blue, Im-PPZ-cyclen/DNA; green, cyclen-PPZ-cyclen/DNA). In Vitro Cytotoxicity Assay. The cytotoxicity of polymers on A 375 and HeLa cell lines was evaluated by MTT assay37, and the branched 25 kDa PEI was used as the control. It is reported that the cytotoxicity of cationic polymers is probably caused by the interactions with the plasma membrane or interactions with negatively charged cell components and proteins.38,39 Hence, as shown in Figure 4, the cytotoxicity increased with an increase of the polymer concentration range with both cell lines tested, which is probably due to increasing positive charge with the increasing of polymer concentration. Among the three poly(organo)phosphazene polymers, ImPPZ-Im displayed the highest cell viability, and Im-PPZ-cyclen showed higher cell viability than cyclen-PPZ-cyclen both in A 375 and Hela cells, which suggested that the cytotoxicity of


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 31

poly(organo)phosphazene as gene vectors strongly depended on the amounts of grafted amine moieties. Hence, when more cyclen molecules were grafted to PPZ to generate cyclen-PPZcyclen, charge density of resulted polymers increased, resulting in lower cell viability compared with Im-PPZ-cyclen. Considering their DNA binding abilities and cell viabilities, Im-PPZcyclen/DNA was selected as representative complex for the following research.

Figure 4. Cytotoxicity of Im-PPZ-Im(■), Im-PPZ-cyclen (●), cyclen-PPZ-cyclen (▼), and branched 25 kDa PEI (▲) at various concentrations for (A) A 375 and (B) HeLa cells, determined by the MTT assay. Particle size, zeta potential and morphology of polymer/pDNA complexes. The particle size and surface charge of polymer/DNA complexes control their cellular uptake.40 A positively charged complex could effectively interact with the anionic proteogylans of the cell membrane and thereby facilitate the adsorption mediated endocytosis of the complex. In general, complex less than 200 nm in size are favorable for cellular uptake through endocytosis.40 The zeta potentials and mean hydrodynamic diameters of Im-PPZ-cyclen/pDNA complex at different weight ratios were determined by dynamic light scattering (DLS) measurement. At the weight ratio of 1:1, the zeta potential was found to be negative as shown in Figure 5, indicating that the amount of Im-PPZ-cyclen was insufficient to condense pDNA completely. As the weight ratio increased, the zeta potentials rose and turned to be positive from the weight ratio


12

Page 13 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of 5:1. The increasing zeta potential can be attributed to the complete self-assembly of Im-PPZcyclen and DNA. Generally, the positively charged complexes can interact with the negatively charged cell membranes, leading to efficient transfection. As shown in Figure 5, at the weight ratio of 1:1, the polymer is inadequate to condense DNA (Figure 5), so that complexes with larger sizes formed. As the weight ratio increased, the particle sizes decreased because more positive charge enhance the DNA condensing capability and produce more compact particles. When the weight ratio raised to 5:1 or higher, the positive charge density of complexes was enough large to condense the DNA, thus the Im-PPZcyclen/pDNA complex with the size in the range of 121-143 nm formed, which was favorable for cellular gene effection.40

Figure 5. Average particle diameters and zeta potentials of Im-PPZ-cyclen/DNA complexes at various weight ratios using dynamic light scattering (DLS) measurement. Complexes were prepared in 10 mM pH 7.4 Tris-HCl buffer. The morphology of Im-PPZ-cyclen/pDNA complexes was further presented using AFM section analysis, as shown in Figure 6. The image of uncondensed pDNA was provided for comparison in Figure 6A, which shows a relaxed, open-loop structure with little twisting or


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 31

fasciculation of the strands. After the addition of Im-PPZ-cyclen (the same conditions as lane 7 in Figure 1C), the AFM image presents a number of globules with diameter of 98±8.3 nm and height of 9.01±0.06 nm, being more compact than the untreated pDNA (Figure 6B and 6C). The AFM data was slightly smaller in comparison with the results obtained by DLS study, which was probably because the sample was observed by AFM in a dry state as compared to a wet state by DLS analysis.

Figure 6. Atomic force microscopy images of (A) pDNA, The insert shows the surface plot delineated by the dashed square in the main panel; (B) Im-PPZ-cyclen/pDNA complexes (w/w = 10:1), The insert shows the surface plot delineated by the dashed square in the main panel; (C) The height of the nanoparticles delineated by the red line in the main panel of (B). The scale of images is 2.0 µm × 2.0 µm for (A) and (B). In vitro transfection experiment. The in vitro transfection efficiency of Im-PPZ-cyclen/DNA complex nanoparticles was investigated on 293 T cells. The optimum condition for transfection on 293 T cells was determined by assessing the gene expression of pEGFP-N3 at various weight


14

Page 15 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ratios in complete medium. The results showed that the efficiency was dependent on the ImPPZ-cyclen/DNA weight ratio (Figure 7), and a bell-shaped dependence of transfection efficiency on Im-PPZ-cyclen concentration was observed (SI Figure S10). This is in agreement with previous reported findings for some other poly(organo)phosphazenes.41,42 The transfection efficiency reached maximum at Im-PPZ-cyclen/DNA weight ratio of 5:1, then decreased with the ratio increasing. A possible explanation was the increase of cell toxicity when the ratio of ImPPZ-cyclen/DNA increased from 5:1 to 10:1-20:1. As a control, naked plasmid DNA barely expressed the reporter, which demonstrated that plasmid DNA without any carrier had very low transfection efficiency.

Figure 7. Images of 293 T cells transfected with naked pDNA, Im-PPZ-cyclen/DNA complex at Im-PPZ-cyclen/DNA weight ratio of 0 (A), 1:1 (B), 3:1 (C), 5:1 (D), 10:1 (E), 20:1 (F), respectively, and Lipofectamine 2000TM at the weight ratio of 5:1 (G) as observed under fluorescence microscope (20 × magnification).

DISCUSSION Although gene delivery has been extensively studied for the past decade, it is necessary to explore more new carriers or modify the existing ones to provide sufficient safety and transfection efficiency in clinical applications. Polyamine as an effective grafter, of which the positive charge on amino groups can facilitate charge interactions with the phosphate backbone


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 31

of DNA, was usually introduced to polymers to generate effective gene delivery carriers. Those grafted polyamine usually has linear or branched structures, while there are a few reports of investigation of cyclic polyamine grafted on polymers evaluated for gene delivery. In fact, on the basis of the structure of cyclic polyamine with relatively concentrated amino groups, valid positive charges were restricted within heterocyclic ring, which may have the potential to reduce cytotoxicity. In addition, lively P-Cl bonds of PDCP makes it easily grafted by some functional moieties to obtain final product. Hence, we introduced cyclic polyamine to PPZ skeleton and evaluate their physicochemical properties and biological performance in the present study. We investigated the influence of the amount of amino in different cyclic polyamine on DNA binding ability which is confirmed by gel retardation assay. As we expected, DNA binding abilities increase with the increasing the amounts of amine. The Im-PPZ-cyclen, with grafted cyclen which has a teraamine structure, is effective for DNA condensation, retarding DNA at polymer/pDNA weight ratio of 10:1 (Figure 1C, Lane 7). In contrast, Im-PPZ-pip and Im-PPZtacn which respectively have pip with diamine structure and tacn with triamine structure, were unable to induce DNA condensation, indicating weak DNA binding ability (Figure 1A and 1B). It seems likely that DNA condensation ability noted using these polymers are mediated by the amounts of amine in cyclic polyamine. For Im-PPZ-cyclen, the cyclen with concentrated amino groups can be effectively protonated and interact with the negatively phosphate backbone of DNA, thus leading to DNA condensation. The compositions and structures of Im-PPZ-cyclen and the previously reported degradable analogue poly(di-DMAEA)phosphazene21 are compared. Both Im-PPZ-cyclen and poly(diDMAEA)phosphazene had the ability to retard pDNA in gel loading well at proper polymer and DNA ratio, respectively. Cytotoxicity of Im-PPZ-cyclen for

A 375 and

HeLa cells are


16

Page 17 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


temperate, when it is 40 µg/mL there are still 50% and 73% cell viabilities It is well known that the cytotoxicity of cationic polymers arises from the introduction of amino/imino groups which can interact with the plasma membrane or negatively charged cell components and proteins.38,39 And reduced cytotoxicity is achieved by delocalizing the positive charge into a heterocyclic ring.43 Hence, reduced cytotoxicity was obtained for Im-PPZ-cyclen due to the introduction of cyclen which can be protonated on a heterocyclic ring compared with poly(diDMAEA)phosphazene with the side group DMAEA which has a classical tertiary amine structure with a permanent positive charge under the tested conditions. Meanwhile, in our previous study, reduced cytotoxicity of polymers grafted with cyclen was observed relative to the polymers grafted with linear amine.26 Hence, it can be concluded that cyclic polyamine may have the potential to lower cytotoxicity due to its centralized structure, which can contribute significantly to gene delivery. The DNA binding ability and biological activity of complexes was investigated in detail. It is demonstrated that the migration of pDNA was completely retarded at the weight ratio of 5:1 by cyclen-PPZ-cyclen (Figure 1E, Lane 7) while the completely retardation of pDNA was achieved at the weight ratio of 10:1 by Im-PPZ-cyclen (Figure 1C, Lane 7), indicating that cyclen-PPZcyclen has higher DNA binding ability than Im-PPZ-cyclen. In addition, the higher binding constant Ksv values of cyclen-PPZ-cyclen in fluorescence quench experiment (Figure 2B) quantitatively demonstrate its higher DNA binding ability compared with that of Im-PPZ-cyclen. It is reported that the cationic polymer/DNA complexes result from a cooperative system between the positive charge of amino groups of cationic polymers and the polyanionic phosphate backbone of nucleic acids via electrostatic interaction. Hence, the higher DNA binding ability for cyclen-PPZ-cyclen results form more grafted cyclen which capture more amounts of positive


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 31

charge compared with Im-PPZ-cyclen, resulting in effective DNA condensation. However, it should be noted that cyclen-PPZ-cyclen has low cell viability compared with Im-PPZ-cyclen. This could be attributed to the introduction of imidazole moieties which was supposed to have weak charge that resulted in weak ability of interaction with DNA for Im-PPZ-cyclen. Meanwhile, it is well known that the imidazole possess a buffering capacity in the endolysosomal pH range, and mediate vesicular escape by a “proton sponge” mechanism.23-25 Hence, it is considered important to maintain the subtle balance between DNA binding ability and biological activity, by substituting the amine moieties.

CONCLUSIONS In this study, we synthesized a family of water-soluble cationic PPZ grafted with cyclic polyamine and imidazole moieties, which could act as promising gene delivery vectors with suitable physicochemical properties and DNA binding activities. The results demonstrated that the efficiency of DNA binding was dominated by the amounts of amino groups in cyclic polyamine, and abundant nitrogen atoms on cyclic polyamine are needed to capture the positive charge to facilitate charge interactions with the phosphate backbone of DNA to induce DNA condensation for gene delivery. Moreover, Im-PPZ-cyclen can effectively protect pDNA from nuclease as well as wrap pDNA into nanoparticles with appropriate size and net cationic charge. In particular, the introduction of cyclen with centralized structures does not result in an increase in cytotoxicity compared with the PPZ grafted with classical tertiary amine. As a result, Im-PPZcyclen could be a promising cationic polymer for efficient gene delivery. Detailed investigations of PPZ grafted with cyclic polyamine and imidazole into the potential for gene delivery are still ongoing; the present work should be of value for design of PPZ-based non-viral vectors.


18

Page 19 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EXPERIMENTAL SECTION Materials and Methods. NH4Cl was dried in a desiccator over P2O5. PCl5 was purified by sublimation. Tetrahydrofuran (THF) was treated with KOH and distilled twice from Na in the presence of benzophenone. Branched polyethylenimine (PEI, 25 kDa) and 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyltetrazo-liumbromide (MTT) were purchased from Sigma-Aldrich (St Louis, MO, USA). Plasmid DNA (pUC 18) (pDNA) and Dnase I were obtained from Takara Biotechnology (Dalian, China). Calf thymus DNA (CT-DNA) was purchased from Huamei Biotechnology Company. Enhanced green fluorescence protein encoding plasmid DNA (pEGFPN3) was purchased from Azanno Biotech Co., Ltd. (Beijing, China). Cells were maintained at 37°C in a humidified and 5% CO2 incubator. All other chemicals and reagents were obtained commercially and used without further purification.

1

H,

13

C and

31

P NMR spectra of

poly(organo)phosphazenes were obtained from Bruker AV-600 NMR spectrometer. The chemical shifts were given relative to tetramethsilane (TMS) or 85% H3PO4 as an external standard. The molecular weight and distribution were determined by gel permeation chromatograph (GPC, Waters 515-2410) with THF as the eluent with flow rate (1 mL/min). The calibrations were Cytochrome C standards (Mw 12327). Preparation of Polymer/pDNA complexes. Polymer/pDNA complexes were prepared as follows: stock solutions of each polymer (Im-PPZ-pip, Im-PPZ-tacn, Im-PPZ-cyclen, cyclenPPZ-cyclen and Im-PPZ-Im, respectively) were prepared by dissolving 1 mg/mL polymer in purified water and stored at -20°C until further use. The stock aqueous solution of each polymer and pDNA were further diluted with purified water to the suitable concentration. An appropriate


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 31

amount of pDNA was added to polymer solution. After the addition of pDNA, solution was immediately mixed for 10s by vortex and incubated for 30 min at R.T. Agarose Gel Retardation Assay. The complex formation between polymers (Im-PPZ-pip, Im-PPZ-tacn, Im-PPZ-cyclen, cyclen-PPZ-cyclen, Im-PPZ-Im, respectively) and pDNA were evaluated by agarose gel electrophoresis experiments. To prepared complexes (10 µL total volume) at different weight ratios, 15 ng of pDNA was mixed with various concentrations of polymer solution and incubated at 37°C. After 12 h incubation, these complexes were loaded on a 1.0% (w/v) agarose gel stained with ethidium bromide (EtBr, 1.0 µg/mL) in TAE buffer solution. Electrophoresis was carried out at 85 V for 45 min in Bio-Rad Electrophoresis system (Bio-Rad, USA), and bands were visualized by UV light and photographed, recorded on an Olympus Grab-IT 2.0 Annotating Image Computer System (Olympus Corporation, Japan). Resistance to DNase I Digestion. The stability of polymer/pDNA complex against nuclease degradation was monitored by DNase I protection assay.28 Polymer/pDNA complex at the weight ratio of 5:1, containing 15 ng of pDNA, was prepared at 37°C for 2 h, and polymer represents Im-PPZ-cyclen and cyclen-PPZ-cyclen, respectively. Then the complex was further incubated at 37°C for 10 units of DNase I. Naked pDNA with 10 units of DNase I served as a positive control. The DNase I reaction was terminated by adding 5 µL of 1m EDTA solution. Finally, the solution was subjected to electrophoresis onto 1.0% (w/v) agarose gel. Fluorescence Quenching Assay. The interaction of polymers to DNA was studied using fluorescence quenching experiments.13 Fluorescence spectra were recorded at room temperature on a Hitachi model F-4500 spectrofluorimeter and corrected for the system response. A 20 mg/mL solution of polymers (Im-PPZ-Im, Im-PPZ-cyclen, and cyclen-PPZ-cyclen, respectively)


20

Page 21 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


was titrated into the DNA-EB solution (60 µg/mL calf thymus DNA solution including 6.2 µg/mL EB) at pH 7.4 10 mM Tris−HCl buffer with 10 mM NaCl. All the samples were excited at 520 nm and the emission was measured at 620 nm. If F0 is the fluorescence intensity (FI) of unintercalated, Fmax is the FI of fully intercalated EB, and Fx is the FI for a given concentration of polymers, then %FI was calculated as %FI = (Fx-F0)/(Fmax-F0). CD Experiments. Circular dichroism (CD) experiments were performed under a continuous flow of N2 (g) using a Jasco-810 spectropolarimeter. A path length cell of 1 cm was used, and all experiments were performed at room temperature. A 20 mg/mL solution of polymer was titrated into the 70 µg/mL DNA solutions (at pH 7.4 10 mM Tris-HCl buffer with 10 mM NaCl), and the polymer represents Im-PPZ-Im, Im-PPZ-cyclen and cyclen-PPZ-cyclen, respectively. The standard scan parameters for all experiments used a wavelength range from 400 to 220 nm. Sensitivity was set at 100 mdeg and a scan speed of 200 nm/min. Three scans were made and computer averaged. Cytotoxicity Assay by MTT. Cytotoxicity of polymer/DNA and branched 25 kDa PEI/DNA complexes were evaluated by MTT assay, and the polymer represents Im-PPZ-Im, Im-PPZcyclen and cyclen-PPZ-cyclen, respectively. A375 and HeLa cells (1 × 104 cells/well) were respectively seeded in 96-well plates for 24 h. Then, different concentrations of polymers were added to the cells with subsequently incubated for 24 h. After that, the medium in each well was replaced with 10 µL of fresh complete medium prior to the addition of 20 µL/well of MTT solution in PBS (5 mg/mL). Cells were incubated for 4 h at 37°C in 5% CO2. Then 150 µL of DMSO was added to dissolve the crystals formed by living cells, and plates were incubated for 30 min at 37°C. The optical intensity was measured at 490 nm using a microplate reader (Beckman DTX 880). The relative cell viability was calculated as cell viability (%) =


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 31

(ODtest/ODcontrol) × 100, where ODcontrol was obtained in the absence of polymers and ODtest was obtained in the presence of polymers or different concentrations. Each value was averaged from three independent experiments. Particle Size and Zeta-Potential Measurements. The polymer/DNA complex was prepared at varying weight ratios from 1 to 10 by adding a solution of polymer of Im-PPZ-cyclen (2 µL, varying concentrations) to a solution of pDNA (8 µL, 15 ng·µL-1), followed by vortexing for 5 s and incubating at 37°C for 30 min. The surface charge and the size of complexes were measured at 25°C with a Zetasizer Nano ZS instrument (Malvern) equipped with a standard capillary electrophoresis cell and dynamic light scattering (DLS, 10 mW He−Ne laser, 633 nm wavelength), respectively. The measurements were performed in triplicate AFM Assay. Atomic force microscope (AFM) imaging was performed in tapping mode on a Digital Instruments multimode NanoScope III having a maximal lateral range of approximately 5 µm. All images were analyzed by tapping in air. High-quality mica sheets (FluorMica) were cut with scissors into squares (1 cm × 1 cm) and attached with superglue to 15 mm round stainless steel sample disks (Ted Pella). Before each use, the mica was freshly cleaved by pulling off the top sheets with tape and then covered with 10 µL of autoclaved AFM buffer (pH 7.4 10 mM Tris-HCl, 1 mM EDTA, 5 mM MgCl2). The surface was precoated with Mg2+ to allow negatively charged DNA to bind. After 5 min, the buffer was rinsed thoroughly with 0.5 mL of distilled water, and the mica was briefly dried under a stream of N2 (g). The DNA sample was diluted with AFM buffer to 2.5 ng/µL, and then 10 µL of diluted sample was dropwise added to the mica surface. After 5 min, the buffer was rinsed thoroughly with 0.5


22

Page 23 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mL distilled water, and the mica was briefly dried under a stream of N2 (g). Each sample was scanned independently three times, and three different areas were chosen in each scan. In Vitro Gene Transfection Experiment. The efficacy of polymers as a non-viral gene delivery vector was evaluated by in vitro gene transfection assay. The 293 T cells were seeded in 24-well plate at a density of 1 × 105 cells/well in 1mL of DMEM medium containing 10% PBS and incubated for 24 h at 37°C under a 5% CO2 atmosphere prior to transfection. When the cells were at 80-90% confluence, the culture medium was replaced with 200 µL of serum-free medium containing 500 µL of complexes (different weight ratios of polymer(Im-PPZcyclen)/DNA complexes). pEGFP-N3 was used as the reporter plasmid to assay the transfection efficiency. After incubation for 6 h at 37°C, the cells received 1 mL of complete medium and incubated sequentially until 48 h post transfection. To assay the expression of polymers, we removed the medium and gently rinsed the cells twice with PBS and collected. The microscopy images were obtained at the magnification of 100 × and recorded using Viewfinder Lite (1.0) software. Control transfection was performed using a commercially available transfection reagent Lipofectamine 2000TM based on the standard conditions specified by the manufactures.

ASSOCIATED CONTENT Supporting Information The synthesis route of Im-PPZ-pip, Im-PPZ-tacn, Im-PPZ-cyclen, cyclen-PPZ-cyclen and ImPPZ-Im; 31P NMR and FT-IR spectra of PDCP; gel permeation chromatography (GPC) and the GPC results of PDCP; 1H, 31P and 13C NMR of new compounds; the protection of pDNA against Dnase I; the transfection efficiency of Im-PPZ-cyclen/pDNA in 293 T cells. This material is available free of charge via the Internet at http://pubs.acs.org.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 31

AUTHOR INFORMATION Corresponding Author *Corresponding author. Tel: +86 10 64413899. E-mail: [email protected] E-mail: [email protected] Author Contributions Chunying Ma and Xiao Zhang performed experiments and calculations. Changguo Du, Baojing Zhao and Chunhua He analyzed data. Chao Li wrote the paper. Renzhong Qiao contributed reagents and analytical tools. §These authors contributed equally. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Support of this research by the National Nature Science Foundation of China is gratefully acknowledged (Nos. 21372024, 21172016, 21572018 and 21232005). Supported by “the Fundamental Research Funds for the Central Universities (No. YS1407).

REFERENCES (1) Nam, J., Nah, J. (2016) Target gene delivery from targeting ligand conjugated chitosan–PEI copolymer for cancer therapy. Carbohydr. Polym. 135,153-161.


24

Page 25 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(2) Opalinska, J. B., and Gewirtz, A. M. (2002) Nucleic-acid therapeutics: basic principles and recent applications. Nat. Rev. Drug Discovery 1, 503-514. (3) Al-Dosari, M. S., and Gao, X. (2009) 3-Nonviral Gene Delivery: Principle, Limitations, and Recent Progress. AAPS J. 11, 671-681. (4) Kim, T., Jang, H., Jere, D., Park, I., Cho, M., Nah, J., Choi, Y., Akaike, T., and Cho, C. (2007) 4-Chemical modification of chitosan as a gene carrier in vitro and in vivo. Prog. Polym. Sci. 32, 726-753. (5) Huang, S., Kamihira , M. (2013) Development of hybrid viral vectors for gene therapy. Biotechnol. Adv. 31, 208-223. (6) Thomas, B., Lentz, S. J., Gray, R., Jude S. (2012) Viral vectors for gene delivery to the central nervous system. Neurobiol. Dis. 48, 179-188. (7) Behr, J. P. (1994) 7-Gene Transfer with Synthetic Cationic Amphiphiles: Prospects for Gene Therapy. Bioconjugate Chem. 5, 382-389. (8) Russ, V., Günther, M., Halama, A., Ogris, M., and Wagner, E. (2008) 8-Oligoethyleniminegrafted polypropylenimine dendrimers as degradable and biocompatible synthetic vectors for gene delivery. J. Controlled Release 132, 131-140. (9) Liu, C., Liu, F., Feng L., Li M., Zhang J., and Zhang N. (2013) The targeted co-delivery of DNA and doxorubicin to tumor cells via multifunctional PEI-PEG based nanoparticles. Biomaterials 34, 2547-2564.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 31

(10) Cavalli R, Bisazza A, Lembo D. (2013) Micro - and nanobubbles: A versatile non-viral platform for gene delivery. Int. J. Pharm. 456, 437-445. (11) Nie, Y., Günther, M., Gu, Z., and Wagner, E. (2011) Pyridylhydrazone-based PEGylation for pH-reversible lipopolyplex shielding. Biomaterials 32, 858-869. (12) Gusachenko, S. O., Kravchuk, Y., Konevets, D., Silnikov, V., Vlassov, V. V., and Zenkova, M. A. (2009) Transfection efficiency of 25-kDa PEI-cholesterol conjugates with different levels of modification. J. Biomater. Sci., Polym. Ed. 20, 1091-1110. (13) Dehshahri, A., Oskuee, R. K., Shier, W. T., Hatefi, A., and Ramezani, M. (2009) 15-Gene transfer efficiency of high primary amine content, hydrophobic, alkyl-oligoamine derivatives of polyethylenimine. Biomaterials 30, 4187-4194. (14) Wakebayashi, D., Nishiyama, N., Yamasaki, Y., Itaka, K., Kanayama, N., Harada, A., Nagasaki, Y., and Kataoka, K. (2004) 16-Lactose-conjugated polyion complex micelles incorporating plasmid DNA as a targetable gene vector system: their preparation and gene transfecting efficiency against cultured HepG2 cells. J. Controlled Release 95, 653-664. (15) Mintzer, M. A., and Simanek, E. E. (2009) Nonviral Vectors for Gene Delivery. Chem. Rev. 109, 259-302. (16) Allcock, H. R., and Fuller, T. J. (1980) 18-Phosphazene High Polymers with Steroidal Side Groups. Macromolecules 13, 1338-1345. (17) Allcock, H. R., Fuller, T. J., Mack, D. P., Matsumura, K., and Smeltz, K. M. (1977) 19Synthesis of Poly[(amino acid alkyl ester)phosphazenes]. Macromolecules 10, 824-830.


26

Page 27 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(18) Allcock, H. R., Fuller, T. J., and Matsumura, K. (1982) 20-Hydrolysis pathways for aminophosphazenes. Inorg. Chem. 21, 515-521. (19) Crommen, J. H. L., Schacht, E. H., and Mense, E. H. G. (1992) 21-Biodegradable polymers I. Synthesis of hydrolysis-sensitive poly[(organo)phosphazenes]. Biomaterials 13, 511-520. (20) Crommen, J. H. L., Schacht, E. H., and Mense, E. H. G. (1992) 22-Biodegradable polymers: II. Degradation characteristics of hydrolysis-sensitive poly[(organo)phosphazenes]. Biomaterials 13, 601-611. (21) Luten, J., van Steenis, J. H., van Someren, R., Kemmink, J., Schuurmans-Nieuwenbroek, N. M. E., Koning, G. A., Crommelin, D. J. A., van Nostrum, C. F., and Hennink, W. E. (2003) 23Water-soluble biodegradable cationic polyphosphazenes for gene delivery. J. Controlled Release 89, 483-497. (22) Yang, Y., Xu, Z., Jiang, J., Gao, Y., Gu, W., Chen, L., Tang, X., and Li, Y. (2008) 24Poly(imidazole/DMAEA)phosphazene/DNA self-assembled nanoparticles for gene delivery: Synthesis and in vitro transfection. J. Controlled Release 127, 273-279. (23) Roufai, M. B., and Midoux, P. (2001) Histidylated Polylysine as DNA Vector: Elevation of the Imidazole Protonation and Reduced Cellular Uptake without Change in the Polyfection Efficiency of Serum Stabilized Negative Polyplexes. Bioconjugate chem. 12, 92-99. (24) Mishra, S., Heidel, J. D., Webster, P., and Davis, M. E. (2004) Imidazole groups on a linear, cyclodextrin-containing polycation produce enhanced gene delivery via multiple processes. J. Controlled Release 116, 179-191.


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 31

(25) Merdan, T., Kopec̆ek, J., and Kissel, T. (2002) Prospects for cationic polymers in gene and oligonucleotide therapy against cancer. Adv. Drug Deliv. Rev. 54, 715-758. (26) Li, C., Tian, H., Rong, N., Liu, K., Liu, F., Zhu, Y., Qiao, R., and Jiang, Y. (2011) 30Chitosan Grafted with Macrocyclic Polyamines on C-2 and C-6 Positions as Nonviral Gene Vectors: Preparation, Characterization, and In Vitro Transfection Studies. Biomacromolecules 12, 298-305. (27) Li, L., Zhao, F. F., Zhao, B. J., Zhang, J., Li, C., and Qiao, R. Z. (2015) Chitosan Grafted with Phosphorylcholine and Macrocyclic Polyamine as an Effective Gene Delivery Vector: Preparation,

Characterization

and

In

Vitro

Transfection

Macromol.

Biosci.

DOI:

10.1002/mabi.201400518. (28) Wang, B., He, C., Tang, C., and Yin, C. (2011) 32-Effects of hydrophobic and hydrophilic modifications on gene delivery of amphiphilic chitosan based nanocarriers. Biomaterials 32, 4630-4638. (29) Carriedo, G. A., García Alonso, F. J., Gómez-Elipe, P., Fidalgo, J. I., García Álvarez, J. L., and Presa-Soto, A. (2003) 33-A Simplified and Convenient Laboratory-Scale Preparation of 14N or 15N High Molecular Weight Poly(dichlorophosphazene) Directly from PCl5. Chem. Eur. J. 9, 3833-3836. (30) Katayose, S., and Kataoka, K. (1998) 34-Remarkable increase in nuclease resistance of plasmid DNA through supramolecular assembly with poly(ethylene glycol)—poly(L-lysine) block copolymer. J. Pharm. Sci. 87, 160−163.


28

Page 29 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(31) Rodríguez-Pulido, A., Ortega, F., Llorca, O., Aicart, E., and Junquera, E. (2008) 35-A Physicochemical Characterization of the Interaction between DC-Chol/DOPE Cationic Liposomes and DNA. J. Phys. Chem. B 112, 12555-12565. (32) Sato, Y.-i., Kobayashi, Y., Kamiya, T., Watanabe, H., Akaike, T., Yoshikawa, K., and Maruyama, A. (2005) 36-The effect of backbone structure on polycation comb-type copolymer/DNA interactions and the molecular assembly of DNA. Biomaterials 26, 703-711. (33) Lepecq, J. B., and Paoletti, C. (1967) 37-A fluorescent complex between ethidium bromide and nucleic acids: Physical—Chemical characterization. J. Mol. Biol. 27, 87-106. (34) Baguley, B. C., and Le Bret, M. (1984) 38-Quenching of DNA-ethidium fluorescence by amsacrine and other antitumor agents: a possible electron-transfer effect. Biochemistry 23, 937943. (35) Shao, Y., Sheng, X., Li, Y., Jia, Z. L., Zhang, J. J., Liu, F., and Lu, G. Y. (2008) 41-DNA Binding and Cleaving Activity of the New Cleft Molecule N,N′-Bis(guanidinoethyl)-2,6pyridinedicarboxamide in the Absence or in the Presence of Copper(II). Bioconjugate Chem. 19, 1840-1848. (36) Ranjbar, B., and Gill, P., (2009) Circular dichroism techniques: biomolecular and nanostructural analyses- a review. Chem. Biol. Drug Des. 74, 101-120. (37) Mosmann, T. (1983) 43-Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63.


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 31

(38) Choksakulnimitr, S., Masuda, S., Tokuda, H., Takakura, Y., and Hashida, M. (1995) 44-In vitro cytotoxicity of macromolecules in different cell culture systems. J. Controlled Release 34, 233-241. (39) Fischer, D., Li, Y., Ahlemeyer, B., Krieglstein, J., and Kissel, T. (2003) 45-In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials 24, 1121-1131. (40) Zauner, W., Ogris, M., and Wagner, E. (1998) 46-Polylysine-based transfection systems utilizing receptor-mediated delivery. Adv. Drug Delivery Rev. 30, 97-113. (41) Jun, Y. J., Kim, J. H., Choi, S. J., Lee, H. J., Jun, M. J., and Sohn, Y. S. (2007) 47-A tetra(llysine)-grafted poly(organophosphazene) for gene delivery. Bioorg. Med. Chem. Lett. 17, 29752978. (42) de Wolf, H. K., Luten, J., Snel, C. J., Oussoren, C., Hennink, W. E., and Storm, G. (2005) 48-In vivo tumor transfection mediated by polyplexes based on biodegradable poly(DMAEA)phosphazene. J. Controlled Release 109, 275-287. (43) Lv, H., Zhang, S., Wang, B., Cui, S., and Yan, J. (2006) Toxicity of cationic lipids and cationic polymers in gene delivery. J. Controlled Release 114, 100-109.


30

Page 31 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Insert for Table of Contents use only Water-soluble Cationic Polyphosphazenes Grafted with Cyclic Polyamine and Imidazole as An Effective Gene Delivery Vector

Chunying Ma†,§, Xiao Zhang†,§, Changguo Du†, Baojing Zhao†, Chunhua He†, Chao Li*,† and Renzhong Qiao*,†,‡


31

Water-Soluble Cationic Polyphosphazenes Grafted with Cyclic

Recommend Documents