Elsevier

Biomaterials

Volume 33, Issue 32, November 2012, Pages 8111-8121
Biomaterials

Serum tolerance and endosomal escape capacity of histidine-modified pDNA-loaded complexes based on polyamidoamine dendrimer derivatives

https://doi.org/10.1016/j.biomaterials.2012.07.032Get rights and content

Abstract

Aiming to aid polyamidoamine (PAMAM, generation 4, PG4) to overcome gene delivery barriers like extrinsic serum inhibition, intrinsic cytotoxicity and lysosome digestion, histidine motifs modified PAMAM was prepared. The histidine activated PAMAM generation 4 (HPG4) was synthesized via aminolysis reaction and characterized by 1H NMR spectrum and MALDI-TOF-MS. Cytotoxicity profiles of HPG4 on MD-MB-231 cells were significantly improved in the form of polymer and polymer/DNA complexes comparing to PG4. The luciferase protein expression level of HPG4 was 20-, 2.7- and 1.2- fold higher than that of PG4, SuperFect and PEI 25k. Most importantly, flow cytometry and gene transfection studies showed that histidine motifs of HPG4 not only acted as enhancer for faster cellular uptake, but also played an important role on enhancing serum tolerance of the system on cellular uptake and transfection. Among the serum concentrations of 10%–50%, HPG4 showed 10–100 folds higher transfection efficiency than PG4. Intracellular fate observation conducted by confocal microscope provided visual and quantitative evidence that endsomal escape efficiency of HPG4 system was higher than that of PG4. Lastly, the endosomal escape mechanism of HPG4 system was analyzed by endosome destabilization and proton pump inhibition treatment. Collectively, compared to PG4/pDNA, HPG4/pDNA showed improvement on cellular uptake, serum tolerance, cytotoxicity profile, and endosomal escape.

Introduction

Polyamidoamine (PAMAM) dendrimer is one kind of cationic polymeric gene delivery vector, which was studied intensively in the last decades. Researchers showed that dendrimers, especially PAMAM dendrimers, allowed efficient transferring in many different cell types and cell lines. PAMAM based dendrimers have been proved as efficient gene delivery vectors, which could be substantiated by the fact that although the utilization of dendrimers as gene delivery vectors is only a few decades time, there are already commercial products available as gene transfection reagents. SuperFect® and PolyFect® are the two commercially available dendrimer deviratives, wherein SuperFect® has fractured dendrimer and PolyFect® has intact dendrimer as their component [1], [2].

PAMAMs possess bio-mimetic covalently fixed structures with interior void and well-defined surface functionality [2]. Primary amine groups on their surface could bind to the DNA and aid in compacting DNA into nanoscale complexes (named as dendriplexes) and eventually promote the cellular uptake of dendriplexes, while the tertiary amine groups, which are buried in the deep core of PAMAMs act as proton sponge and enhance endosomal escape of DNA. The sufficiency of primary amine groups on the surface makes it easy to be activated and modified by other functional motifs for functionality improvement. In comparison with other cationic polymers like polyethylenimine (PEI), PAMAM has its own superiority. That is its small size, the diameter of PAMAM generation 4 (G4) is only 45 Å [3], which leads it to be rapidly secreted into the urine to minimize exposure time-dependent carrier toxicity.

However, PAMAM dendrimer still has a long way to go before it enters clinical applications. The major limitations for its in vivo applications are including the followings: 1. Cytotoxicity of PAMAM dendrimer is relatively high [4]. 2. Transfections conducted in serum conditions would be compromised [5], indicating that PAMAM is not an ideal carrier for in vivo gene delivery. In the case of in vivo gene delivery, the negatively charged plasma proteins in the blood may neutralize the positively charged polymer/DNA complexes, causing aggregation of particles and decrease of cellular uptake mediated by charge interaction with cell membrane [6]. 3. PAMAM is still not efficient enough to escape from endosomes/lysosomes to release DNA in cytosol. Though PAMAM is regarded as proton sponge polymer, its proton sponge effect is correlated with cytotoxicity. The buffering capacity increased with an increase in molecular weight/generation, whereas the cytotoxicity appears to increase with increasing dendrimer generations [7].

Although PAMAM is not easy to circumvent these barriers, its ability to functionalize terminal groups and structures provides us endless possibilities to solve all the problems. Modification of PAMAM with histidine residues might be one of the solutions to assist PAMAM to overcome the barriers indicated above.

The imidazole group of histidine is a weak base with a pKa around 6 that has the ability to absorb proton when the pH of the environment drops below 6 [8]. The lone pair of electrons on the unsaturated nitrogen of imidazole ring provides pH dependent amphoteric properties. This phenomenon can induce membrane fusion or membrane permeation in an acidic medium. Moreover, the accumulation of histidine residues inside acidic vesicles can induce osmotic swelling and membrane disruption, and eventually vesicular escape, which lead to enhanced transfection.

The proof of concept has been demonstrated by several reported polymers and lipids modified with imidazole/histidine motifs. Histidylated polylysine (HpK) with 50% histidyl residues caused a dramatic increase by 3–5 orders of magnitude of transfection efficiency of polylysine/DNA complexes, which probably ascribed to the enhancement of endosomal escape capacity [9]. Davis et al. had developed a b-cyclodextrin based polycation (CDP) [10]. An imidazole modification (CDPim) has been applied on this polymer in order to introduce the pH-buffering elements to the system [11]. It was found that CDPim complexes were almost 10 times more effective than CDP complexes, and this type of imidazole group containing polymer has been continuously proved for successful pDNA and siRNA transfection [12].

In addition, it was reported that histdine/imidazole containing polymer-based or lipid-based carriers were resistant to digestion/disruption by serum and heparin, which promoted efficient transfection in the presence of serum [13]. Midous P. showed that peptides containing histidine functional residues were sufficient to transfect cells in vitro even in the presence of high amount of serum (up to 50%) [14].

The aim of this study was to facilitate PAMAM dendrimer to overcome the gene delivery barriers as above indicated by engineering a histidine activated PAMAM. In this study, we introduced histidine functional groups to the surface amino groups of PAMAM generation 4 (PG4) via aminolysis reaction. The degrees of substitution of histidine were measured by 1H NMR and MALDI-TOF-MS. The physicochemical properties of the dendrimer/DNA dendriplexes were evaluated by determination of the particle size, zeta potential and SEM, as well as DNA binding ability. MTT assay was employed to determine whether the cytotoxicity profile would be improved by histidine modification. The gene transfection processes were examined by luciferase assay, flow cytometry, and confocal laser scanning microscopy in order to prove the enhancement of gene transfection of histidine activated PAMAM was due to enhanced serum-tolerant ability, increased cellular uptake and improved endosomal escape capacity.

Section snippets

Materials

Starburst® PAMAM dendrimer G(4.0) surface: 100% NH2 was purchased from Dendritech®, Inc. (Midland, MI). l-histidine methyl ester dihydrochloride, 2-hydroxypyridine, chloroquine diphosphate salt, bafilomycin A1 from Streptomyces griseus and 4′ 6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma–Aldrich. LysoTracker Red-DND-99 was purchased from Invitrogen. Renilla luciferase assay system was purchased from Promega Co. (Madison, WI). The other materials and solvents were

1H NMR analysis

Histidine conjugation of PAMAM G4 (PG4) was performed by aminolysis method in anhydrous DMSO. The primary amine groups of PG4 were utilized to aminolyze the ester group of histidine methyl ester. Histidine-PAMAM G4 (HGP4) was characterized by 1H NMR and MALDI-TOF-MS.

The 1H NMR spectra were shown in Fig. 1. The signal assignments of pure PAMAM PG4 (A), l-histidine methyl ester dihydrochloride (B) and histidine-PAMAM HPG4 (C) were shown in detail. In the case of HPG4 (C), the protons at δ7.0 and

Conclusion

In this study, histidine activated generation 4 PAMAM (HPG4) synthesized via aminolysis was explored as a non-viral vector for gene delivery. We demonstrated that modification with histidine motifs improved the cytotoxicity profile and in vitro gene transfection of native PMAMA. Flow cytometry results showed that HPG4/pDNA complexes possessed higher cellular uptake kinetics than that of PG4/PDNA systems, which was probably due to more favorable interactions between histidine residues and

Acknowledgment

The authors greatly acknowledge the financial support from the National Nature Science Foundation of the People's Republic of China (30870618/C10020). The authors also greatly acknowledge the lab support of the Key lab on Assisted Circulation of Ministry of Health, China.

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