In this work, we investigated the synergistic cytotoxic actions of the chemotherapeutic (ST), combined with gold-sensitized γ-radiation, on human hepatocellular carcinoma (HepG2) cells. For this purpose, hexagonal gold nanoparticles were prepared and used as a novel radiosensitizer to enhance the radiobiological response of HepG2 cells. Nanoporous alginate/CaCO3 hybrid was prepared as a biocompatible/biodegradable nanocarrier for physical encapsulation of the hydrophilic hexagonal gold nanoparticles (HG) and the hydrophobic drug (ST). The proposed combination regimen was designed to target key pathways in a characteristically synergistic or an additive manner.
3.1 Hexagonal gold nanoparticles
Size, shape and coating of the gold nanoparticles determine their ability of cellular internalization and radiosensitization efficacy [15,27]. In the present study, a facile procedure for synthesis of gold hexagonal nanocrystals (HG) is ascribed. The radiosensitization effect of HG was used. Chitosan was used to reduce Au3+ ions to atomic gold, where D-(+)-glucono-1,5-lactone was used to dissolve chitosan polymer instead of acetic acid. This led to formation of lactone-capped hexagonal nanocrystals (HG). The ruby-red color of colloidal gold (Fig. 1) is characteristic for the excitation of surface plasmonic resonance of HG. Extinction spectra of aqueous lactone-capped HG colloid showed an extinction spectrum at 536 nm (Fig. 1). Low magnification TEM images (Fig. 1) showed hexagonal gold nanocrystals of average diameter of about 28 nm, while high magnification TEM images of HG (Fig. 1) showed high degree of crystallinity. As predicted from dynamic light scattering (DLS) measurements, the particles have a mean hydrodynamic radius of 34.2±4.5 nm indicating the presence of thin layer coat of about 6 nm (Table 1). According to z potential measurements, HG NPs have a negative surface charge of -14.62 mV (Table 1) and this suggests the lactone capping on the surface of the gold hexagons. We likely ascribe the formation of hexagonal nanocrystals to the relatively slow growth of gold crystals in the viscous lactone medium. Apparently, lactone capping instead of chitosan capping contribute to the formation of the hexagonal-shaped nanocrystals. Generally, the proposed surfactant-free procedure for preparation of HG is more favored than the surfactant-based method [28], and more facile than other previously described procedures [10, 29].
3.2 Alginate/CaCO3 polyelectrolyte nanoparticles
This study aims to investigate the cytotoxic actions of gold hexagons-sensitized radiation in synergism with sorafenib tosylate against human hepatoma HepG2 cells. The key parameter in such combinatorial model is the choice of a nanocarrier capable for incorporating and co-delivery of both the hydrophobic drug (sorafenib tosylate) and the hydrophilic radiosensitizer (hexagonal gold nanoparticles). Alginate hybrid is capable for loading different therapeutics regardless of their surface charge and hydrophilicty [30, 31]. Nanoporous alginate/CaCO3 hybrid platform is significant in that the hydrophilic anionic alginate sites can be loaded with the hydrophilic gold hexagons, while the nanosized porous can be loaded with the hydrophobic drug (ST). Divalent cations like Ca2+can induce interchain association with G units of the alginate polymer forming junction zones (Fig. 2). After addition of Na2CO3, carbonate anions tend to deprive the coordinated calcium cations as CaCO3, while the polymer chains prevent the precipitation of CaCO3. Thus, Ca2+ rich sites will be condensed in the core of the nanoparticle with formation of nanosized pores, whereas Ca2+ deficient sites will be enlarged in size due to repulsion between the anionic carboxylate groups (Fig. 2). In a previous work [32], the hydrophobic drug PTX was loaded in alginate hybrid followed by loading of the hydrophilic drug (DOX) and thus low amounts of DOX could be loaded because some nanoporous have been occupied by PTX. However, in this work, we aimed to achieve a simultaneous loading of the hydrophobic drug and the hydrophilic drug to avoid low loading of the hydrophilic drug in the previously described two steps loading process [32]. In this work, we proposed a one step procedure for production of the hydrophilic HG and the hydrophobic ST loaded alginate hybrid nanoparticles suitable as a combinatorial (chemo-radio) model for treatment of HepG2 cells.
The physicochemical properties of the prepared nanoprobes were studied by TEM, DLS, zeta-potential analyzer and DSC. TEM images showed spherical nanoparticles with mean diameter of 230, 265, 290 and 295 nm for empty ALG, ST-ALG, HG-ALG and HG-ST-ALGNPs, respectively (Fig. 3). The high magnification TEM image (Fig. 3 inset) shows porous structure of the prepared hybrid and hexagon-shaped gold nanoparticles.
Table 1. Data of hydrodynamic diameter and zeta potential of HG NPs, blank nanoparticles and drug loaded nanoparticles. The data corresponds to the average of five independent batches and are presented as mean ± SD
Formulation
|
HD ± SD (nm)
|
PDI
|
ζ ± SD (mV)
|
HG NPs
|
34.2 ± 4.5
|
0.211
|
- 12.35 ± 2.4
|
Empty ALG NPs
|
345.4 ± 4.2
|
0.205
|
-20.1 ± 1.9
|
ST-ALG NPs
|
366.1 ± 3.2
|
0. 271
|
-12.4 ± 0.6
|
HG-ST NPs
|
390.9 ± 2.9
|
0.282
|
-13.6 ± 0.9
|
HG-ST-ALG NPs
|
405.8 ± 6.2
|
0.296
|
-14.5 ± 1.2
|
Data of HD and zeta potential represent the mean values ± SD, n=3. Abbreviations ALG, a alginate/CaCO3 hybrid nanoparticles, EE, encapsulation efficiency, HD, the average hydrodynamic diameter estimated by DLS, HG, Hexagonal gold nanoparticles, PDI, polydispersity index, ST, sorafenib tosylate, SD, standard deviation, ζ, zeta potential.
Table 2. Drug loading content and encapsulation efficiency of the drug loaded nanoparticles. The data corresponds to the average of five independent batches and are presented as mean ± SD
|
DL (mg/g)± SD
|
EE (%) ± SD
|
Formulation
|
HG
|
ST
|
HG
|
ST
|
ST-ALG NPs
|
----
|
59±3
|
----
|
42.6 ± 3.6
|
HG-ALG NPs
|
71±4
|
----
|
58.4± 2.6
|
----
|
HG-ST-ALG NPs
|
64±5
|
55±4
|
47.8± 4.2
|
39.65 ± 3.7
|
Note, Data represent the mean values± standard deviation (SD), n=3. Abbreviations ALG, alginate/CaCO3 hybrid nanoparticles, DL, drug loading, EE, encapsulation efficiency, HG, hexagonal gold nanoparticles, NPs, nanoparticles, ST, sorafenib tosylat.
DSC measurements is an important tool in drug delviery systems to inspect the nature of the encapsulated drug (amorphous or crystaline) and the possible physicochemical interactions between the drug and the the recipients used for design of the formulations [33, 34]. DSC measurements of pure ST (A), sodium alginate polymer (B), lyophilized empty ALG NPs (C) and HG-ST-ALG NPs (D) were carried out from room temperature to 400 °C (Fig. 5). The DSC thermogram of pure ST showed a sharp melting endotherm band at 241.2 °C (Fig. 5A) coincides with reported for sorafenib tosylate (240-243 °C) indicating purity of the drug [33]. The DSC thermogram of sodium alginate polymer showed an endothermic peak centered at 88°C and an exothermic peak centered at 256 °C corresponding to polymer dehydration and degradation, respectively [35]. The DSC thermogram of lyophilized empty ALG/CaCO3 hybrid NPs exhibited a sharp endothermic band at 185 °C and a broad exothermic band from (260 °C to 295 °C). The endothermic band is probably assigned to cleavage enthalpies (breakage) of calcium-carboxylate bonds within the calcium rich sites of the complex forming the so called “egg-box” structure [35]. The broad exothermic band probably corresponds to degradation cross-linked alginate hybrid. The shift of this band to higher temperature, compared to that of alginate polymer, indicates that the cross-linked hybrid is more resistive to thermal degradation than the polymer. Lyophilized HG-ST-ALG NPs exhibited a DSC thermogram similar to that of empty ALG NPs, except the broadening of the endothermic peak.
3.3 In vitro release study
Release profiles of ST revealed a sustained release from both ST-ALG and HG-ST-ALG nanoparticles within the first 72 hours (Fig. 6). For ST release in neutral media (pH 7.4), the driven mechanism of release is drug diffusion. Release of ST from both formulations in neutral media is slow and shows no burst release, probably due to the hydrophobicity of ST. A moderate release of ST is observed in acidic medium with optimum release of 62.1 % and 50.3 %, from ST-ALG and HG-ST-ALG nanoparticles, respectively (Fig. 6). A similar release profile was reported for release of the hydrophobic drug paclitaxel from alginate/CaCO3 hybrid nanoparticles (ALG). Compared to release in neutral medium, the released amounts of ST are greater in acidic medium (pH 5.2) from both ST containing formulations. This release behavior is adequate for cancer treatment due to the well known acidic extracellular tumor environment [36,37]. It is well known that that the acidic conditions accelerate the breaking down of particle structure and resulting in fast diffusion of drug from the interior of the nanoparticles [38]. A steady and optimal release of ST was reached after 48 h.
3.4 In vitro anticancer efficiency
In this work, IC50 of sorafenib tosylate (ST) free drug on human hepatocarcinoma HepG2 cells of 18.9 µg/ml was estimated from the dose response curve. This value is comparable to the previously reported value 17.5 µg/ml [39]. Free drug samples were prepared by dissolving sorafenib in dimethyl sulfoxide (DMSO) and stored as stock solution at -20°C. IC50 of ST in ST-ALG, HG-ST-ALG and were found to be 12.7 and 14.3 µg/ml, respectively. It can be concluded that inclusion of ST in alginte nanoparticles significantly reduced the IC50 values of ST, compared to the free drug. This evidences the crucial role of the nanocarrier in the cellular internalization of the drug. To verify the cytotoxic effects of the proposed model on human hepatocarcinoma HepG2 cells, the cells were cultured and incubated with formulations containing different concentrations of sorafenib tosylate (0-24 µg/ml) followed by γ-irradiation with selected radiation doses (0, 3 and 6 Gy) (Fig. 7A-C). According to gold analysis, the coressponding gold concentrations were found to be in the range of (0 - 25.2 µM). HepG2 cells were incubated with the formulations for 48 h before processing MTT assay (Fig. 7A-C). The cell viability results are summarized in the next two parts.
3.4.1 Treatment without external radiation
Cell viabilities obtained after incubation of HepG2 cells with the prepraed nanoprobes without subsequent external radiation represent the pure chemical cytotoxicity of the formulations. As seen in Fig. 7A, The results can be summarized as follows:
- Empty alginate nanoparticles (ALG) showed high biocompatibility to HepG2 cells with viability percentage more than 90%. Treatment of HepG2 cells with ST-ALG NPs showed a significant toxicity on HepG2 cells in a concentration dependent manner (Fig. 7A) and this evidences the efficient release and cellular internalization of ST nanoparticles. This is in accordance with the previous reports on the increment in cell cycle inhibition of cancer drugs when loaded in polymeric nanoparticles [40].
- HG-ALG NPs are nearly non-toxic to HepG2 cells and about 90% of the cells remain viable after treatment with the highest gold concentration. This is mainly ascribed to biocompatibility of HG nanoparticles at the tested concentrations (Fig. 7B) This may be attributed to elevated released amounts of HG from HG-ALG NPs.
- HG-ST-ALG NPs are significantly toxic to HepG2 cells and this toxicity increases gradually with increasing the concentration of both ST and HG in the nanoparticles (Fig. 7C). It is noteworthy to mention that the synergistic effects of HG and ST are not observed in this set of experiments where no external radiation is applied. More than 40 % of HepG2 cells could survive even at the highest concentrations of both ST and HG.
3.4.2 Treatment with external radiation
To verify the cytotoxic effects of the combinatorial (chemo-radio) model, HepG2 cells were incubated with the prepared formulations at the same previously tested concentratios of HG and/or ST followed by γ-radiation (3 and 6 Gy) using Cs-137 source. The results of cell viability assay are illustrated in Fig. 7B,C. It can be concluded that:
- Incubation of HepG2 cells with empty ALG NPs followed by irradiation with 3 and 6 Gy showed moderate cytotoxicity and more than 60% of the cells remain viable. Since ALG NPs are nearly non-ttoxic to HepG2 cells in absence of external radiation (Fig. 7A), this cytoxicity is mainly attributed to the radiobiological actions on HepG2 cells and generation of reactive oxygen species [17].
- Incubation of HepG2 cells with gold containing nanoparticles (HG-ALG or HG-ST-ALG) followed by γ-radiation (3 or 6 Gy) results in a significant reduction of the cell viability compared to irradiation without prior incubation with gold formulations. This effect increases with increasing of the concentration of the gold radiosensitizer. This evidences that gold hexagonal nanoparticles efficiently enhances the radiobiological response of HepG2 cells (Fig. 7B,C).
- Incubation of HepG2 cells with ST-ALG NPs followed by radiation doses of 3 or 6 Gy resulted in cytotoxicities higher than the coressponding values obtained for each individual treatment (Fig. 7). These results, together with the markedly increased cytotoxicity observed with increasing ST concnetration, confirms the synergistic cytotoxic actions of ST and the non-sensitized ionizing radiation on HepG2 cells.
- Incubation of HepG2 cells with gold-sensitized combined treatment model (HG-ST-ALG + radiation doses of 3 or 6 Gy) resulted in the highest cytotoxicity values among all other modalities (Fig 7). This confirms both the radiosensitization enhancement effects of HG and the synergetic cell killing actions of ST and the gold sensitized radiotherapy. The cytotxicity of this model increases with increasing the molar concentration of both ST and HG.
Generally, it can be concluded that both the hydrophilic radiosensitizer (HG) and the hydrophobic chemotherapeutic (sorafenib tosylate) could be efficiently released from the hybrid nanoporous alginate/CaCO3 and can be efficientlt internalized into HepG2 cells resulting in significant and synergestic cytotoxic effects. Eventually, the gold-sensitized combinatorial model (HG-ST-ALG + radiation dose of 6 Gy) efficeiently reduce the cell viabilty of HepG2 cells to less than 15%.