Cationic cross-linked polymers containing labile disulfide and boronic ester linkages for effective triple responsive DNA release

Highlights

• A pH, redox and ATP responsive cationic cross-linked polymers having reversible boronic ester linkages had been engineered.

• Degree of crosslinking was established by ¹¹B NMR spectra.

• More effective DNA condensation resulting in the formation of nano-sized polyplexes by cross-linked polymers compared to linear polymers.

• Triple-responsive CLPs can be potentially efficient non-viral DNA carriers.

Abstract

Disruption of DNA carriers triggered by intracellular bio-stimulants has been broadly considered as most convenient strategy for efficient DNA delivery. In this direction, we have designed and synthesized pH, redox and ATP responsive cationic cross-linked polymers (CLPs) having disulfide and reversible boronic ester linkages. These CLPs also contain folate groups that are known for their targeting capability towards cancer cells. Biophysical studies showed that these cationic CLPs exhibited more effective DNA condensation in comparison to cationic linear polymers resulting in the formation of nano-sized polyplexes with sufficient positive zeta potentials and good colloidal stability at neutral pH (∼7.4). More interestingly, the polyplexes prepared from these CLPs have the ability to selectively release complexed DNA under conditions similar to those prevalent in cancer cells such as acidic pH, ATP rich surroundings or presence of glutathione, as revealed by ethidium bromide exclusion assay, agarose gel electrophoresis, AFM measurements, etc. Therefore, these cross-linked polymers have high potential of being effective non-viral gene delivery vehicles.

Introduction

Gene therapy offers an interesting and effective technique to cure various genetic diseases by transporting genetic substances into target cells by vectors to displace defective genes or substitute lost genes [[1], [2], [3]]. This gene therapy can be successfully achieved by developing efficient and risk-free gene delivery system [4,5]. Nonviral vectors, such as polycations [[6], [7], [8], [9]], dendrimers [10,11] are promising substituents to viral vectors because of their less toxicity, low immunogenicity, easy production, availability of versatile chemical modifications, etc. Among the nonviral delivery vehicles, cationic polymers undergo complex formation with negatively charged DNA which are commonly referred as polyplexes [12,13]. Therapeutic efficacy of a DNA delivery system involving polyplexes largely relies upon efficient condensation or packing of DNA, fruitful transportation to target cells, entry through cell-membrane and effective de-condensation etc [4,5]. For effective delivery of DNA to nucleus, polyplexes should have superior stability before they arrive at the desired cells [14]. Most importantly, they should be protected from degradation and protein adsorption on the surface of polyplexes in the blood stream. Likewise, gene transfection efficiency may also be hampered by insufficient and untimely DNA release from the polyplexes [7]. Additionally, removal of undesired degraded polymers and unutilized polyplexes from the cells are also important. For all these reasons, researchers have been focusing on innovative strategies to meet all these requirements for achieving improved gene delivery [15,16].

It has been well known that introduction of hydrophilic polymers such as poly(ethylene glycols) (PEG) enhances water-solubility, biocompatibility, colloidal stability and targeting capability of the polyplexes [15]. Moreover, PEG shells on the surface of the polyplexes prevent protein adsorption and aggregation, which help in increasing the blood circulation time of the polyplexes [17]. Also, in recent years, the receptor targeted DNA delivery has gained a tremendous research interest by which the number of therapeutic doses of DNA to specific cells can be reduced. Among various receptors, folate receptor (FR) is remarkably expressed on the surface of many cancer cells, but this expression of FR is restricted in case of normal tissues [18]. The affection of folic acid (FA) for FR also boosts the rate of internalization. Therefore, conjugation of FA to the polyplexes can potentially enable the latter to target the FR on the cancer cell membrane and enhance the transfection efficacy by receptor-mediated endocytosis [[19], [20], [21]].

The capability of a gene delivery vehicle also depends on the architecture, molecular weight, cationic charge density and bioresponsivity of polycations [[22], [23], [24], [25]]. Especially, polycations having high molecular weight (MW) provide stronger gene packing capabilities compared to their low-MW analogues [26]. For example, a high MW polyetheleneimine (PEI) (25 kDa) causes significant cell-cytotoxicity by disturbing the cell membrane although it has higher transfection efficacy [26]. In contrast, PEI with low molecular weight (2 kDa) exhibits minimal cytotoxicity but diminished gene transfection capability [27].

It is highly challenging to design novel carriers of sufficiently high MW and charge density for effective DNA condensation along with the ability to undergo instant degradation into low molecular weight counterparts triggered by intracellular lower pH and higher glutathione (GSH) concentration to promote rapid release of genetic materials [[26], [27], [28]]. In the context of designing optimum polycations, we have relied on a phenylboronic acid (PBA) derivative in the present study, to get control over various parameters including extracellular stability and efficient DNA release from the carriers [29,30]. Under slightly basic conditions (pH∼8 to 9), PBA, a Lewis acid, is proficient in formation of reversible boronic ester linkages with molecules having cis-diol such as dopamine, sugars [30]. However, these covalently coupled linkages between PBA and cis-diol can be destabilized at higher concentrations of ATP because the ribose moieties of ATP have a greater affinity towards PBA. Therefore, intracellular higher ATP concentrations can trigger the disintegration of PBA-diol linkages, thus facilitating rapid release of DNA from such PBA-diol containing carriers [31]. In addition, intracellular acidic environment (in cancer cells) can also split the PBA-diol coupling due to acid-labile properties of boronic acid-diol linkages. Taking this into consideration, we have developed a novel catechol and methacrylopropyltrimethyl ammonium chloride (MAPTAC) containing cationic cross-linked polymers (CLPs) using multifunctional PBA based cross-linker (DTBA) by formation of boronic-ester linkages between catechol and PBA. Here, we anticipated that this reversibly cross-linked polycations would offer strong DNA packing to ease cellular entry of formed polyplexes and would subsequently split spontaneously in intracellular compartment on exposure to acidic environment and/or higher concentration of GSH and/or ATP leading to the formation of low-MW fragments facilitating a rapid release of DNA.