Controllable synthesis and characterisation of palladium (II) anticancer complex‐loaded colloidal gelatin nanoparticles as a novel sustained‐release delivery system in cancer therapy

Abstract

Over the past few years, there have been several attempts to deliver anticancer drugs into the body. It has been shown that compared to other available carriers, colloidal gelatin nanoparticles (CGNPs) have distinct properties due to their exceptional physico‐chemical and biological characteristics. In this study, a novel water‐soluble palladium (II) anticancer complex was first synthesised, and then loaded into CGNPs. The CGNPs were synthesised through a two‐step desolvation method with an average particle size of 378 nm. After confirming the stability of the drug in the nanoparticles, the drug‐loaded CGNPs were tested for in vitro cytotoxicity against human breast cancer cells. The results showed that the average drug encapsulating efficiency and drug loading of CGNPs were 64 and 10 ± 2.1% (w/w), respectively. There was a slight shift to higher values of cumulative release, when the samples were tested in lower pH values. In addition, the in vitro cytotoxicity test indicated that the number of growing cells significantly decreased after 48 h in the presence of different concentrations of drug. The results also demonstrated that the released drug could bind to DNA by a static mechanism at low concentrations (0.57 µM) on the basis of hydrophobic and hydrogen binding interactions.

1 Introduction

Colloidal drug delivery systems (CDDSs) are in the form of particles or vesicles with nanometre dimensions made of different biomaterials such as natural/synthetic polymers, liposomes or multiple emulsions [1]. These colloidal drug carriers are basically require for successful carriage of loaded drugs into desire sites [2]. Transportation of drugs to the target sites could always minimise undesirable toxic effects by decreasing the amount of drugs that have to be administered to attain a therapeutic response [3]. This extremely selective method can significantly decrease the systemic side effects [2]. It has been frequently reported that CDDSs such as solutions of micelles, vesicles, liquid crystal dispersions and nanoparticle dispersions containing small particles in size ranges between 10 and 400 nm display a great capability for target delivery of anticancer drugs [4, 5]. The goal of developing these formulations is to achieve applicable systems with enhanced DL and release behaviours. Nanoparticles emerge as one of the main tools in cancer research because of their superior advantages for targeted delivery of drugs and also extra potential for cancer diagnosis [6]. Some studies have demonstrated that the main goals are increasing their stability in the biological environment, developing DL/targeting and also improving their interactions with biological barriers [7]. It has been reported that the key problem in the use of nanoparticles are related with their cytotoxicity and biodegradation products. Therefore, one of the most important concerns associated with the use of nanoparticles is their biocompatibility [8]. For this purpose, gelatin has been used in the preparation of nanoparticles for drug delivery applications as a natural polymer with non‐toxic nature and hydrophilic properties. This polymer has long history of safe use in pharmaceuticals, cosmetics and food products with FDA approval for human consumption. Moreover, some studies have offered using this natural polymer for designing drug delivery systems in nanometre scales for fighting cancer cells due to its special properties such as biodegradability and non‐immunogenicity [9].

On the other hand, cisplatin is one of the most widely used antitumor drugs in the treatment of a variety of solid tumours [10]. This drug has commonly been used in different biomedicine applications due to its effects against testicular, non‐small‐cell lung, head and neck and cervical cancers. In addition, cisplatin is one of the most effective agents in cancer chemotherapy in the clinic. However, its clinical use has some restraints due to its severely toxic side effects, such as acute nephrotoxicity and chronic neurotoxicity [11]. Therefore, a rational method to overcome intrinsic or acquired resistance and severe side effects of cisplatin could be the preparation of platinum complexes. Previous studies have reported that due to chemistry similarity of palladium to platinum, palladium complexes demonstrated significant antitumor activity compare with cisplatin with less side effects [11]. Additionally, it has been offered that palladium complexes are expected to have less kidney toxicity than cisplatin [12]. Therefore, in the present study, the synthesis of palladium (II) anticancer complex (Pd(II)ACC) of 2,20‐bipyridine derivative was first reported. Then, colloidal gelatin nanoparticles (CGNPs) were used as carriers for Pd(II)ACC. The in vitro antitumor activity of released Pd(II)ACC was examined against human breast cancer cells. Furthermore, we described the DNA‐binding, thermodynamic parameters and evaluation of binding modes of released Pd(II)ACC. These parameters may explain the interaction mechanisms of this type of complex with DNA of the cells and probable side effects of this anticancer agent.

2 Experimental

2.1 Materials

Palladium (II) chloride anhydrous, sodium chloride, sodium hydroxide and potassium bromide were obtained from Fluka (Switzerland). Piperidine and bromoacetic acid were purchased from Aldrich (UK). 2,2′‐Bipyridineand Tris–HCl [tris(hydroxymethyl) amino methane hydrochloride] were obtained from Merck (Germany). Gelatin type A from porcine skin (GelA, Sigma G2625, 175 Bloom), glutaraldehyde (25% solution, Grad I, Sigma), HCl and acetone purchased from Samchun Co. South Korea. Calf thymus DNA and ethidium bromide (EB) were obtained from Sigma Chemical Co. (USA) and used as received. All reagents and solvents were of analytical reagent grades and double‐distilled water (DDW) was used all along.

2.2 Methods

2.2.1 Synthesis of Pd(II)ACC

The Pd(II)ACC, [Pd(bpy)(pip‐Ac)]NO₃ (which bpy is 2,2′‐bipyridine, pip‐Ac is 1‐piperidineacetato), has been synthesised according to a procedure explained in our previous publication [13]. Briefly, after the preparation of the basic structure of [Pd(bpy)Cl₂] [14], [Pd(bpy)(H₂ O)₂](NO₃)₂ was prepared, to the clear yellow filtrate containing above aqua complex (1.5 mmol), a solution of 1.5 mmol (0.247 g) 1‐piperidineacetate sodium salt in 10 mL water was slowly added with stirring under dark. Then, the clear yellowish orange solution obtained was evaporated at 35–40°C and the achieved solid was washed three times with acetone. Then, the solid residue was dissolved in 30 mL of methanol–acetonitrile (1:1, v/v) mixture at 30–35°C and then filtrated [13].

2.2.2 Preparation of CGNPs

CGNPs were prepared by a two‐step desolvation method [15]. In this experiment, 1.25 g of gelatin was dissolved in 25 mL of DDW by heating to 50°C, and 25 mL of acetone was added to the solution. The supernatant was discharged and the gel‐like precipitate was redissolved in 25 mL of preheated water, and pH was adjusted to 2.5. Then, under constant stirring at 50°C, 75 mL of acetone was added dropwise to the solution. The white milk‐like solution started to form when 65–70 mL of acetone was added into the mixture. After 10 min, 0.5 mL of 8% glutaraldehyde was added to the stirring mixture, followed by an overnight stirring at room temperature and 700 rpm. The particles were purified by centrifugation and redispersion in acetone (70%). After the last redispersion, the acetone was evaporated in a water bath at 50°C, and the nanoparticles were stored at 2–8°C [16].

2.2.3 Preparation of Pd(II)ACC‐loaded CGNPs

To prepare Pd(II)ACC‐loaded CGNPs, 50 µL of Pd(II)ACC stock solution 5 mM was added to a flask containing 500 µL CGNPs solution (6.67 mg/ml) and incubated at 37°C under magnetic stirring (100 rpm) for 24 h. Then, the solution was centrifuged several times and supernatant replaced with DDW. The encapsulation efficiency (EE) of Pd(II)ACC‐loaded CGNPs was determined by centrifuging at 11,000 rpm for 1 h. The supernatant was filtered with 0.2 µm membrane filters. The absorbance of the filtered aliquot was measured at 312 nm by using a UV/Visible Spectrophotometer (1800, Shimadzu, Kyoto, Japan). The percentage of DL and EE were determined by[17]

$$\mathrm{DL}(\text{\%})=\mathrm{weight}\mathrm{of}\mathrm{Pd}(\mathrm{II})\mathrm{ACC}\mathrm{in}\mathrm{CGNPs}/\mathrm{weight}\mathrm{of}\mathrm{CGNPs}\times 100$$ (1)

$$\mathrm{EE}(\text{\%})=\mathrm{weight}\mathrm{of}\mathrm{Pd}(\mathrm{II})\mathrm{ACC}\mathrm{in}\mathrm{CGNPs}/\mathrm{weight}\mathrm{of}\mathrm{feeding}\mathrm{Pd}(\mathrm{II})\mathrm{ACC}\times 100$$ (2)

2.3 Characterisation

2.3.1 Size and size distribution

To determine the average particle size of the synthesised nanoparticles a photon correlation spectroscopy was applied based on dynamic light scattering, by a Zetasizer, model HSA 3000 (Malvern, Worcestershire, UK). For analysing the size of nanoparticles, 1 mL of the nanoparticle suspension was dispersed in 50 mL DI water. Measurements were carried out at 25°C at a light‐scattering angle of 90°. The mean particle size and polydispersity index were determined [18].

2.3.2 Morphological parameters

The morphology of the nanoparticles were examined using a Zeiss EM900 transmission electron microscope (TEM) at a voltage of 80 kV. The TEM sample was ready by adding the aqueous CGNPs solutions onto a carbon‐coated copper grid stabilised with evaporated carbon film and then the CGNPs were negatively stained. The sample was air dried at room temperature before loading into the microscope [18]. In addition, the morphology of Pd(II)ACC‐loaded CGNPs was determined by scanning electron microscopy (SEM). The samples were dipped into liquid nitrogen for 10 min, and then freeze‐dried for 24 h in a freeze drier, EMITECH, model IK750, Cambridge, UK. The sample was fixed on the aluminium stub and coated with gold palladium by Polaron machine model SD515, EMITECH, Cambridge, UK, at 20 nm coating thickness [19]. Finally, the sample was examined under SEM using Stereo scan model S360 brand SEM – Leica Cambridge, Cambridge, UK.

2.3.3 Fourier‐transforms infrared analysis

Fourier‐transforms infrared (FTIR) spectroscopy was employed to record the spectrum of CGNPs, Pd(II)ACC and Pd(II)ACC‐loaded CGNPs by an infrared spectrophotometer (Perkin Elmer, MA, USA). The samples were made according to KBr‐pellet method. The scanning range was 4000–400 cm⁻¹ at a resolution of 1 cm⁻¹ [20].

2.3.4 In vitro release of Pd(II)ACC from the nanoparticles

The in vitro release of Pd(II)ACC was performed using the dialysis method [21]. In brief, 2 mL of Pd(II)ACC‐loaded CGNPs solution was filled in dialysis bags (12 kDa, Sigma) and dialysed against 20 mL of Phosphate‐buffered saline (PBS) (10 mM/L at pH 7.4 and 6.5) retained at 37°C and stirred at 100 rpm. Then, 5 mL solution was withdrawn at varied time intervals and replaced with fresh PBS of same pH to mimic sink conditions [17]. The Pd(II)ACC concentration in each sample was measured at 312 nm using a UV/Visible Spectrophotometer (1800, Shimadzu, Kyoto, Japan).

2.3.5 In vitro antitumor experiments

The MTT assay is dependent on the cleavage and conversion of the soluble yellowish MTT to the insoluble purple formazan by active mitochondrial dehydrogenase of living cells. The T47D human breast cancer cell line was kept in an Roswell Park Memorial Institute (RPMI) 1640 medium complemented with 10% heat‐inactivated foetal calf serum and 2 mM l ‐glutamine, streptomycin and penicillin (5 µg/ml), at 37°C under a 5% CO₂ /95% air atmosphere. In this study, the clear stock solution (2 mM, in deionised water) was sterilised by filtering through sterilising membrane (0.1 nm) and then different concentrations of the sterilised complex (0–0.8 mM) was added to harvested cells. Then, harvested cells were seeded into 96‐well plates (2.9 × 10⁴ cell/mL) with different concentrations of the sterilised complex and incubated for 48 h. At the end of incubation, 25 µL of MTT solution (5 mg/mL in PBS) was added to each well containing fresh culture media. The insoluble formazan produced was then dissolved in solution containing 10% sodium dodecyl sulfate (SDS) and 50% dimethylformamide (DMF) (under dark condition for 2 h at 37°C), and optical density (OD) was read against the reagent blank with a multi‐well scanning spectrophotometer (ELISA reader, Model Expert 96, Asys Hitchech, Austria) at a wavelength of 570 nm [22]. Absorbance is read as a function of concentration of converted dye. The OD value of study groups was divided by the OD value of untreated control and presented as a percentage of the control (as 100%) [23].

2.3.6 Spectroscopic studies on DNA interaction

In this study, a constant amount of DNA (21.4 μM) was titrated with different concentrations of Pd(II)ACC (50–100 μM) in a total volume of 2 mL. The absorption was read after each addition of Pd(II)ACC at 260 nm. The experiment was repeated multiple times to achieve consistent consequences. In addition, different concentrations of the Pd(II)ACC (0.45–0.87 mM) were added to DNA solution (60 µM) and EB (2 µM) for fluorescence studies. The samples were excited at 471 nm and the emission spectra were observed between 540 and 700 nm with 5 nm slit widths at different temperatures of 27 and 47°C [24, 25].

3 Results and discussion

3.1 Evaluation of morphology and particle size of the nanoparticles

The CGNPs prepared by the above‐mentioned two‐step desolvation method [16]. It has been reported that the size and size distribution are important characteristics of a nanoencapsulation product used for drug delivery applications because of the dependence of drug release rate on these properties [26]. The zetasizer analysis and TEM of CGNPs (Fig. 1) showed a mean size of almost 378 nm with a very low polydispersity 0.07. Furthermore, SEM micrographs of Pd(II)ACC‐loaded CGNPs is shown in Fig. 2. As shown in this figure, no cracks or heterogeneity could be seen on the Pd(II)ACC‐loaded CGNPs surfaces. Moreover, the SEM images showed that Pd(II)ACC‐loaded CGNPs were larger than unloaded CGNPs as a result of DL into the nanoparticles.

Fig. 1.

(a) Particle size distribution analysis, (b) Transmission electron microscopy of CGNPs

Fig. 2.

(a) Low, (b) High‐magnification SEM micrographs of Pd(II)ACC‐loaded CGNPs

3.2 Chemical‐bonding characteristics

The synthesised CGNPs, Pd(II)ACC and Pd(II)ACC‐loaded CGNPs were characterised by FTIR to explore the chemical bonding characteristics of each element in the final system. As it can be seen in Fig. 3 a, a strong bond can be observed at 3333 cm⁻¹, which was due to N–H stretching of amine groups. In addition, the small peaks ∼2959 cm⁻¹ were detectable for C–H stretching of methyl and methylene groups. There were also two functional groups at 1652 and 1548 cm⁻¹ which are responsible for the existence of C=O stretching of amide groups and N–H stretching vibrations, respectively [17].

Fig. 3.

FTIR spectra of

(a) CGNPs, (b) Pd(II)ACC, (c) Pd(II)ACC‐loaded CGNPs

Fig. 3 b shows the FTIR spectrum of Pd(II)ACC. As it can be seen, the main characteristics peaks of Pd(II)ACC are around 1158, 1324 and 1353 cm⁻¹ attributed to C–N stretching of aliphatic groups, C–N stretching of aromatic amine groups, and symmetric stretching of nitro groups, respectively. It is also suggested that the aldehyde groups (–CHO) of the cross‐linker could react with amino groups of gelatine, leading to the formation of an imine linkage (CH=N), which is detectable with a small peak around 1458 cm⁻¹ [13]. The FTIR spectrum of the synthesised Pd(II)ACC‐loaded CGNPs was also recorded and the obtained spectrum is illustrated in Fig. 3 c. In comparison of the transmittance spectra between the CGNPs, Pd(II)ACC and Pd(II)ACC‐loaded CGNPs, it could be observed that the characteristic absorption peaks corresponding to both of the drug and synthesised nanoparticles were also appeared to the final product.

3.3 In vitro release of Pd(II)ACC from the nanoparticles

The in vitro release of Pd(II)ACC from nanoparticles was examined by dynamic dialysis method [15], in PBS of pH 6.5 and 7.4 to simulate the cancer tissue and normal body environment, respectively [27] (Fig. 4 a). As it can be observed, there is a slight shift to higher values of cumulative release, when the samples were tested in a lower pH value. For example, after 4 h of release for the sample tested in pH 6.5, the Pd(II)ACC‐loaded CGNPs showed a release around 91.89 ± 2.63% which was higher than that of samples tested in pH 7.4 (around 78 ± 1.53%). It has also been shown that after a burst effect in the first hours of immersing in PBS, there was a slow release behaviour for both samples. Moreover, the average drug encapsulating efficiency and DL of CGNPs were 64 and 10 ± 2.1% (w/w), respectively. It has been suggested that the swelling behaviour of several of natural polymers such as gelatin is responsive to pH. In addition, under suitable conditions of temperature and pH, the linear polymers form helices in areas stabilised by extensive hydrogen bonding. These helices perform as cross‐links holding the amorphous areas together. This protein with minimal surface charge at its isoelectric point (pI) shows extensive swelling at a pH away from their pI because of the increase of high surface net‐charge and improved electrostatic repulsive force [28]. The drug release behaviour from hydrophilic polymers is mostly influenced by the rate of water uptake, drug dissolution and the rate of polymer matrix erosion or degradation [29]. In our samples, by further immersing in PBS solution, a lower amount of drug released to the environment. It can be explained by the surface adsorbed drug that release easily from the surface of nanoparticles, while after few hours, the release behaviour is basically due to the entrapped drug from the nanoparticles into the environment [17].

Fig. 4.

(a) Release profile of the drug from the CGNPs in PBS and pH = 6.5 and 7.4, (b) The growth suppression activity of the released drug on T47D cells, (c) Fluorescence emission spectra of interacted DNA in the absence and presence of different concentrations of the released drug at 27 and 47 °C, (d) Stern–Volmer curve for quenching of DNA by released Pd(II)ACC in 20 mM NaCl solution, 20 Mm Tris–HCl at 27 and 47°C and pH 7.0, (e) Van't Hoff plot for the binding of released Pd(II)ACC with DNA

It is known that the nanoparticles which are used in cancer therapy should be able to go through the systemic circulation, spread in the tumour tissue, passage across the microvessels and penetrate into the cancer cells [7]. However, irregular blood stream, high interstitial pressure, acidification of tumour cells microenvironment and the absence of an appropriate lymphatic system may usually lead to sub‐therapeutic uptake of nanoparticles. It has been also frequently reported that the significant hydrostatic pressure gradient across the cancer cells limits the dispersion of drugs into the cells [30]. Therefore, CGNPs has illustrated maximum release of Pd(II)ACC at acidic environment rather than physiological pH, which might be useful for achieving better results.

The effect of Pd(II)ACC on human breast cancer cells (T47D human cell line) was evaluated by methylthiazolyl tetrazolium assay (MTT assay). In this experiment, different concentrations of Pd(II)ACC, ranging from 0 to 0.8 mM of the stock solution (2 mM), were used in the culture medium of the tumour cells for 48 h. The 50% cytotoxic concentration (IC₅₀) [31] value of Pd(II)ACC was 0.42 mM. As it can be seen in Fig. 4 b, the number of growing cells significantly decreased after 48 h in the presence of different concentrations of the drug. This result proposes that Pd(II)ACC can be potentially useful as a antitumor agent.

Electronic absorption spectroscopy is an important method to study the binding modes of anticancer drugs with DNA [32]. The UV–Vis spectrum analysis of the interaction of DNA with Pd(II)ACC at temperatures of 27 and 47 °C was investigated. It was observed that the absorbance of DNA at 260 nm should increase with increasing amount of metal complex.

This profile has shown that the concentration of metal complex in the centre of transition, [L]_1/2, is analogous to [L]_1/2 values of binding of similar complexes [Pd/Pt(bpy)(mor‐dtc)]NO₃ and is less than values of crocin and crocetin, with DNA [33, 34]. As a result, the drug released from the Pd(II)ACC‐loaded CGNPs can efficiently interact with DNA at low concentrations (0.57 µM). According to our previous explanations, the released dose of drug from the Pd(II)ACC‐loaded CGNPs corresponds to the effective dose of Pd(II)ACC on DNA. Moreover, if Pd(II)ACC is administered as an antitumor agent, low doses of this drug can be used which have less side effects.

In this study, fluorescence spectroscopy was used to provide a better understanding on the interactive effects of the released drug from the Pd(II)ACC‐loaded CGNPs with DNA molecules. The obtained results demonstrated that no specific fluorescence characteristics were detected for the released Pd(II)ACC in aqueous solution or in the presence of calf thymus DNA. Some studies have reported the nature of the interaction of EB with nucleic acids [35, 36]. Fig. 4 c shows the emission spectra of DNA‐EB system in the absence and presence of the released Pd(II)ACC. As it can be seen, with the addition of Pd(II)ACC, a notable reduction in fluorescence of DNA‐EB system was observed that showing the complex bind to DNA [37].

The quenching mechanisms are usually categorised into dynamic quenching and static quenching, which can be recognised by their diverse in viscosity and temperature or by lifetime comparison. The dynamic quenching result from collision diffusion between the fluorophore and quencher, and higher temperatures will cause larger fluorescence quenching constants. In contrast, the static quenching is triggered by the formation of a ground‐state complex, and higher temperatures are likely to result in the reduced stability of bound complexes and less values of the static quenching constants [38]. Dynamic or static quenching can be identified by analysing the fluorescence data with the Stern–Volmer (3) [39]:

$$F_0/F=1+k_{\mathrm{q}}{\tau }_0\left[\mathrm{L}\right]=1+K_{\mathrm{SV}}\left[\mathrm{L}\right]$$ (3)

F ₀ and F are the DNA fluorescence intensities in the absence and in the presence of Pd(II)ACC, respectively. k _q is the quenching rate constant of the biomacromolecule; τ ₀ is the average lifetime of the molecule without any quencher (for most biomolecules, τ ₀ is about 10⁻⁸ s). K _SV is the Stern–Volmer quenching constant and [L] is the total concentration of released Pd(II)ACC. The plots of F ₀ /F on the quencher concentration ([L]) are shown in Fig. 4 d and the K _SV values of the released Pd(II)ACC are simulated in Table 1. As it has been shown in Table 1, the values of K _SV decreased with increasing of temperatures, which demonstrated that the quenching mechanism was most likely a static quenching process. In addition, the quenching constant, k _q, can be calculated based on k _q  = K _SV /τ ₀, which the mentioned k _q value is greater than the maximum scatter collision‐quenching constant (2.0 × 10¹⁰ M⁻¹ s⁻¹). This shows that the main quenching mechanism is started by static process [40].

Table 1.

Binding parameters for the possible interaction of Pd(II)ACC on DNA

T, °C	K SV  × 103, (M)−1	k q  × 1010, (Ms)−1	K b  × 103, (M)−1	n	ΔG, kJ/mol	ΔH, kJ/mol	T ΔS, J/mol K	R 2 b
27	0.88 ± 0.2a	8.8 ± 0.2	1.01 ± 0.02	1.71 ± 0.06	−0.025 ± 0.02	1.82 ± 0.22	6 ± 0.07	0.98
47	0.66 ± 0.2	6.6 ± 0.2	1.06 ± 0.01	1.37 ± 0.08	−0.147 ± 0.01	1.82 ± 0.22	6 ± 0.07

^a Standard deviation.

^b The correlation coefficient for the Van't Hoff plot.

For static quenching, the next equation was employed to acquire different binding parameters. The binding constant (K) and number of binding sites (n) were counted according to (4) [41]:

$$\mathrm{Log}\left[\left(F_0-F\right)/F\right]=\log K_{\mathrm{b}}+n\log \left[\mathrm{L}\right]$$ (4)

F and F ₀ are the florescence intensity with and without the presence of the released Pd(II)ACC, respectively. A chart of log[(F ₀  − F)/F] versus log[L] provided a straight line using least square analysis whose slope was equal to n (binding site number) and the intercept on Y ‐axis to log K _b (K _b  = binding constant). As it has been revealed in Table 1, the values of K _b were increased with increasing the temperatures due to an increase in the stability of Pd(II) complex–DNA systems. The value of n was around 1, which indicated that there was one binding site for the released Pd(II)ACC on the DNA molecular [42].

Since temperature is dependent on binding constant, a thermodynamic process was deliberated to be reliable for the formations of a complex. Therefore, the thermodynamic parameters dependent on different temperatures (27 and 47°C) were examined to further describe the interaction forces between the release drug and DNA molecules. The interaction forces between small molecules with macromolecules mainly comprise electrostatic force, hydrogen bonds, van der Waals force and/or hydrophobic interaction force. The thermodynamic parameters of binding interactions are the main indications to define the type of binding forces. If the enthalpy change (ΔH °) does not vary considerably over the temperature range studied, then its value and that of entropy change (ΔS °) can be determined from the Van't Hoff equation as

$$\ln K=-\mathrm{\Delta }{H}^{\circ }/RT+\mathrm{\Delta }{S}^{\circ }/R$$ (5)

$$\mathrm{\Delta }{G}^{\circ }=-RT\ln K_{\mathrm{b}}$$ (6)

R is gas constant and K is binding constant. The enthalpy change (ΔH °) and entropy change (ΔS °) were achieved from the slope and intercept of the linear Van't Hoff plot based on log K versus 1/T [see Fig. 4 e]. The values of ΔH °, ΔS ° and ΔG ° are listed in Table 1. As it can be seen, the negative value of ΔG ° in the interaction process can be related to a spontaneous reaction. Furthermore, the formation of the drug‐DNA complex was mainly enthalpy driven accompanied by positive enthalpy (ΔH °) and entropy (ΔS °). Consequently, the thermodynamic parameters for the interaction of the released Pd(II)ACC and DNA can be described on the basis of hydrophobic interaction and hydrogen binding [24, 43, 44]. According to the mentioned evidence, released Pd(II)ACC is suitable for cancer therapy and the designed system is an efficient controlled delivery system for cancer drugs.

4 Conclusion

In the present study, we have focused on the synthesis and characterisation of colloidal nanoparticles loaded with a new anticancer agent (Pd(II)ACC)). Gelatin was successfully employed to prepare Pd(II)ACC‐loaded nanoparticles <400 nm using an optimised two‐step desolvation method. To investigate the effects of the synthesised palladium complex on DNA‐binding properties and cytotoxic activities, we selected 1‐piperidine as the ligand to design and synthesise one new water‐soluble palladium (II) complex, [Pd(bpy)(pip‐Ac)]NO₃. The cytotoxicity of the complex was examined against T47D human breast cancer cell line. The absorption and fluorescence spectra studies on the interaction of the Pd(II)ACC with DNA demonstrated that the complex interacted with DNA cooperatively at low concentrations. Thermodynamic parameters, enthalpy change (ΔH °) and entropy change (ΔS °) were calculated according to the related fluorescent data and Van't Hoff equation. The results of this study suggested that the complex interacted with DNA, probably via a hydrophobic interaction and hydrogen binding, and also it lay a foundation for the rational design of new agents to probing and targeting nucleic acids.