Electrochemical examination of ability of dsDNA/PAM composites for storing and releasing of doxorubicin
Graphical abstract
Introduction
Hydrogels made of oligonucleotides have become more attractive recently [1], [2], [3]. The introduction of oligonucleotides into a regular-polymer hydrogel lattice offers a possibility of combining the unique properties of both constituents and creates a novel biomaterial. Hydrogels are crosslinked polymeric networks that can absorb significant amounts of aqueous- and organic-solvent solutions, up to 95% content. Some hydrogels possess the unique ability to undergo the volume phase transition (swelling–shrinking) triggered by a change in external physicochemical factors such as ionic strength, temperature, pH and a change in the oxidation number of the electroactive chemical groups of the gels [4], [5], [6], [7], [8]. The influence of the novel environmental stimuli on the physicochemistry of hydrogels is still the subject of new investigations [9].
A few procedures for the synthesis of DNA-hydrogel lattices were proposed: firstly, the noncovalent absorption of oligonucleotides from their solutions and secondly, the covalent linking of oligonucleotides with the polymer chains via various chemical groups [10]. The covalent attachment of DNA to various matrices is explored for the construction of novel devices and can be used in biosensors and implants [11], [12]. The noncovalent attachment of oligonucleotides was applied in the development of novel gene delivery methods, e.g. in the delivery of siRNA in the targeted gene therapy [13].
A number of materials were suggested for systemic delivery of pharmaceutical compounds. The list of proposed drug carriers includes: polymeric micelles, liposomes, metallic nanoparticles, quantum dots, carbon nanotubes, graphene sheets, dendrimers, and pH- and temperature-sensitive polymers [14], [15], [16], [17], [18], [19], [20]. Temporal and spatial control of drug delivery for an improvement in the efficacy of the drug administration and limitation of the side effects is the major goal in the design of hydrogel-based drug delivery systems. The search, among others, continues for the best biocompatible and stimuli-responsive (thermo-, photo-, pH-, ionic strength-, magnetic field- and redox-response), “smart” polymer-based systems. Polyelectrolytes ensured ultrahigh loading capacity [21], [22], alginate cellulose beads degraded drastically in the presence of microflora (colon) [23], and appropriately modified polymeric gels reacted to pH, temperature and magnetic field in the surrounding environment [24], [25], [26], [27]. If various polymer-based delivery systems are compared in terms of their place of action, it can be seen that pH-responsive polymer carriers are mostly applicable for cancer treatment. Thermo-responsive polymer systems are mainly desired for tissue engineering and local radiotherapy, while redox-responsive carriers can be useful for cancer therapy and diagnostic. Photo-responsive polymer carriers are used to release drugs by irradiation with light and therefore are useful for e.g. skin cancer treatment. Recent challenges involve design of environmental multi-responsive drug delivery systems where each response stimulates another [28], [29].
Recently, the design of smart DNA-based nanocomposite materials, useful for cell recognition and controlled drug delivery, got some attention. These materials may have several advantages: 1) the synthetic oligonucleotides from the composites may better interact with the overexpressed genetic material characteristic for tumor cells, 2) there is a possibility of anchoring a higher amount of the drug to the oligonucleotides while it remains less toxic, and 3) they can enhance the permeability and retention effect and cause bigger accumulation of the drug in the cancer cells compared to normal cells [30], [31], [32], [33], [34]. Also, DNA-based hydrogels possess unique properties of the bonding by intercalation, due to the presence of dsDNA, and the encapsulation of organic molecules in the intramolecular cavity. In the case of DNA-based composites a possibility of longer, controlled treatment, after the intercalation of the drug into the dsDNA strand, appears. Independently, the enhanced charge transfer in dsDNA could stimulate the light- or thermo-based generation of reactive oxygen species (ROS) needed for more efficient elimination of tumor cells, as in the case of ROS active polymers [35].
The polyacrylamide (PAM) hydrogel was selected in our work as the matrix for immobilization of DNA. PAM is an insulating, polar and soluble in water polymer that is widely studied in pharmacy, agriculture, biochemistry and the genetics [36], [37]. PAM is commonly used for oligonucleotide sequencing in conventional gel electrophoresis and microelectrophoresis techniques: especially for high resolution of short DNA sequences [38], [39]. In addition, the PAM lattice is not thermoresponsive; thus, an increase in temperature will affect only dsDNA structure. In our recent work, we presented the influence of the shrinking process of hydrogels based on N-isopropylacrylamide on the structural change and electroactivity of entrapped dsDNA [40].
dsDNA is a good material for storing planar compounds/drugs due to the intercalation process. The intercalation can be successfully monitored electrochemically, see e.g. [41]. The best way for releasing an intercalated compound from dsDNA is to denature the dsDNA molecule. This, however, requires an inconveniently high temperature. It has been shown recently that to lower the temperature for the denaturation process a redox transformation of the intercalator can be used; simply the redox state of the intercalator will determine the state of DNA [42]. In fact, a slow release of the intercalated drug/compound may start at temperatures much lower than that corresponding to the denaturation process. This expectation is based on our earlier data on the voltammetric electrooxidation of guanine in a DNA strand: usually the oxidation current is enhanced already at temperatures slightly higher than circa 30 °C. This effect was interpreted in terms of relaxation of the dsDNA helix distinctly away of the denaturation-process temperature [43], [44]. Square wave voltammetry (SWV), a very sensitive and fast technique, was used for monitoring the kinetics of the drug release process. This technique was especially useful in monitoring the trace levels of drugs of relatively short life-time [45]. To our best knowledge, the storing and releasing of a drug from the DNA/PAM gels have not been discussed so far. Similarly the direct electrochemical monitoring of anodic response of DNA entrapped in PAM hydrogels has not been reported. We focused on the release of doxorubicin (Dox) stored in dsDNA entrapped in PAM. The efficiency and kinetics of the release were investigated using a constant and pulsing temperature. The properties of the DNA/PAM composites can be useful in such applications as: drug release, components of implanted devices, parts of biosensors or logic switching devices in microfluidic systems and miniaturized analytical nanosystems.
Section snippets
Reagents
Double stranded Calf Thymus DNA (dsDNA, ctDNA) was purchased from Fluka. The content of GC pairs in dsDNA was 48.2 ± 0.5%. Polyacrylamide (PAM) lattice constituents: acrylamide (AA, 99%), N,N′-methylenebisacrylamide (BIS, 99.5%), ammonium persulfate (APS, 98%), N,N,N′,N′-tetramethylenediamine (TEMED, 99%) and intercalator, ethidium bromide (EtBr), were purchased from Sigma Aldrich. Doxorubicin hydrochloride (Dox) was purchased from LC Laboratories (Woburn, USA) and its solubility was determined
Synthesis of electroresponsive DNA/PAM composites
Synthesis of the lattices with entrapped oligonucleotides was done by free radical polymerization reaction. Fig. 1A presents a scheme of the synthesis of a typical PAM network. As it is known, the amide group from PAM can interact via hydrogen bonding with nucleic bases [47], [48]. The purification of the lattices was a crucial step, since we noticed that APS and TEMED gave anodic current responses located near the anodic response of dsDNA; see inset in Fig. 1B [42], [49], [50]. Finally direct
Conclusions
We have shown that successful immobilization of dsDNA can be done in a PAM network without necessity of formation of covalent bonds between dsDNA and the polymer net. Further, the PAM lattice with embodied dsDNA molecules allowed efficient accumulation of doxorubicin. In fact, alone PAM could also accumulate some Dox. Since the examined drug possesses a planar aromatic-ring system, it can simply intercalate into the dsDNA helix; this process was found to lead to an increase in the denaturation
Acknowledgments
Support of this work by NCN grant no.: 2012/05/D/ST5/03464 is thankfully acknowledged.
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Ewelina Zabost and Wioletta Liwinska contribute as first Authors to the work.