Dual-functionalized calcium nanocomplexes for transfection of cancerous and stem cells: Low molecular weight polycation-mediated colloidal stability and ATP-mediated endosomal release

https://doi.org/10.1016/j.jiec.2018.03.028Get rights and content

Highlights

  • Calcium based gene delivery carriers having low molecular weight polycations improved colloidal stability.

  • ATP was introduced within the delivery systems to enhance an endosomal escape.

  • The Ca2+/ATP-pDNA/PC NCs were used for stem cell gene delivery.

Abstract

To overcome colloidal instability of calcium phosphate nanoparticles in gene delivery, colloidally stable and endosomolytic Ca2+-based pDNA nanocomplexes (NCs) were designed by a surface coating of biocompatible polycations (PCs; low molecular weight branched polyethyleneimine [bPEI], protamine sulfate and ε-polylysine) and the addition of natural and endosomolytic ATP, respectively. Without remarkable cytotoxicity and colloidal instability, Ca2+/ATP-pDNA/bPEI1.8 kDa NCs having [bPEI1.8 kDa] = 3.6 μg showed 5.8-fold and 4.4-fold higher transfection efficiencies than bPEI25 kDa/pDNA NCs in HepG2 cells and dental pulp stem cells, respectively. In conclusion, pH-sensitive endosomolytic ATP and Ca2+-based gene complexes could be potentials as effective and safe gene delivery vectors in various cells.

Introduction

Non-viral vectors (e.g., polymer, lipid, biometal or their combinations) for gene delivery carriers have attractive biological characteristics such as a non-immunogenicity, low toxicity and potent specific cell targetability [1], [2], [3], [4]. Among various non-viral delivery systems, calcium phosphate (CaP) nanoparticles (NPs), which are constructed by electrostatically interacting Ca2+ ion with PO43− ion, are one of the widely used gene delivery vehicles due to their excellent biocompatibility, biodegradability and gene-encapsulating capacity [5], [6], [7], [8], [9]. Moreover, the preparation processes of CaP NPs are straightforward with low-cost materials [10]. However, a serious drawback of these techniques is the difficulty in controlling the preparation conditions such as components’ concentrations, pH, temperature and technician’s skills [11], [12], [13], [14], resulting in poor reproducibility of CaP NPs in size and colloidal stability compared with other lipid- or polymer-based transfection NPs. Especially, the sizes of CaP NPs grow with the time, leading to a steep drop in transfection efficacy due to the unfavorable effect of the increased size on cellular internalization [15], [16], [17].

To achieve improved transfection efficiency of CaP NPs, the NPs without irregular particle growth have been prepared by applying various strategies (e.g., surface coating, optimizing of stoichiometry between calcium and phosphate ions and microemulsion). Among them, surface coating using cationic lipids, natural derived polysaccharides and high molecular weight cationic polymers has been used for inhibiting NP growth. At first, despite of limitations in colloidal stability, fabrication reproducibility and cytotoxicity, using cationic lipids (e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE], 1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC], 1,2-dioleoyl-3-trimethylammonium-propane [DOTAP], and so on) is an attractive method due to ease in scale-up and size control [18], [19], [20] and chemical modification [21], [22], [23], [24]. In general, chitosan as a natural cationic polysaccharide is widely used in biomedical fields due to its excellent biocompatibility, excellent biodegradability and minor immune response [25], [26], [27]. However, its low aqueous solubility in physiological pH has triggered to design chitosan derivatives. These modified chitosans such as 3,4-dihydroxy-l-phenylalanine (DOPA) modified chitosan, PEG grafted carboxymethyl chitosan and glutamine conjugated oligochitosan were also applied to coat the surface of CaP NPs. In the cases of polycations (PCs) such as branched polyethylenimine (bPEI) and poly(l-lysine), their intrinsic gene condensing capability and tailor-made biofunctions have made much interest in PCs as gene carriers [28], [29], [30], [31], [32], [33].

In this study, we designed PC-stabilized Ca2+ ion-gene nanocomplexes (NCs). In the NCs, the core components were negatively-charged pDNA and positively-charged Ca2+ ions. Additionally, PCs and adenosine triphosphate (ATP) were introduced for improving colloidal stability and endosomal escape of the NCs, respectively. Unlike typical components of CaP NPs, the designed NCs were prepared by electrostatic interaction between Ca2+ ions and the phosphate groups in pDNA, resulting in the formation of Ca2+/pDNA NCs and the method could improve pDNA content in the NCs. In addition, it has been already reported that calcium phosphate induces osteogenic differentiation of dental pulp stem cells (DPSCs) [34]. Therefore, calcium ion based gene delivery carriers could deliver the genes to stem cells for osteogenic differentiation.

Furthermore, in this study, to avoid unfavorable size growth of the NCs, three potent PC candidates such as low molecular weight (LMW) bPEI (bPEILMW), protamine sulfate (PS) and ε-polylysine (ε-PL) were used as surface coating materials. First, bPEI has primary, secondary and tertiary amines with a theoretical ratio of 1:2:1. Its primary amines condense genetic materials (e.g., pDNA and siRNA), resulting in the protection of genes from nucleases [35], whereas secondary and tertiary amines buffer decrease in pH, leading to osmotic shock-mediated endosomal destabilization [36]. These characteristics of bPEI allow its use as a golden transfection polymer. However, high molecular weight (HMW) bPEIs having molecular weights (MWs) of 25 kDa (bPEI25 kDa) and 750 kDa (bPEI750 kDa) were not suitable for clinical usage in gene delivery carriers due to their severe cytotoxicity. Thus, instead of HMW bPEIs (i.e., bPEI25 kDa and bPEI750 kDa), various bPEILMW derivatives having MWs of 0.8 kDa (bPEI0.8 kDa), 1.2 kDa (bPEI1.2 kDa) and 1.8 kDa (bPEI1.8 kDa) were selected due to the lower cytotoxicity of the latter than the former [35], [37]. Second, PS is a cationic and naturally occurring polypeptide with arginine-rich sequence. The characteristics endow both nuclear targeting and intranuclear transcription activities because of its arginine-rich sequence [38] as well as gene condensing ability [39]. Especially, PS is an FDA-approved PC adjuvant for a vaccine stabilizer and an antidote of heparin [40], allowing the use of PS in blood. Third, ε-PL is a linear homopolymer of l-lysine with ε-amine group-α-carboxyl group linkages, and its cationic property allowed to interact with genes. Especially, ε-PL was selected because it is an FDA-approved PC preservative for food [41], [42], allowing its use for oral administration. Selected PC candidates have lower cytotoxicities (e.g., bPEILMW, PS, ε-PL) and FDA-approval (e.g., PS, ε-PL) which represented safe materials.

In addition, endosomolytic ATP was incorporated into PC-stabilized Ca2+/gene NCs to facilitate endosomal escape and express improved transgene efficiencies because the secondary phosphate groups of ATP cause a proton-buffering effect in endosomal pH range [43], [44]. Conclusively, this study constructed Ca2+/pDNA/PC NCs and Ca2+/ATP-pDNA/PC NCs and their physicochemical and biological characteristics were investigated in terms of size, zeta-potential, gene condensation, transfection efficiency and cellular uptake.

Section snippets

Materials

bPEI1.2 kDa and bPEI1.8 kDa were received from Polysciences, Inc. (Warrington, PA, USA). bPEI0.8 kDa, bPEI25 kDa, PS (MW 5–10 kDa), ATP disodium salt hydrate, calcium chloride (CaCl2), Dulbecco’s Modified Eagle’s Medium (DMEM), sodium bicarbonate, d-glucose, Ca2+-free and Mg2+-free Dulbecco’s phosphate buffered saline (DPBS), 4-(2-hydroxy-ethyl)-1-piperazine (HEPES), fetal bovine serum (FBS), penicillin-streptomycin antibiotics, trypsin-EDTA and dimethyl sulfoxide (DMSO) were purchased from

Results and discussion

This study introduced two functions such as improved colloidal stability and facilitated endosomal escape into PC-stabilized Ca2+ ion/gene NCs. To understand well each function considered, the investigation first focused on the surface coating of PCs and then moved forward the introduction of endosomolytic ATP. That is, this study evaluated first Ca2+/pDNA/PC NCs and then Ca2+/ATP-pDNA/PC NCs in terms of physicochemical and biological viewpoints.

Conclusions

In this study, we designed low molecular PCs, Ca2+ and ATP based nanocomplexes for gene delivery systems. To improve the colloidal stability of conventional calcium based gene delivery systems, low molecular weight polycations (e.g. bPEILMW, PS and ε-PL) were used to shield the calcium ion/gene complexes. Shielding by PCs like bPEILMW, PS and ε-PL produced stable Ca2+/ATP-pDNA/PC NCs. As a result, ATP-pDNA containing NCs showed enhanced transfection efficiencies with non-toxic characteristics

Conflict of interest

The authors declare no competing financial interest.

Acknowledgements

This study was supported by Bio & Medical Technology Development Program, the National Research Foundation of Korea (NRF) grants funded by Ministry of Science and ICT (NRF-2014M3A9B6034225 and NRF-2017M3A9F5028608). Also, the study was supported by BK21PLUS grant of NRF funded by the Korean government (Ministry of Education) (22A20130012250).

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