Research paperSemi-interpenetrated, dendritic, dual-responsive nanogels with cytochrome c corona induce controlled apoptosis in HeLa cells
Graphical abstract
Dendritic thermoresponsive nanogels loaded with cytochrome c corona induce controlled apoptosis upon sample cooling.
Introduction
Nanogels (NGs) are three-dimensional crosslinked nanoparticles that are able to absorb a large amount of water. They constitute a class of new nanocarriers for the loading and improved release in situ of hydrophilic cargoes (both small molecules or macromolecules) in controlled conditions. Their ease of functionalization with dyes or inorganic imaging agents allows them to be used as powerful diagnostic agents, in an effort to combine therapeutic efficacy and diagnostics in one single theranostic macromolecule [1], [2]. Their nanosized dimensions allow for an increased residence time in the bloodstream by avoiding excretion from the kidneys [3], as well as for enhanced skin [4] and follicular penetration [5] or improved mucosal adhesion [6]. Stimuli-responsive NGs are capable of changing their physicochemical properties upon exposure to external conditions such as temperature, pH, reducing conditions, light, and magnetic field, among others. These “smart” materials offer new promising perspectives for the development of next generation agents for the controlled delivery of drugs [7], [8]. Thermoresponsive polymers exhibit a reversible phase transition, caused by the hydrophobic aggregation upon heating of their aqueous solutions to temperatures above their lower critical solution temperature (LCST). Poly(N-isopropylacrylamide) (pNIPAM) undergoes this phase transition at around 33 °C [9]. Thermoresponsive NGs based on N-isopropylacrylamide (NIPAM) are one of the main candidates for biomedical research due to their reversible phase transition at a temperature of 33 °C, close to the body temperature [10]. pNIPAM NGs crosslinked with dendritic polyglycerol (dPG) [11], were published by our group, showing a versatility for their use in biomedical applications. The copolymerization of pNIPAM with poly(N-isopropylmethacrylamide) (pNIPMAM) or hydrophilic comonomers, such as acrylamide or acrylic acid, helps tuning the LCST values to a more desired transition temperature, closer to 37 °C [12], [13]. The use of dPG as a macromolecular crosslinker helps improving the biocompatibility of pNIPAM, as well as avoiding the pNIPAM-driven NG aggregation above the LCST, thus ensuring the colloidal stability of dPG-pNIPAM NGs under physiological conditions [11]. The high swelling abilities of NGs can be exploited to achieve high loadings of bioactives [14]. This property, combined with the abrupt shrinkage of the thermoresponsive network upon temperature change, gives NGs unique properties for the controlled release of small drugs, as well as biomacromolecules, in a controlled fashion.
Dendrons are molecules with regular branching points and symmetrical tree-like structures. At their extremity, the repeated end groups may induce multivalency, leading to an exponential increase in binding affinities [15], [16]. In some cases though, multivalency is counteracted by steric crowding which may hinder the multivalent effects, leading to a binding saturation point, in an anti-cooperative fashion [17], [18], [19]. The tuning of the generation (number of repeated branchings) allows the regulation of the hydrophilic/hydrophobic balance of the dendron [20]. In this way, the binding affinity towards guest molecules can be tuned, whether these are small molecules, proteins, dendrons, or polymers [21]. Newkome-type dendrons/dendrimers are a class of molecules that are biocompatible, non-toxic and are used to create polymers with multifunctional acidic units, useful for promoting host-guest interactions with therapeutically relevant moieties [22], [23], [24], [25], [26], [27]. Among this class of dendrons, 4-acryloylamine-4-(carboxyethyl)heptanodioic acid (ABC) and its copolymer NGs with pNIPAM have been developed by our group and have been proven to provide sustained release of cisplatin at lysosomal conditions [28]. Following a similar approach, we used ABC in this study to generate semi-interpenetrated NGs with dendritic multifunctional units that can be used as anchoring points for the binding of therapeutic proteins. Semi-interpenetrated polymer networks (SIPNs) are composed by more than one polymeric network that are physically entangled, although not covalently bound to each other. Due to the entanglement, the different networks cannot be separated by simple mechanical stress, but rather only by chemical reactions. This approach is useful for the fabrication of polymer composites that retain the physicochemical properties of the individual independent networks and maintain stable polymer compositions. SIPN NGs can be synthesized by a two-step process, in which the secondary network is being polymerized inside the preformed NG. By controlling the synthetic parameters of the polymerization, the resulting SIPN concentrations can be modified in order to get chain lengths that can be optimized to achieve maximum SIPN surface exposure upon shrinkage of the thermoresponsive NG scaffolds. Our group has developed dPG-pNIPAM SIPN NGs with polymerized 2-acrylamido-2-methylpropane sulfonic acid (AMPS) or dimethylaminoethyl methacrylate (DMAEM) as vectors for the efficient delivery of doxorubicin to the resistant KB-V1 cell line [29]. Their incorporation into honeycomb films for controlled bovine serum albumin (BSA) delivery was investigated and described by our group in a follow-up study [30]. Their copolymeric NG analogues were reported in another case study for the sustained delivery of doxorubicin and methotrexate [31]. In another report, we employed dPG-PNIPAM SIPN NGs with poly(aniline) as secondary network, a near infrared - active polymer to add photothermal reactivity to the NGs [32]. Several other examples of SIPN NGs can be found in literature, showing a broad range of applications for this synthetic approach, for the development of next generation nanoparticles for biomedical applications [33], [34], [35], [36], [37].
The use of protein drugs has advantages over conventional chemotherapy agents, due to their inherent lower toxicities combined with high specificities. The presence of a protein corona has a profound impact on the biological efficacy of nanoparticles, and by designing tailor-made protein corona one could boost the therapeutic efficacy of the nanoparticles [38], [39], in a synergistic effort to combine efficient nanosized vectors to therapeutic proteins [40]. Cytochrome c (cyt c) is a small, 12 kDa heme protein with an isoelectric point (pI) of 10 and is therefore positively charged at a physiological pH of 7.4. In cells, cyt c is loosely associated to the inner membrane of the mitochondria and is involved as an intermediate in the electron transport chain. If released into the cytosol, cyt c triggers apoptosis by binding to apoptosis activating factor-1 (Apaf-1). This in turn is responsible for the activation of caspase-9 to initiate the intrinsic apoptotic cascade [41], [42]. While cyt c itself is membrane-impermeable, recent studies involved the use of nanoparticles (NPs) for sustained cytosolic release of cyt c. Different nanoparticle-based systems have been evaluated for the activation of apoptosis following cyt c release: mesoporous SiO2 NPs [43] or a combination of end-capped cyt c and encapsulated doxorubicin [44], crosslinked cyt c – based NPs [45] or redox-sensitive hyaluronic acid NGs [46], [47]. To our knowledge however, none of these studies investigate the temperature-dependent cyt c activation for controlled induction of apoptosis.
In this work, we describe the synthesis of dendritic SIPN NGs, based on thermoresponsive dPG-pNIPAM NGs, with pABC as a SIPN (Fig. 1). This arrangement forms NGs which maintain the original thermoresponsiveness of pNIPAM, together with dPG to provide colloidal stability at a temperature higher than the pNIPAM LCST (33 °C), combined with pH-sensitive dendritic pABC or monofunctional control poly(acrylic acid) (pAA) as SIPN. We characterized the NGs via UV–VIS, nuclear magnetic resonance spectroscopy (NMR), dynamic light scattering (DLS) and acid-base titration. These NGs were loaded with cyt c at 37 °C, to boost the preferential formation of a corona around the collapsed NG, which was mediated by binding pABC or pAA on the NG surface. The subsequent swelling of the NGs at 25 °C was investigated in relation with its ability to disrupt the binding, thereby triggering the release of cyt c. The temperature-controlled delivery of cyt c was evaluated in vitro in HeLa cells, where the ability of the cyt c-laden NGs to induce apoptosis by controlled intracellular delivery of cyt c was probed. In this way, we combined dendritic NG design with the use of a thermal trigger in order to achieve full control on the reactivity of a therapeutic protein. Thus, we introduce a model system for the development of NG-based therapeutics for the treatment of superficial tumors or skin diseases, where local cooling may be efficiently applied.
Section snippets
Materials and methods
All materials were purchased from Sigma, except 2,2-azobis(2-methyl-N-(2-hydroxyethyl)-propionamide) (VA-086), which was purchased from Wako Ltd.
The syntheses of dPG-PNIPAM NGs and ABC have been previously published [11], [28]. Briefly, for a partial acrylation of dPG, a solution of acryloyl chloride (0.22 mL, 2.7 mmol) in dry DMF (4 mL) was added to a solution of dPG (2 g, 10 kDa, 27.03 mmol OH groups) and triethylamine (0.24 mL, 1.72 mmol) in DMF (60 mL) at 0 °C. The reaction was then allowed
Synthesis and characterization of SIPN nanogels
The dPG-pNIPAM NGs and ABC were synthesized according to procedures published previously by our group [11], [28]. In a common procedure for NG semi-interpenetration, the lyophilized dPG-pNIPAM NGs were soaked in a concentrated aqueous solution of ABC or acrylic acid (AA). ABC or AA were present in a concentration range of 0.3–2.6 mg mL−1 in the NGs solution (10 mg mL−1). After purification by ultracentrifugation, the SIPN formation occurred via radical polymerization of the acrylic monomer
Conclusions
In this work, we synthesized SIPN NGs by a simple one-pot strategy, to achieve NGs with repetitive dendritic charged units, which were increasingly exposed to the NG surface above 35 °C. By exploiting negative charges at 37 °C, we were able to form a reversible binding of cyt c by electrostatic pairing with pABC. The stability of the cyt c – NG complex was investigated in relation with the NG swelling in vitro, where a boost in the release of cyt c was observed for SIPN pABC NGs when cooled
Acknowledgements
We gratefully acknowledge financial support from the Bundesministerium für Bildung und Forschung (BMBF) through the NanoMatFutur award (ThermoNanogele, 13N12561), DFG-CONICET, CONICET, Alexander von Humboldt foundation, the Focus Area NanoScale of the Freie Universität Berlin (http://www.nanoscale.fu-berlin.de), and the Sonderforschungsbereich 1112 (http://www.sfb1112.de), Project A04.
Declaration of interest
None.
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