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DOI: 10.1039/D4NJ02391A (Paper) New J. Chem., 2024, 48, 15620-15632

Ajwain-assisted synthesis of oxalipalladium nanoparticles for colorectal cancer treatment: enhanced anticancer activity and protein interaction profiling†

Fatemeh Goli‡ a, Adeleh Divsalar *a, Milad Rasouli‡ *bc and Hamid Gholami d
aDepartment of Cell and Molecular Sciences, Faculty of Biological Sciences, Kharazmi University, Tehran, Iran. E-mail: divsalar@khu.ac.ir
bEndocrinology and Metabolism Research Institute, Tehran University of Medical Sciences, Tehran, Iran. E-mail: miladrasouli@outlook.com
cDepartment of Physics, Kharazmi University, Tehran, Iran
dDepartment of Biochemistry, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

Received 23rd May 2024 , Accepted 19th August 2024

First published on 19th August 2024

Abstract

In this study, we employed Ajwain seed extract to synthesize oxalipalladium (OX) NPs and systematically investigated their physicochemical properties and biological activities. Characterization studies revealed that the OX NPs exhibited an average size of approximately 31.2 nm with a stable zeta potential of −26 mV, indicating colloidal stability conducive to drug delivery applications. We confirmed the homogeneous and spherical nature of the NPs, with FTIR spectra highlighting the presence of functional groups consistent with OX and Ajwain extract. Notably, OX NPs demonstrated potent anticancer activity against HCT116 colon cancer cells, inducing dose-dependent apoptosis. Compared to free oxaliplatin, OX NPs exhibited enhanced cytotoxicity. Flow cytometry analysis further elucidated the apoptotic pathway induced by OX NPs, confirming their efficacy as anticancer agents. Additionally, investigation into the molecular interactions between human serum albumin (HSA) and NPs revealed structural alterations in the protein upon interaction with the ligand. Analysis using three-dimensional fluorescence spectroscopy revealed alterations in the surroundings of Tyr and Trp residues, suggesting the influence of nanoparticles (NPs) on the protein's structural integrity. In a nutshell, our study contributes to the advancement of natural product-based synthesis procedures and therapeutic strategies for colorectal cancer treatment.

1. Introduction

Cancer denotes the unbridled proliferation of cells wherein apoptosis is hindered.1 Colorectal cancer stands as a leading cause of mortality worldwide,2 characterized by a multifaceted pathogenesis influenced by diverse factors.3 These factors encompass dietary and lifestyle elements as well as genetic predispositions.4 Typically, colorectal cancer initiates with the formation of a polyp, which subsequently progresses into a primary adenoma – a benign tumor measuring less than 1 cm in size.5,6 These adenomas may advance into larger lesions known as advanced adenomas, ultimately culminating in colorectal cancer.5,7

Numerous cancer treatments exist, each accompanied by its own set of limitations and notable side effects.8,9 Conventional chemotherapy drugs operate by swiftly eradicating dividing tumor cells.10 However, these drugs also impact healthy and normal cells in the body that exhibit a high rate of division, including bone marrow cells, macrophages, gastrointestinal epithelial cells, and hair follicle cells, disrupting their normal functioning.10–13 The primary challenge with chemotherapy drugs lies in their inability to discern cancer cells from normal cells,13–15 thereby precipitating numerous side effects. Moreover, to mitigate these adverse effects, drug dosages are often reduced, potentially resulting in treatment delays or cessation.10

The existence of P-glycoprotein (P-gp), a prominent multidrug-resistant protein, impedes the penetration and accumulation of chemotherapy drugs within cancer cells.16 Notably, the expression of this glycoprotein is elevated on the surface of cancer cells,16 where it serves as a drug export pump, fostering resistance to anticancer medications in tumor cells.16,17 Given the challenges outlined above, nanotechnology has sparked a significant shift in the targeted identification of cancer cells and tissues.18 NPs are engineered to target cells through diverse modifications, including alterations in size, shape, and physical as well as chemical properties.14,19–21 They possess the ability to pinpoint cancer cells through active or passive targeting mechanisms.8,21

Nanotechnology represents one of the most dynamic realms of contemporary scientific research,18 exerting a profound influence across diverse domains of human endeavor.22 A pivotal pursuit within nanotechnology involves the development of economical and ecologically sound methodologies for fabricating metal nanoparticles (NPs) endowed with distinctive attributes.23 Various approaches exist for synthesizing NPs, encompassing physical, chemical, biological, and hybrid techniques.23,24 However, physical and chemical methods entail the use of high-energy radiation and stabilizing agents that pose risks to human health.25 Consequently, biosynthesis emerges as a singular, low-energy, one-step degradation method for crafting biocompatible and non-hazardous NPs.26 Over the past decade, significant attention has been directed toward NP biosynthesis employing plant extracts as an efficacious, straightforward, and fitting approach.19 Prior investigations have underscored the pivotal role of plant metabolites – comprising sugars, terpenoids, polyphenols, alkaloids, phenolic acids, and proteins – in mediating the biodegradation of metal ions and their subsequent conversion into NPs, along with stabilizing the resultant NPs.27

Ajwain (AJW), or Carum capticum, belongs to the Apiaceous family, which grows in the eastern regions of India, Iran, and Egypt.28,29 This plant has many medicinal properties that can be used to treat abdominal tumors, gastrointestinal disorders, asthma, colds, and sore throats.30,31 It is also very valuable as an antifungal, bacterial, and body resistance enhancer.31 The active ingredients in AJW include the six main chemical compounds: thymol (49%), γ-terpinene (30.8%), p-cymene (15.7%), β-pinene (2.1%), myrcene (0.8%), and limonene (0.7%). Thymol is a monoterpene compound that gives the plant seeds an aromatic aroma.28,32 Thymol, a monoterpene compound prevalent in AJW seeds, imparts the characteristic aromatic aroma to the plant. Furthermore, it exerts a concentration-dependent inhibition on the peroxidation of liposome phospholipids.32

Human serum albumin (HSA) is the most abundant protein in human plasma and has a high binding capacity for various endogenous and exogenous substances, including drugs. By binding to HSA, NPs can leverage the natural transport mechanisms of HSA, enhancing the delivery and stability of therapeutic agents in the bloodstream.33,34 The interaction with HSA can prolong the circulation time of NPs, increasing the likelihood of reaching the target site and enhancing bioavailability and therapeutic efficacy. Furthermore, HSA has a natural affinity for tumor tissues due to the overexpression of albumin-binding proteins such as gp60 and SPARC in many tumors, which can be exploited to achieve targeted delivery of NPs to tumor sites. This property enhances the concentration of the therapeutic agent at the target site while minimizing systemic side effects. Additionally, the interaction between NPs and HSA can facilitate a controlled release of the drug, reducing peak plasma concentrations and minimizing potential side effects.33–35 A previous study has developed multitargeted palladium (Pd) agents that bind to HSA, exhibiting significant cytotoxicity and multimodal action against tumor growth.36 Furthermore, studies have shown that nanoparticles can efficiently target tumor cells, improve drug solubility and circulation time, and reduce toxicity.37,38 These findings provide a foundation for our study on OX NPs synthesized using Ajwain seed extract and underscore the importance of HSA interactions in optimizing drug delivery, aiming to overcome drug resistance and enhance anticancer activity.

The anticancer potential of AJW and its bioactive compounds has been explored across various types of cancer, encompassing both cancer cell lines and animal models. This study aims to propose a natural, low-risk, and efficacious treatment approach for targeting colorectal cancer cells. To this end, the aqueous extract of AJW was initially prepared, followed by the synthesis of OX NPs via green chemistry methodology utilizing AJW extract. Subsequently, the physicochemical characteristics of the synthesized NPs were assessed. Finally, the cytotoxic effects of the synthesized NPs were evaluated and compared with those of AJW extract on the colorectal cancer cell line HCT116.

2. Materials

The HCT116 cell line was obtained from the Pasteur Institute in Tehran. Cells for cell culture were procured from Gibco, while flasks and microplates were purchased from Greiner. Other materials were sourced from Sigma-Aldrich unless specified otherwise.

3. Methods

3.1. Synthesis of OX NPs

3.1.1. Preparation of AJW seed extract. To initiate the green chemistry method for NP preparation, AJW plant extraction was the initial step. A total of 50 grams of AJW seeds were ground.39 The crushed seeds were then combined with 150 cc of deionized water and incubated for 6 h at 55 °C with agitation at 70 rpm in an incubator shaker.39 The solution obtained was filtered using Whatman filter paper, and the resulting extract was stored at −20 °C for future experiments.

Subsequently, an OX solution was prepared at a concentration of 2 mM. Next, 1 mL of the solution was added to 5 mL of the AJW seed extract, maintaining a ratio of 1[thin space (1/6-em)]:[thin space (1/6-em)]5. To reduce palladium ions, the solution was placed in an incubator shaker for 24 h at a temperature of 50 °C with agitation at 200 rpm in the dark.40 After incubation, the solution underwent centrifugation at 30[thin space (1/6-em)]000 rpm for 30 min. The resulting precipitate was dissolved in 1 cc of distilled water and then subjected to freezing for 24 h to facilitate drying.

3.2. Characterization of synthesized NPs

3.2.1. Measurement of NP size and surface charge. The size and zeta potential (ZP) of the NPs were determined using the DLS technique provided by Brookhaven Instruments Corporation. DLS serves as a suitable, rapid, and precise method for measuring particle size within the sub-micron range.41 In this technique, particle size is assessed based on Brownian motion. To conduct the measurement, the NPs were dissolved in 1 cc of deionized water. To ensure the uniformity of the NP solutions, the sample was vortexed for 10 seconds and subsequently subjected to ultrasonication for 10 min. The test was carried out at a temperature of 25 °C.
3.2.2. SEM study. Scanning electron microscopy (SEM) was utilized to observe the ultimate shape of the NPs. In this investigation, around 50 μL of the sample solution containing NPs was deposited onto a platinum pan, and the samples were scrutinized on these plates subsequent to drying using an SEM (model: Leo 440i UK, Britain).41,42
3.2.3. AFM study. Atomic force microscopy (AFM), conducted using Veeco Instruments, was utilized to ascertain the morphological characteristics and topography of the NP surface. The NPs were dispersed onto the lamel surface and allowed to dry. To achieve this, the device's linear scanning rate was configured to 2 Hz. The measurements were conducted in non-contact mode. The obtained results were analyzed using IP Image Analysis 2.1 software.42
3.2.4. FTIR spectroscopy. A Fourier transform infrared spectroscopy instrument (FTIR, Avatar 370, Thermo Nicolet, USA) was used to examine the functional groups existing in OX NPs, OX, and Ajwain seed powder. The infrared spectrum was recorded within the 400–4000 cm−1 range with a resolution of 4 cm−1.43

3.3. Cell line experiment

3.3.1. Cell culture. The HCT116 cells were cultured in T25 flasks filled with high-glucose Dulbecco's Modified Medium (DMEM) culture medium, supplemented with 10% FBS (fetal bovine serum) and 1% penicillin–streptomycin, within an incubator set at 37 °C with 5% CO2. The medium was refreshed every two days to maintain cell viability. Upon reaching 80% confluence within the flask, the cells were subcultured.
3.3.2. Treatment of HCT116 cells with drug groups. HCT116 cells were seeded into 96-well plates at a density of 5000 viable cells per well. To facilitate cell attachment to the bottom of the wells, they were incubated for one day under the aforementioned conditions. Subsequently, OX NPs, OX, AJW extract, and oxaliplatin at concentrations ranging from 0 to 150 mM, along with phosphate-buffered saline (PBS) as a control, were added to the wells. The plates were then incubated for 24 and 48 h. To ensure the reliability and consistency of the results, each concentration was tested in triplicate.
3.3.3. MTT assay. The MTT colorimetric method was employed to assess the impact of OX NPs on cancer cell growth and proliferation. In summary, 10 μL of MTT solution at a concentration of 0.5 mg mL−1 was added to each well containing the cells. Subsequently, the cells were incubated on a shaker for four h at 37 °C, shielded from light. Following incubation, 150 μL of dimethyl sulfoxide (DMSO) was introduced to each well and gently shaken for 10 min to ensure complete dissolution. The optical density of the solution in each well was then measured using an ELISA reader at a wavelength of 570 nm. Under these conditions, the absorption or optical density (OD) correlates with the amount of MTT converted to formazan. Ultimately, the cell survival rate was calculated using eqn (1):44
 
image file: d4nj02391a-t1.tif(1)
where Abs sample denotes the absorption of cells incubated with the NP suspension, and Abs control signifies the absorption of cells solely incubated with culture medium. The IC50 value was then calculated, representing the drug concentration at which 50% cell growth inhibition was observed compared to the control sample. The MTT assay was performed at both 24 and 48 hours to capture the time-dependent cytotoxic effects of the treatments.
3.3.4. Annexin V assay. For this experiment, the cells (the treatment group) were treated with OX NPs at the IC50 concentration for 24 h, while one group served as the control without treatment. Free oxaliplatin was used as a positive control. Following treatment, the cells were detached using trypsin and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cell pellet was washed with PBS, followed by two washes with binding buffer (150 mM NaCl, 10 mM HEPES, 1.8 mM CaCl2, KCL mM, and 1 mM MgCl2).45

Following incubation in darkness with annexin V-FITV antibody (5 μL in 100 μL binding buffer) at 4 °C for 15 min, the cells were washed with binding buffer, and the supernatant was removed. Subsequently, 490 μL of binding buffer and 10 μL of propidium iodide (PI) (10 mg mL−1 in PBS) were added to the cell pellet. Finally, the percentage of apoptosis was determined using the FACS caliber cytometer (Becton Dickinson, San Diego, CA, USA) with Partec FloMax software (USA).45,46

3.5. Fluorescence measurements

The Carry model fluorescence spectrometer was utilized to examine changes in the intrinsic fluorescence intensity of HSA resulting from interaction with various concentrations of NPs. Protein excitation was conducted at 295 nm, and emission spectra were recorded for all samples in the absence and presence of different NP concentrations (ranging from 0 to 9.5 μMs) across the range of 300–500 nm. The experiments were conducted at temperatures of 25 and 37 °C.

3.6. Three-dimensional fluorescence studies

A JASCO spectrophotometer (FP 6200) was employed to acquire three-dimensional (3D) fluorescence spectra. The experimental configuration included recording the excitation wavelength ranging from 220 to 350 nm and the emission spectrum from 220 to 500 nm. Following this, 30 μL of NPs at a final concentration of 5.82 μM were added to 1 mL of HSA (21 mM).

4. Results and discussion

4.1. DLS and ZP results

Particle size and surface charge were determined using DLS at 25 °C. The results indicate that the average size of NPs containing OX is less than 100 nm. Although the size of colorectal cancer cells ranges between 400 and 600 nm, NPs need to be smaller than 400 nm to effectively serve as drug delivery carriers. This characteristic facilitates their preferential accumulation and penetration into tumors, enhancing cell viability and minimizing leakage. The obtained NPs exhibit an average size of 31.2 nm, which aligns with the optimal size for targeted drug delivery. Additionally, the polydispersity of NPs is 0.323, indicating a uniform and homogeneous distribution of NPs (Fig. S1, ESI†).

Another crucial aspect to consider in selecting NPs for drug delivery to the target site is the ZP of the NPs. ZP serves as a vital parameter determining the physical properties of NP formation, which is crucial for drug delivery to the chosen target site.35 The results of the ZP measurement indicate that the NP possesses a ZP of −26 mV. As depicted in the observations (Fig. S2, ESI†), the NP demonstrates exceptional stability and acceptable stability levels. Higher particle loads result in increased repulsive forces between the particles, thereby enhancing the stability of the suspension.

4.2. FTIR spectroscopic analysis

The structural properties of the synthesized NPs were investigated using FTIR spectroscopy to elucidate the relationship between their structure and performance. This spectral range provides insights into biomolecules with diverse functionalities within the system.47 In the current study, FTIR spectra of oxaliplatinum (OX) solution, aqueous extract of AJW, and synthesized OX NPs were recorded. As depicted in Fig. S3 (ESI†), the absorption peaks within the range of 475.38 cm−1 to 3424.95 cm−1 correspond to functional groups such as alkanes, free hydroxyl groups, alcohols, phenols, alkyl halides, and esters. These findings are consistent with the molecular structure of OX.

Additionally, as depicted in Fig. S3 (ESI†), the absorption spectra of the synthesized OX NPs exhibit peaks at 1567.73 cm−1, indicative of type 1 amine functional groups, 804.54 cm−1 associated with aromatics, and 1005.01 cm−1 corresponding to alkenes. In Fig. S3 (ESI†), the absorption spectrum of AJW extract reveals peaks at 1745.96 cm−1, 1459.06 cm−1, 11[thin space (1/6-em)]646.62 cm−1, and 718.98 cm−1, representing the presence of ester functional groups, alkanes, type 1 amines, and aromatics, respectively. Consequently, the spectra related to the type 1 amine group and aromatics in the OX NPs, absent in the OX solution, indicate that the AJW extract encompasses the OX NPs.39,48

4.3. FE-SEM and AFM analysis

Morphological changes and particle size distribution can be assessed through the utilization of FE-SEM images. The FE-SEM images depict OX NPs with predominantly homogeneous and spherical (colloidal) structures (Fig. 1a). The particle size measures approximately 35 nm, aligning well with the NP sizing results obtained using DLS.
image file: d4nj02391a-f1.tif
Fig. 1 (a) FESEM images of OX NPs and their particle size distributions; (b) AFM two-dimensional images of OX NPs; (c) AFM topographic three-dimensional image of OX NPs.

AFM analysis was conducted to evaluate the morphological characteristics, two-dimensional images, and topography of the NP surface, as illustrated in Fig. 1b and c.49 As depicted in the images, the synthesized NPs exhibit a spherical, homogeneous, and stable structure, with a size determined to be approximately 20 nm. These findings align with the results obtained from SEM and DLS.

4.4. MTT analysis

To assess the cytotoxicity of NPs synthesized via the green chemistry method and compare them with OX without encapsulation (a free drug), AJW extract, and oxaliplatin, an MTT assay was conducted to measure the growth and proliferation of HCT116 cells. The IC50 values, representing the concentrations of the aforementioned compounds that induce 50% mortality in cells, were calculated as presented in Table 1. The MTT assay was performed at both 24 and 48 hours to capture the time-dependent cytotoxic effects of the treatments. The choice of a 48 h incubation period for OX NPs was based on the need to observe the extended cytotoxic potential of nanoparticles, as some effects may manifest more clearly over longer periods. Additionally, this extended incubation period allowed for a more comprehensive comparison with free oxaliplatin, as nanoparticle formulations can exhibit enhanced and sustained cytotoxic effects.
Table 1 IC50 values of compounds in HCT116 cell lines
Materials IC50 at 24 h IC50 at 48 h
OXPd 600 ± 12 μM —
OXPd NPs 8.4 ± 0.1 μM 6.9 ± 0.36 μM
OXPd 1100 ± 25 μM —
Extract 166.5 ± 14 g L−1 —

As depicted in Fig. 2a–d, OX NPs elicit cell death in this cell line in a dose- and time-dependent manner. Elevating the concentration of the drug complex notably diminishes cell growth. Likewise, the growth inhibition curves in HCT116 cells for the mentioned three scenarios also demonstrate inhibition dependent on concentration.


image file: d4nj02391a-f2.tif
Fig. 2 MTT test results after treatment of HCT116 cells with (a) OX NPs after 24 (●) and 48 (○) h of incubation; (b) OX solution (free drug) after 24 h of incubation; (c) oxaliplatin solution after 24 h of incubation; (d) with AJW extract after 24 h of incubation.

The IC50 results revealed that the encapsulated form of the drug exhibited a lethal effect at much lower doses compared to the free form of the drug. The IC50 value for the NPs synthesized via the green chemistry method after 24 h of incubation was calculated to be 8.4 μM. In contrast, the IC50 value for oxaliplatin in its free form was 1100 μM, indicating approximately 131 times higher cytotoxicity for the encapsulated NPs. Essentially, with a concentration approximately 131 times lower than the synthetic NPs, the encapsulated NPs have the capability to induce cell death in this particular category of cancer cells.

4.5. Annexin V analysis

Typically, phosphatidylserine resides in the inner layer of the membrane within living cells. One of the events occurring in apoptosis involves the movement of phosphatidylserine from the inner layer of the cell membrane to its surface. This translocation can be easily detected by staining with the fluorescent marker fluorescein isothiocyanate (FITC).50,51

Quantitative analysis was conducted using flow cytometry, which divides cells into four quadrants. Q1 represents necrotic cells (annexin V−/PI+), Q2 indicates delayed apoptosis (annexin V+/PI+), Q3 represents primary apoptosis (annexin V+/PI−), and Q4 denotes live cells (annexin V−/PI−) (Fig. 3).51 As depicted in Fig. 3a, in the control sample, the majority of cells are alive and exhibit no migration to other areas. Conversely, in the sample treated with OX NPs (Fig. 3b), some cells displayed migration to primary apoptosis, secondary apoptosis, and necrosis due to induction of cell death. Therefore, the aforementioned results indicate that the type of cell death induced by NPs in HCT116 colon cancer cells is apoptotic.


image file: d4nj02391a-f3.tif
Fig. 3 Flow cytometry results. (a) Control sample (untreated cells); (b) sample treated with oxali-palladium nanoparticles after 24 h of incubation.

4.6. Fluorescence quenching results

Fluorescence quenching measurement is a potent spectroscopic technique used to explore the structure, dynamics, protein–ligand interactions, and interaction properties of protein molecules in solution.52 The intrinsic fluorescence of proteins arises from the presence of amino acids such as tryptophan, phenylalanine, and tyrosine in their structure. According to previous crystallographic studies, HSA contains a tryptophan residue located at position 214 along the polypeptide chain.53,54

Fig. 4a and b illustrate the changes in the fluorescence emission spectrum of HSA in the presence of different concentrations of NPs at 25 °C and 37 °C. The intrinsic fluorescence intensity of HSA diminishes progressively with increasing concentrations of NPs at both ambient and physiological temperatures, suggesting a clear interaction between the NPs and HSA. Additionally, Fig. 5 illustrates that as the concentration of NPs increases, a significant decrease in maximum protein fluorescence emission is observed at both temperatures under study. Finally, at higher concentrations of the NPs, the extent of this reduction diminishes and reaches a saturation state.


image file: d4nj02391a-f4.tif
Fig. 4 Fluorescence emission spectra of HSA in the absence and presence of different concentrations of NPs at (a) 25 °C and (b) 37 °C.

image file: d4nj02391a-f5.tif
Fig. 5 Changes in maximum fluorescence intensity of HSA with different concentrations of NP complex at different temperatures of 25 (●) and 37 °C (○).

The protein fluorescence quenching mechanism can occur as dynamic, static, or simultaneous static and dynamic quenching.52 Both quenching mechanisms depend on temperature and viscosity. Dynamic quenching occurs when a collision happens between the fluorophore and the quencher, whereas static quenching involves the formation of a complex between the fluorophore and the quencher.52,55 To determine the quenching mechanism, the Stern–Volmer equation (eqn (2)) was utilized:

 
F0/F = 1 + Ksv[Q](2)
where F0 and F represent the fluorescence intensity in the absence and presence of the NPs (quencher), respectively. [Q] denotes the total quencher concentration, and Ksv signifies the Stern–Volmer dynamic quenching constant.

The Stern–Volmer curve used to analyze the protein fluorescence quenching mechanism is depicted in Fig. 6a. The Stern–Volmer curve appears non-linear, suggesting that the quenching values under study may result from a combination of static and dynamic quenching. Therefore, the modified Stern–Volmer equation was employed to ascertain the type of quenching mechanism and the quenching constant of the protein's interaction with NPs at both temperatures (eqn (3)).56

 
F0/ΔF = F0/F0 − F = 1/faKsv × 1/[Q] +1/fa(3)
where fa is the fraction of initial fluorescence accessible to the quencher. The data obtained based on eqn (3) are presented in Table 2. As depicted in Fig. 6b, the values of fa and Ksv can be calculated from the X-intercept and slope values of this graph, respectively.52 According to the analysis of the Stern–Volmer curve and Table 2, the quenching constant value increases with an increase in temperature. This indicates that the mechanism of interaction between the protein and the NPs involves more than just dynamic quenching.


image file: d4nj02391a-f6.tif
Fig. 6 (a) The Stern–Volmer plots of the interaction between NPs and HSA at two temperatures of 25 °C (●) and 37 °C (○); (b) changes in F0/(F0 − F) against 1/[Q] according to eqn (2) at different temperatures of 25 °C (●) and 37 °C (○); the log F0 − F/F against log[NPs] at different temperatures of 25 (●) and 37 °C (○).
Table 2 Binding parameters of the interaction of HSA with NPs at 25 and 37 °C temperatures
Temperature (°C) K SV × 103 (M−1) n K a × 103 (M−1) ΔG° (kJ mol−1) ΔH° (kJ mol−1) ΔS° (J mol−1 K−1)
25 196 ± 10.6 0.50 ± 0.09 0.199 ± 0.13 −13 ± 1.1 −9.73 ± 2.7 11.4 ± 0.18
37 620 ± 14.2 0.44 ± 0.11 0.0947 ± 0.07 −11 ± 0.9

4.7. Binding and thermodynamic parameters analysis

Based on the results of fluorescence quenching, eqn (4) was utilized to determine the binding constant parameters (K) and the number of binding sites (n) of NPs with the protein.55
 
log(F0 − F)/F = log[thin space (1/6-em)]K + n[thin space (1/6-em)]log[Q](4)
where K is the binding constant and n is the number of binding sites on the protein for the NPs. The plots of log(F0 – F)/F versus log[Q] exhibit linearity (Fig. 6c). Furthermore, based on the slope and X-intercepts in Fig. 6c, the values of binding sites (n) and NP binding constants (Table 2) can be calculated at both 25 and 37 °C.

Generally, the forces that act between small molecules and macromolecules include electrostatic forces, van der Waals forces, hydrophobic interactions, and hydrogen bond formation.52,57 To determine the type of interaction between NPs and HSA, thermodynamic parameters such as enthalpy (ΔH°) and entropy (ΔS°) are utilized. For this purpose, the vaEn't Hoff equation was employed (eqn (5)):

 
ln[thin space (1/6-em)]K = −ΔH°/RT + ΔS°/R(5)
where R represents the gas constant, T denotes the absolute temperature, and K signifies the binding constant for the interaction of HSA with the NPs.

In addition, the free energy change (ΔG°) is determined from the following equation (eqn (6)):

 
ΔG° = −RT[thin space (1/6-em)]ln[thin space (1/6-em)]K = ΔH° – TΔS°(6)

Thermodynamic parameters for protein–NP binding are summarized in Table 2. A negative ΔG° indicates that protein interactions occur spontaneously.

Previous studies indicate that interactions between ligands and proteins are categorized into three groups based on thermodynamic parameters: if ΔS° > 0 and ΔH° > 0, the primary force is hydrophobic forces; if ΔS° < 0 and ΔH° < 0, hydrogen bonds and van der Waals forces are the main forces; and finally, if ΔS° > 0 and ΔH° < 0, the primary force is electrostatic interaction.52,58–60 Thermodynamic parameters for protein–drug binding are summarized in Table 2. A negative ΔG° indicates that protein interactions occur spontaneously.

4.8. Analysis of three-dimensional fluorescence spectra

To determine the structural changes of the protein during interaction with the ligand, a 3D fluorescence spectroscopy technique was employed. The contour maps of free HSA and HSA-NPs are depicted in Fig. 7 and Table 3. There is a distinct region in the contour map associated with free HSA (Fig. 7a). In this diagram, peak 1 corresponds to an excitation wavelength of around 280 nm and an emission wavelength of about 340 nm, commonly linked to Trp and Tyr residues.61 The findings from the contour maps reveal a decrease in the fluorescence intensity of peak 1 following the introduction of NPs. This reduction in peak 1 implies alterations in the microenvironment surrounding Tyr and Trp residues and aromatic amino acids due to the protein's interaction with NPs. Additionally, the emergence of peak 2, attributed to the presence of aromatic amino acids and the uniform characteristics of the polypeptide backbone structures in the protein-free state, suggests a modification in the protein's backbone structure upon adding NPs. The interaction between NPs and HSA can facilitate a controlled drug release, reducing peak plasma concentrations and minimizing potential side effects. Understanding the structural changes in HSA upon binding to NPs provides insights into the binding affinity and release profile of the drug, which are critical for optimizing therapeutic efficacy.33–35 Our findings indicate that the interaction between OX NPs and HSA leads to structural alterations in the protein, as evidenced by fluorescence quenching and three-dimensional fluorescence spectroscopy. These alterations in the microenvironment surrounding Tyr and Trp residues suggest a significant impact of NPs on the protein structure, which may influence the binding and release properties of the drug. The interaction between OX NPs and HSA enhances drug delivery and therapeutic efficacy. Previous literature on HSA–platinum(II) agent nanoparticles highlights the role of HSA in optimizing therapeutic behavior and inhibiting tumor growth through multimodal action.62 Our findings align with these studies, suggesting that OX NPs can similarly leverage HSA interactions to enhance their anticancer activity and overcome multidrug resistance. Our study has several limitations. First, although OX NPs demonstrated enhanced cytotoxicity against cancer cells, we did not evaluate their toxicity on normal cells. Future studies should include cytotoxicity assays on normal cell lines, such as fibroblasts or epithelial cells, to assess the selectivity and safety profile of the nanoparticles. Second, while we explored the interaction between OX NPs and HSA, further mechanistic studies are needed to fully understand the pathways involved in nanoparticle uptake, intracellular trafficking, and apoptosis induction. Third, the stability studies conducted in this research were limited to a few months. Long-term stability assessments under various storage conditions are necessary to ensure the practical applicability and shelf life of OX NPs. Fourth, although we employed several characterization techniques, additional methods such as XRD and TGA could provide further insights into the nanoparticles' crystalline structure and thermal stability.
image file: d4nj02391a-f7.tif
Fig. 7 Contour diagrams of three-dimensional fluorescence spectra of HSA free (21 μM) (a) HSA + NPs (NP concentration = 150.7 μM) (b), T = 25 °C, pH = 7.4.
Table 3 Three-dimensional fluorescence spectral characteristics of HSA free, HSA + NPs. T = 25 °C, pH 7.4
System Peak 1 Peak 2
λ ex/λem Δλ Intensity λ ex/λem Δλ Intensity
HSA free 280/350 70 673 —/— — —
HSA + NPs 280/345 65 58 340/460 120 32

5. Conclusions

Using Ajwain seed extract, we successfully synthesized Oxalipalladium (OX) NPs and thoroughly investigated their physicochemical properties and biological activities. Our findings reveal that the synthesized OX NPs exhibit favorable characteristics for drug delivery, with an average size of approximately 31.2 nm, facilitating effective permeability and cellular uptake. Moreover, the stable ZP of −26 mV ensures the colloidal stability of the NPs, which is crucial for their biomedical applications. The anticancer activity of OX NPs against HCT116 colon cancer cells was particularly noteworthy. Our results demonstrate that OX NPs induce apoptosis in a dose- and time-dependent manner, outperforming free oxaliplatin in terms of cytotoxicity. The enhanced cytotoxicity of OX NPs can be attributed to their ability to overcome multidrug resistance mechanisms, potentially offering a promising strategy for combating colorectal cancer. Furthermore, our study sheds light on the molecular interactions between HSA and NPs, elucidating structural changes in the protein upon interaction with the ligand. Through 3D fluorescence spectroscopy, we observed alterations in the microenvironment surrounding Tyr and Trp residues, indicating a significant impact of NPs on the protein structure. Further research in this direction holds promise for addressing key challenges in transferring the nanoplatforms for potential clinical applications.

Data availability

The data supporting the findings of the study can be obtained from the corresponding author upon reasonable request.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

The authors thank the Research Council of Kharazmi University for their financial support.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4nj02391a
‡ These authors equally contributed to the study.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2024