Bioreducible Fluorinated Peptide Dendrimers Capable of

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Bioreducible Fluorinated Peptide Dendrimers Capable of Circumventing Various Physiological Barriers for Highly Efficient and Safe Gene Delivery Xiaojun Cai, Rongrong Jin, Jiali Wang, Dong Yue, Qian Jiang, Yao Wu, and Zhongwei Gu* National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan 610064, P. R. China S Supporting Information *

ABSTRACT: Polymeric vectors have shown great promise in the development of safe and efficient gene delivery systems; however, only a few have been developed in clinical settings due to poor transport across multiple physiological barriers. To address this issue and promote clinical translocation of polymeric vectors, a new type of polymeric vector, bioreducible fluorinated peptide dendrimers (BFPDs), was designed and synthesized by reversible cross-linking of fluorinated low generation peptide dendrimers. Through masterly integration all of the features of reversible crosslinking, fluorination, and polyhedral oligomeric silsesquioxane (POSS) core-based peptide dendrimers, this novel vector exhibited lots of unique features, including (i) inactive surface to resist protein interactions; (ii) virus-mimicking surface topography to augment cellular uptake; (iii) fluorination-mediated efficient cellular uptake, endosome escape, cytoplasm trafficking, and nuclear entry, and (iv) disulfide-cleavage-mediated polyplex disassembly and DNA release that allows efficient DNA transcription. Noteworthy, all of these features are functionally important and can synergistically facilitate DNA transport from solution to the nucleus. As a consequences, BFPDs showed excellent gene transfection efficiency in several cell lines (∼95% in HEK293 cells) and superior biocompatibility compared with polyethylenimine (PEI). Meanwhile BFPDs provided excellent serum resistance in gene delivery. More importantly, BFPDs offer considerable in vivo gene transfection efficiency (in muscular tissues and in HepG2 tumor xenografts), which was approximately 77-fold higher than that of PEI in luciferase activity. These results suggest bioreducible fluorinated peptide dendrimers are a new class of highly efficient and safe gene delivery vectors and should be used in clinical settings. KEYWORDS: physiological barriers, reversible cross-linking, fluorination, peptide dendrimers, gene delivery

1. INTRODUCTION

multiple, optimal distinguished features which could collectively overcome these barriers.21−23 Peptide dendrimers, as a promising class of macromolecules, have drawn continuous interest in recent years because they do not share the general characteristics of typical dendrimers24−26 (such as a well-defined architecture, low polydispersity, high density of surface charges, and highly adaptable surface chemistry) but rather have certain unique properties, such as proteinlike molecular architecture, good biocompatibility, biodegradability, and resistance to proteolytic digestion.27−29 These superior properties make them well suited to various biomedical applications, especially since they are regarded as inherent and versatile nanocarriers for gene delivery.30−32 However, their commercial and clinical use is still far from being realized because of some practical problems, including the fact that it is more difficult to synthesize high-generation

Gene therapy is considered to be one of the most promising therapeutic strategies for the treatment of various chronic conditions, such as cancer and genetic disorders.1−5 The key challenge in successful gene therapy relies on the development of safe and efficient delivery vehicles and methods.6,7 While viral vectors can mediate highly efficient gene transfer (≥95%),8 they pose lots of issues such as genotoxicity, immunogenicity, limited loading capacity, and inablility to achieve large-scale production and long-term gene therapy.9 The challenges faced with viral vectors have inspired the development of nonviral vectors based on polymers,10−13 liposomes,14,15 and nanoparticles.16,17 These vectors, however, are less efficient than viral vectors, since they are incapable of efficiently overcoming the numerous extra- and intracellular barriers, including serum stability, cellular uptake, endosome escape, cytoplasm trafficking, nuclear localization, polyplex disassembly, and DNA release.18−21 Therefore, efforts need to be more focus on the design of a multifunctional polymeric vector that possesses © XXXX American Chemical Society

Received: November 27, 2015 Accepted: February 18, 2016

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DOI: 10.1021/acsami.5b11545 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Schematic illustration for preparation of BFPDs and biomedical applications of BFPDs polyplexes. (A) Chemical structure of POSS corebased G2 poly(L-lysine) dendrimers (OAS-G2-Lys). (B) Peripheral amino groups of OAS-G2-Lys partially fluorinated by heptafluorobutyric anhydride. (C) Reversible cross-linking of FG2-Lys using different amount of DSP. (D) BFPD complexation with DNA to form BFPD polyplexes. (E) BFPD polyplexes for intratumoral gene delivery. (F) BFPD polyplexes efficiently transport across the numerous intracellular barriers, including: (i) cellular internalization, (ii) endosomal escape, (iii) cytoplasmic trafficking to the nucleus, (iv) polyplex disassembly and DNA release in the perinuclear regions, and (v) nuclear entry of free DNA.

high-generation dendrimers are usually required to utilize the full effect of fluorination, and these are largely associated with synthetic challenges as mentioned earlier. The fluorinated dendrimers that have been subject to the greatest number of studies are the derivatives of poly(amidoamine) (PAMAM) and poly(propylenimine) (PPI). These are toxic and nondegradable, which may hinder their clinical application. In this way, fluorinated peptide dendrimers may have more clinical utility due to their superior biocompatibility and biodegradability. Inspired by these lessons, it is hypothesized that a rational chemical design may allow the development of a new generation of polymeric vectors via reversible cross-linking of fluorinated peptide dendrimers, and these dendrimers may have several advanced features that could exactly resolve the issues related to specific physiological barriers. To validate this, bioreducible fluorinated peptide dendrimers (BFPDs) were designed and synthesized by reversible crosslinking of fluorinated low-generation peptide dendrimers (Figure 1). This vector was built through the integration all of the features of polyhedral oligomeric silsesquioxane (POSS) core-based peptide dendrimers, fluorination, and reversible cross-linking. It may have lots of properties: (i) inactive surface to resist protein interactions; (ii) virus mimicking surface topography to enhance cellular uptake;40,41 (iii) fluorinationmediated efficient cellular uptake, endosome escape, cytoplasm trafficking, and nuclear localization to facilitate intracellular delivery of gene cargo; (iv) disulfide cleavage-mediated efficient polyplex disassembly and DNA release that allows DNA transcription; (v) ease of preparation, good biocompatibility, and biodegradability, which are of particular importance in its use in industrial, commercial, and clinical settings. To the best of our knowledge, this is the first report of a polymeric vector that is capable of addressing most of the specific physiological

peptide dendrimers than to synthesize other macromolecules; hence, the high cost of production largely hinders its industrialization and commercialization.33,34 Besides, highgeneration dendrimers have a relatively high transfection efficacy; however, they are usually accompanied by more severe cytotoxic effects on transfected cells than low-generation ones.35,36 More importantly, just like other nonviral vectors, the existing peptide dendrimers are still not efficient enough to overcome the numerous biological barriers. Therefore, there is an urgent need to develop a simple synthetic methodology that could addresses the challenges related to its synthesis and to resolve the paradox between transfection efficacy and cytotoxicity for peptide dendrimers-based gene delivery vectors; as well as to equip them with lots of advanced features to circumvent the numerous biological barriers. Reversible cross-linking of low-generation dendrimers has recently emerged as a versatile strategy for the preparation of efficient, biocompatible gene carriers. This is because of the ease of preparation, low cost, and the fact that its transfection efficiency is comparable to or better than that of classic highgeneration dendrimers.31,34 More importantly, the cross-linked dendrimers can be easily equipped with various functional ligands via simple surface functionalization of low-generation dendrimers, such as targeting moieties, lipids, etc. Among which, fluorination has attracted significant attention from researchers in chemistry and materials science because of its superior performance on addressing some of the major biological barriers of dendrimers,13,37,38 including limited serum resistance capability, poor cellular uptake efficacy and limited endosomal escape ability. It is noteworthy that the fluorinated dendrimers can achieve efficient gene transfection at extremely low N/P ratios, thus remarkably reducing the cytotoxicity of dendrimers on the transfected cells.39 However, B


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Figure 2. Biophysical properties of BFPD-11 polyplexes. (a) Gel retardation assay of BFPD-11 polyplexes with different BFPD-11/pDNA weight ratios (containing 0.2 μg pDNA/well). (b) Size distribution. (c) AFM images (left) top view; (right) size profile along with red lines and 3D view. (d) SEM images. (e) TEM images of BFPD-11 polyplexes.

group were first synthesized according to a previously reported method.27 Then their peripheral amino group were partially functionalized by heptafluorobutyric anhydride in methanol.39 Finally, disulfide bond containing linker [(3,3′-dithiodipropionic acid-di(N-succinimidyl ester (DSP)] was used to crosslink the fluorinated G2-Lys (FG2-Lys) to form BFPDs.33 The detailed synthesis and characterization of G2-Lys can be found in the Supporting Information (Figures S1−S4). Previous reports have demonstrated that the degree of fluorination plays a key role in the biological efficacy of fluorinated dendrimers, and the optimal transfection efficacy is usually achieved when the fluorination degree is around 50%. 37,38 However, considering that cross-linking reactions consume large numbers of amino groups, which in turn increases the degree of fluorination, hence, no more than half of the peripheral amino groups of each G2-Lys were fluorinated by heptafluorobutyric acids (which represent a ≤50% fluorination degree for G2-Lys)

barriers for nonviral gene transfection, which may be suitable for use in basic research experiments and clinical applications.

2. RESULTS AND DISCUSSION 2.1. Synthesis and Characterization of Bioreducible Fluorinated Peptide Dendrimers. To prepare BFPDs, POSS core-based poly(L-lysine) dendrimer served as model peptide dendrimer. They have all of the features of dendrimers and several advantages over typical dendrimers, including more terminal functional groups at low generations, well-defined globular molecular architecture that mimics the biological functions of globular proteins such as capsid proteins, minimal nonspecific interactions with tissues, and good biodegradability and biocompatibility.27,42 The synthesis route for BFPDs is shown in Figure 1A. Briefly, POSS core-based generation 2 poly(L-lysine) dendrimer (G2-Lys) with 32 peripheral amino C


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Figure 3. Transfection efficacies of BFPDs in (a) HEK293, (b) HepG2, and (c) HeLa cell lines. Effects of BFPD formulations (BFPD-12, BFPD-11, and BFPD-21) and BFPD/pDNA weight ratios (8, 10, 15, and 20) on the gene transfection efficiency of BFPDs were evaluated by luciferase assay (a1, b1, and c1). The luciferase activity is here expressed relative luciferase light units per milligram of protein (RLU per mg protein) (means ± SD, n = 5, *P < 0.05, **P < 0.01, BFPD-11 polyplexes vs BFPD-12 and BFPD-21 polyplexes at a weight ratio of 10). The optimal gene transfection efficiency (percentage of GFP-positive cells) of each BFPDs in (a2) HEK293, (b2) HepG2, and (c2) HeLa cell lines were determined by FACS (mean ± SD, n = 3, *P < 0.05, **P < 0.01, BFPD-11 polyplexes vs BFPD-12 and BFPD-21 polyplexes at a weight ratio of 10).

BFPD-11 polyplexes measured by atomic force microscopy (AFM) (Figure 2c) were consistent with the results of DLS and scanning electron microscopy (SEM) (Figure 2d). TEM images clearly show that BFPD-11 (Figure 2e), BFPD-12 (Figure S10a2), and BFPD 21 (Figure S10b2) polyplexes have a rough surface topography, similar to the envelope-spike structure of some viruses, such as HSV. This unique surface topography is a desirable characteristic for enhancing both binding toward genetic molecules and cellular uptake efficacy of polyplexes.40 It is well-known that the particle size, surface morphology, and zeta potential of polyplexes strongly influence cellular uptake/ intracellular trafficking, DNA release and subsequent transfection efficiency,12 therefore, the appropriate particle size and unique surface topography of BFPDs polyplexes may greatly enhance the gene delivery efficiency of BFPDs. In addition, it is worth noting that the polyplex size was almost unchanged after incubation in DMEM medium with 10% FBS for 24 h (Figure S11), indicating the high stability BFPDs polyplexes. The biological efficacy of BFPDs was assessed on HEK293, HepG2, and HeLa cell lines using pGL-3 and pEGFP as reporter genes. As shown in Figure 3, the gene transfection efficacy of BFPDs varied across different cell types. Greater transfection efficiency was observed in HEK293 cells. The transfection efficacy of BFPDs was found to be closely related

via controlling the feed ratio of heptafluorobutyric anhydride to G2-Lys. The average number of heptafluorobutyric acids modified on each G2-Lys is about 12, as determined by elemental analysis and GPC analysis (Figure S5), and the degree of fluorination of G2-Lys was 38%. Three molar ratios of DSP to FG2-Lys (2:1, 1:1, and 1:2) were utilized to optimize the synthesis of BFPDs. The products were designated as BFPD-21, BFPD-11, and BFPD-12, respectively. Assessment of its 1H NMR (Figure S6) and GPC spectra (Table S1) confirmed that its synthesis was indeed successful. 2.2. Biophysical and Biological Properties of BFPD Polyplexes. As shown by transmission electron microscopy (TEM), BFPDs presented a well-defined spherical shape with diameters of 25 ± 5 nm, which agreed with an average size of 29 nm by dynamic light scattering (DLS) measurement (Figure S7A). The zeta potential of BFPDS measured by DLS was ∼27 mV. BFPDs can form stable polyplexes with DNA with a weight ratio of >4 (Figure 2a and Figure S8), these polyplexes are positively charged (14 ± 4 mV) with a mean diameters of 20 μg·mL−1, whereas the cell viability of BFPD-11 was always maintained at >80% throughout the tested range of concentrations in both HEK239 (Figure S15b), HepG2 (Figure S15c), and HeLa (Figure S15d) cells. Collectively, these results clearly suggest that BFPD-11 is an efficient and safe vector suitable for the introduction of both reporter genes and functional genes into various commonly used cells. 2.3. Gene Transfection Mechanisms of BFPD Polyplexes. As demonstrated earlier, BFPD-11 showed a much higher transfection efficiency than nonfluorinated BPD-11 and PEI, thus indicating that fluorination and reversible crosslinking play essential roles in the transportation of polyplexes across various extra- and intracellular barriers. To further validation this finding, gene transfection mechanisms of BFPD11 polyplexes, including cellular uptake, endosomal escape, cytosolic trafficking, polyplex disassembly and release of DNA, and serum resistance were investigated in HEK293 and HepG2 cells, with PEI and BPD-11 serving as controls. Because the plasma membrane is the first barrier for the intracellular delivery of DNA polyplexes, the cellular uptake profiles of BFPD-11, PEI, and BPD-11 polyplexes were first investigated by confocal laser scanning microscopy (CLSM) and fluorescence-activated cell sorting (FACS) analysis. As shown in Figure 5a, after 2 h incubation, PEI and BPD-11 polyplexes with slight red fluorescence (Cy3 labeled pDNA) were observed in HepG2 cells. While more fluorescent dots were found to be attached to the cell surfaces after treatment E


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Figure 5. Cellular uptake and intracellular trafficking of PEI, BFPD-11, and BPD-11 polyplexes. (a) CLSM images of the cellular uptake of PEI, BFPD-11, and BPD-11 polyplexes in HepG2 cells at different points in time. The scale bars correspond to 25 μm. Cy3-positive cells in (b1) HEK293 and (c1) HepG2 cells at different points in time were determined by FACS. Mean fluorescence intensity (MFI, Cy3-labeled DNA) in (b2) HEK293 and (c2) HepG2 cells at different points in time were determined using FACS. Data are expressed as the mean ± SD, (n = 3), *P < 0.05, **P < 0.01, BFPD-11 polyplexes vs PEI and BPD-11 polyplexes at the same experimental points in time.

with BFPD-11 polyplexes. More obvious distinctions were observed after 4 h incubation, at this point PEI and BPD-11 polyplexes had attached to the cell surfaces, but most BFPD-11 polyplexes were not only on the cell surface but also within cells or perinuclear regions, which was indicative of their faster and more efficient cellular uptake. FACS analysis further confirmed the efficient cellular uptake of BFPD-11 polyplexes. For example, at 4 h, the cellular uptake efficiency (percentage of Cy3-positive cells) of BFPD-11 polyplexes in HEK293 (Figure 5b1) was 98.5%, which was slightly higher than that of PEI (87.8%) and much higher than that of BPD-11 (72.7%) in the corresponding cells. In addition, the mean fluorescence intensity (MFI) of Cy3-labeled pDNA in BFPD-11-polyplextreated cells (Figure 5b2 and c2) remained higher than that of PEI- and BPD-11-polyplex-treated cells. Taken together, these results clearly indicate the BFPD-11 polyplexes underwent faster and more extensive cellular uptake. Previous studies have shown that the particle size, surface charge, and topography of

DNA polyplexes play major roles in determining the efficiency and pathway of cellular uptake,43−45 and polyplexes of similar particle size, surface charge, and topography may have comparable cellular uptake efficiencies. Nevertheless, BFPD11 and BPD-11 polyplexes with similar particle size, surface charge, and topography (Figures S10 and S16) showed different cellular uptake efficiencies. The most likely reason for this phenomenon is its fluorination, which not only improves the affinity of BFPD-11 polyplexes to the cell membrane but also their ability to cross the lipid bilayer of the cell membrane.46 After successful cellular internalization, endosomal escape is the most important determinant of the intracellular fate of DNA polyplexes.47,48 Figure 6a shows that PEI polyplexes with inherent proton-sponge effect can successfully escape endosomes (stained by Lyso tracker green (Invitrogen, Carlsbad, CA, USA)) after 4 h of incubation with HepG2 cells, unlike BPD-11 polyplexes, which remained entrapped within endoF


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Figure 6. Endosome escape abilities of PEI, BFPD-11, and BPD-11 polyplexes. (a) CLSM images of HepG2 cells treated with PEI, BFPD-11, and BPD-11 polyplexes for 2 and 4 h, respectively. The scale bars correspond to 25 μm. HEK293 and HepG2 cells were pretreated with bafilomycin A1 or sucrose, followed by incubation with PEI, BPD-11, and BFPD-11 polyplexes for 48 h. The luciferase activity of PEI, BPD-11, and BFPD-11 polyplexes in (b) HEK293 and (c) HepG2 cells was determined by luciferase assay, and the data are expressed as the mean ± SD, (n = 5), *P < 0.05, **P < 0.01, compared to cells pretreated with bafilomycin A1 or sucrose.

Figure 7. Release of DNA from BFPD-11 and PEI polyplexes after incubation with different concentrations of heparin (0−1.2 μg·μL−1) (a1 and b1) or different concentrations of heparin supplemented with 5 mM GSH (a2 and b2) for 2 h. PEI and BFPD-11 polyplexes were prepared at weight ratios of 1.3/1 and 10/1, respectively, which are the optimized weight ratios in the gene transfection experiments.

markedly increased the endosomal escape ability of poly(Llysine) dendrimer polyplexes because most BFPD-11 polyplexes had successfully escaped from the endosomes and were then scattered across the perinuclear region within a 4 h

somes due to the limited ability of poly(L-lysine) dendrimer polyplexes to escape the endosome. This was because all amino groups of lysine had already been protonated under physiological conditions (i.e., pH = 7.4).49 Fluorination G


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Figure 8. Fluorescent microscopy images of HEK293 (a1) and HepG2 (b1) cells transfected with PEI (w/w = 1.3/1) or BFPD-11 polyplexes (w/w = 10/1) for 48 h. PEI and BFPD-11 was treated with 5 mM GSH or left untreated for 30 min prior to complexation with EGFP gene. The scale bars correspond to 100 μm. Luciferase expression and cytotoxicity after treatment with different amounts of BSO, an inhibitor of GSH synthetase, in HEK293 (a2) and HepG2 (b2) cells. The data are expressed as the mean ± SD, (n = 5), *P < 0.05, **P < 0.01, compared to cells without BSO treatment.

incubation period. The efficiency of the endosomal escape of BFPD-11 polyplexes was further confirmed by endosomal acidification inhibition or promotion testing.50 Figure 6b and c show that the addition of bafilomycin A1, an inhibitor of endosomal acidification, significantly decreased the transfection efficiency of PEI, BFPD-11, and BPD-11 polyplexes. However, supplementation with sucrose, a lysosomotropic agent that accelerates the escape of polyplexes from acidic vesicles, markedly increased the transfection efficiency of BPD-11 polyplexes but did not further improve the transfection efficacy of BFPD-11 and PEI polyplexes, suggesting that the endosomal escape ability of BFPD-11 polyplexes was strong enough for efficient endosomal escape due to the fluorination effect. Upon escape from endosomes and relocation to the cytoplasm, the DNA polyplexes need to be further transported into the nucleus, which is itself a limiting step in gene delivery.51,52 As shown in Figures 5a and 6a, after 4 h of incubation, a large number of BFPD-11 polyplexes were scattered across the perinuclear region but only a small number of PEI and BPD-11 polyplexes clustered within the perinuclear region. These results suggest that the diffusion coefficient of BFPD-11 polyplexes in the cytoplasm was much higher than that of PEI and BPD-11 polyplexes, which is essential to minimizing DNA degradation in the cytoplasm and enhancing nuclear import of DNA. For DNA transcription to occur, polyplexes are likely to dissociate for DNA release via counter polyion exchange reactions with charged biomacromolecules, such as protein and RNA species.15 Figure 7a1 shows that incubation with heparin (1.2 μg·μL−1) for 2 h allows a remarkable amount of DNA to be released from the BFPD-11 polyplexes (w/w = 10) via the counter polyion exchange reactions between BFPD-11 and heparin. DNA release profiles

were significantly enhanced after coincubation with 5 mM GSH for 2 h (Figure 7a2) due to the reductive cleavage of disulfide bond-induced polyplex and the release of DNA (Figure S17b). Taken together, these results clearly suggest that BFPD-11 polyplexes could be efficiently dissociated in the intracellular milieu because various types of negatively charged macromolecules, and high GSH levels (approximately 2−10 mM) were present in the intracellular milieu as essential cellular components, which favors successful gene transfer and expression. To investigate the role of reversible cross-linking in gene transfection, BFPD-11 was first incubated with 5 mM GSH before complexation with DNA, and EGFP expression was measured by fluorescence microscopy. As shown in Figure S15a, GSH-treated BFPD-11 restored its DNA binding capacity at the weight ratio of 10, but presented extremely low transfection efficacy in both HEK293 and HepG2 cells (Figure 8a1 and b1), suggesting that the disulfide bonds had been reduced, and reversible cross-linking played an important role in gene transfection. To further confirm whether the disulfide bond cleavage in BFPD-11 was relevant to high gene transfection efficiency, a transfection experiment was performed in the presence of DL-buthionine sulfoximine (BSO), a glutathione-depleting agent.53,54 Because depletion of GSH can lead to cell death, toxicity testing was first performed. As shown in Figure 8a2, BSO treatment was found to be toxic to HEK293 cells, exposure to 200 μM of BSO resulted in 72% cell viability. However, BSO was found to be much less toxic to HepG2 cells, with at least 87% of cells remaining viable, even at a BSO concentration as high as 200 μM (Figure 8b2). Noteworthy, BSO treatment did not affect luciferase expression of PEI polyplexes, whereas that of BFPD-11 polyplexes H


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Figure 9. Serum-resistance of BFPD-11 polyplexes on luciferase gene transfection. Luciferase expression was examined in HEK293 (a) and HepG2 (b) cells with PEI (w/w = 1.3/1), BFPD-11 (w/w = 10/1), and BPD-11 (w/w = 10/1) polyplexes in DMEM medium containing 0%, 10%, 30%, and 50% FBS, respectively. The data are expressed as the mean ± SD, (n = 5), *P < 0.05, **P < 0.01, compared to luciferase transfection in the presence of 10%, 30%, and 50% FBS.

markedly decreased in a dose-dependent manner, suggesting that the reduction of disulfide bonds in BFPD-11 was abruptly decreased with the inhibition of GSH. These results confirmed the importance of disulfide cleavages in BFPD-11 during gene transfection, and the regulation of the release of pDNA through controlled dendrimer degradation is critical to efficient gene delivery. Development of efficient and biocompatible gene delivery vehicles requires highly stable polyplexes that circulate in the blood.55,56 To investigate whether BFPD-11 polyplexes have serum resistance capacity, gene transfection were conducted in the presence of 10%, 30%, and 50% serum using PEI and BPD11 polyplexes as control. Figure 9 shows that the gene transfection efficiency of PEI polyplexes in HEK293 and HepG2 cells was significantly affected by serum; for example, these were about 18-, 72-, and 226-fold lower in HEK293 cells (Figure 9a) and 12-, 50-, and 460-fold lower in HepG2 cells (Figure 9b) in the presence of 10%, 30%, and 50% serum, respectively. The reduction in transfection efficiency of PEI polyplexes may be attributed to its instability in serum due to its high surface charges.57 The gene transfection efficiency of BFPD-11 polyplexes was slightly affected in the presence of 10% and 30% FBS, and only slightly decreased in the presence of 50% FBS. The superior serum resistance of BFPD-11 polyplexes can be ascribed to the fluorination, reversible crosslinking and unique features of POSS core, which respectively provides an inactive surface for BFPD-11 against serum proteins, a good stabilization effect against counter polyanion exchange,15,58 and the highly compact and symmetrical, which minimizes nonspecific interactions with serum proteins.32 2.4. In Vivo Gene Transfection via Intramuscular and Intratumoral Injection. Because of the superior performance shown by BFPDs in addressing the several sophisticated extraand intracellular barriers, it was here hypothesized that BFPDs may be utilized for in vivo gene delivery and cancer therapy. To validate this hypothesis, in vivo gene transfection of pCMV-βgal and pSC-Luc by BFPD-11 was conducted in BALB/c mice via intramuscular injection. As expected, more intense and darker blue colors were observed in the muscular tissue treated with BFPD-11 than in those treated with PEI and BPD-11 (Figure 10a). This suggested that β-galactosidase was upregulated by the presence of BFPD-11 polyplexes. The quantitative results of luciferase activity (Figure 10b) further confirmed that the in vivo gene transfection efficiency of BFPD-11 polyplexes was relatively high, which was about 1.6 × 106 RLU·mg−1 of protein, whereas those of PEI and BPD-11 were only 3.6 × 104 and 2.1 × 104 RLU mg−1 protein, respectively. The transfection efficacy of BFPD-11 polyplexes

Figure 10. Transfection efficacy of pCMV-β-gal (a) and pSC-Luc (b) in mouse muscles after intramuscular injection of PEI, BFPD-11 and BPD-11 polyplexes for 4 days. Each polyplexes (containing 10 μg pDNA) were prepared at their optimal weights ratios. The data are expressed as the mean ± SD, (n = 5), **P < 0.01, compared to transfection efficiency of BFPD-11 polyplexes.

was approximately 45- and 77-fold higher than that of PEI and BPD-11 polyplexes, respectively. To assess the preliminary applications of BFPD-11 in cancer therapy, the intratumoral efficiency of BFPD-11 in functional gene (p53) transfection was investigated. Before transfection, its enrichment and retention behavior were monitored in HepG2 tumor-bearing nude mice because those of DNA polyplexes in tumor sites play a crucial role in intratumoral gene transfection. As shown in Figure 11a, the fluorescence signal (Cy5.5-labeled BFPD-11) of BFPD-11 polyplexes was only visible in tumor tissues from 2 to 48 h, and it showed no significant reduction. However, the fluorescence signal of PEI polyplexes significantly decreased in a time-dependent manner, which can be attributed to the relative instability of PEI polyplexes in vivo, in where they may be aggregated rapidly and forms large aggregates, which largely hinders their tumor penetration and result in their continual elimination from the I


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Figure 11. (a) Fluorescence images of nude mice bearing HepG2 tumor xenografts at 2, 4, 8, 24, and 48 h after intratumoral injection of PEI or BFPD-11 polyplexes. (b) Ex vivo fluorescence intensity of organs 48 h after intratumoral injection. (c) Expression of p53 mRNA in HepG2 tumor xenografts transfected with PEI, BFPD-11, and BPD-11 polyplexes for 48 h (means ± SD, n = 3, *P < 0.05, **P < 0.01). (d) Western blotting analysis of p53 protein expression in HepG2 tumor xenografts transfected with BPD-11, PEI, and BFPD-11 polyplexes for 48 h. For mRNA and Western blot analysis, tumors treated with PBS were served as control.

tumor site by the lymphatic system. The ex vivo fluorescence images (Figure S18) and semiquantitative fluorescence intensity of organs (Figure 11b) also indicated that almost all of the BFPD-11 polyplexes were located in the tumor tissue, while PEI was rapidly excreted by the mice. These results clearly showed that BFPD-11 was enriched in the tumor site for an extended period of time, which is a highly desirable feature for long-term gene therapy. Furthermore, a much higher p53 mRNA expression level (Figure 11c) and p53 protein expression level (Figure 11d) were detected in the HepG2 tumor xenografts of the BFPD-11 group versus the PEI and BPD-11 group. For example, the p53 mRNA level in HepG2 tumor xenografts transfected by BFPD-11 were about 2.8-fold and 1.7-fold higher than those of transfected by BPD-11 and PEI, respectively. HE staining of TA muscles showed BFPD-11 polyplexes have excellent biocompatibility, and no pathological changes (such as inflammatory reaction or necrosis focus) were detected 24 h after treatment (Figure S19). However, the administration of PEI polyplexes clearly resulted in inflammatory cell infiltration and plasmolysis. These results collectively suggest that BFPDs are safe and efficient gene delivery vectors and they may be suitable for use as an alternative to viral vectors, and may be used in in vivo gene delivery, particularly in cases involving intramuscular and intratumoral routes.

overcome the complex physiological problems that can prevent gene delivery. These advanced features include (i) nanoglobular architecture of POSS core-based peptide dendrimers that create a virus-mimicking surface topography that facilitates cellular uptake, (ii) fluorination provides an inactive surface that resists protein interaction, (iii) fluorination-mediated cellular uptake, endosome escape, cytoplasm trafficking, and nuclear localization facilitate intracellular delivery of gene cargos, and (iv) disulfide cleavage-mediated polyplex disassembly and the release of DNA allow DNA transcription and vector degradation. This novel vector also prevents the need for time-consuming synthesis procedures and has excellent biocompatibility and biodegradability. These features render this novel vector highly efficient and biocompatible both in vitro and in vivo. It also shows superior efficacy and biocompatibility to the commercial transfection reagent, PEI. These results indicate that this novel vector is a promising alternative to viral vectors and may be suitable for use in basic research experiments and in clinical applications.

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ASSOCIATED CONTENT

S Supporting Information *

3. CONCLUSIONS In summary, a new type of polymeric vector was here shown to be capable of safe and highly efficient gene delivery via reversible cross-linking of fluorinated low-generation peptide dendrimers. Because of its unique POSS core-based peptide dendrimers, fluorination, and reversible cross-linking chemistry, this novel vector has several advanced features that can

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b11545. Additional characterization data of BFPD dendrimers and biophysical as well as biological properties of BFPD polyplexes (PDF) J


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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel./Fax: +86-028-85410336 (0653). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (NSFC, 51133004, 81361140343, and 51503131), Joint Sino-German Research Project (GZ756 and GZ905), the Department of Science and Technology from Sichuan Province (2013FZ0003), and the China Postdoctoral Fund (2014M552359 and 2015T80977).

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Bioreducible Fluorinated Peptide Dendrimers Capable of

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