Newly isolated sporopollenin microcages from Cedrus libani and Pinus nigra as carrier for Oxaliplatin; xCELLigence RTCA-based release assay

Sporopollenin-mediated control drug delivery has been studied extensively owing to its desirable physicochemical and biological properties. Herein, sporopollenin was successfully extracted from C. libani and P. nigra pollens followed by loading of a commonly known anticancer drug Oxaliplatin. Drug loading and physicochemical features were confirmed by using light microscopy, FT-IR, SEM and TGA. For the first-time, real-time cell analyzer system xCELLigence was employed to record the Oxaliplatin loaded sporopollenin-mediated cell death (CaCo-2 and Vero cells) in real time. Both the release assays confirmed the slow release of oxaliplatin from sporopollenin for around 40–45 h. The expression of MYC and FOXO-3 genes has been significantly increased in CaCo2 cell and decreased non-cancerous Vero cell confirming the fact that sporopollenin-mediated control release of oxaliplatin is promoting apoptosis cell death preventing the spread of negative effects on nearby healthy cells. All the results suggested that C. libani and P. nigra can be suitable candidates for the slow delivery of drugs.


Introduction
Designing and production of drug carrier systems for slow and targeted release to prevent the premature burst release of the content in the bloodstream represents an ever-evolving research and application area in biomedical science [1,14,40,47]. In recent years with the advancements in nanotechnology, huge efforts have been made to design carrier systems exhibiting the desired structural and chemical properties [10,18,19]. Nano/micro-vehicles tested so far for the controlled release of anticancer drugs include liposomes, water-soluble polymers, dendrimers, vesicles [41], polymeric nanoparticles [16,20,21,34], and some inorganic materials [5,15,17,35]. All the tested drug carriers had their pros and cons. For example, the encapsulation capacity of liposomes and nanoparticles has been reported around 10% [31,38,48]. Besides, obtaining the nanocarriers with desired geometry and functional properties requires expensive processes in large time scales [7]. In this regard, the quest has been underway for producing alternative macro/nano/microcarriers which offer several properties such as non-toxic, non-reactive with load, economical, and biodegradable. In nature, a variety of biomaterials are already present which have the application capabilities in different fields. These natural materials have been evolved in much longer time scales, thus ensuring the fidelity and physicochemical properties of such materials. Among many examples, polen comes with excellent features such as excellent elasticity, size uniformity, homogeneity in pore sizes, physical and chemically resistance, UV shielding ability, and antioxidant ability [11,28].
Since the last decade, researchers have actively focused on the development and modification of plant pollen as a delivery vehicle for many active ingredients such as drugs [12,24,43], active compounds [27]. Considering the up-to-date research related to the utilization of sporopollenin for the controlled delivery of anticancer drugs, researchers are conducting the in vitro release assays using different buffer solutions to simulate the intestine and stomach environment and to investigate the release effect of the drug from carrier [3,13,29]. However, the release assays have not been conducted using novel real-time cell analyzer tools such as xCELLigence to get a more accurate perspective of the controlled release in cancer cells. To bridge this gap in the literature, besides the conventional release assay (simulated pH systems), the current study was mainly designed to get an inclusive insight of the sporopollenin-based release in cancer cells by using a recently developed tool known as a real-time cell analyzer. Most of the anticancer drugs exhibit problems like poor water solubility, rapid blood clearance, low tumor selectivity, and severe side effects for healthy tissues [31]. For this purpose, several delivery systems have been tested for the controlled release of anticancer drugs. As is known that in apoptosis the cell dismantled in rather a controlled way from inside minimizing the damage and disruption to the surrounding healthy cells. The resulting debris is then removed through phagocytes [36]. While in necrosis the abrupt dismantling of cells releases the debris into the surrounding healthy cells [4]. Considering these facts, it is fathomable that the slow release of the therapeutic drug may have a direct link with cell apoptosis. In the current study, two reference genes i.e., c-Myc and FOXO3 have been selected to assess the effect of sporopollenin-mediated slow release of Oxaliplatin on cell death by apoptosis. c-Myc gene plays a regulatory role in cell proliferation and cellular metabolism by inducing apoptosis [26]. Another gene we selected in this study, FOXO3, promoting apoptosis through the expression of genes responsible for cell death [9]. Thus, by determining the level of expression of both genes, we have determined the possible relationship of sporopollenin to apoptosis.
In the current study, the sporopollenin of C. libani and P. nigra were tested as a control release vehicle for a common anticancer drug known as Oxaliplatin. Pinus and Cedrus belong to Pinaceae family. Around, 80 species of the Pinus genus that distributed from the Northern hemisphere to the Tropics [32]. There are five naturally grown species in our country. 10-20 m tall, evergreen trees with 2-3 or 5 needle leaves coming out of the shoots [33]. Male cones are located at the bottom of small, yellow, young shoots, and female cones are located green, close to the tip. Pinus nigra pollen is monad, heteropolar and bilateral symmetry. The pollen shape is equilateral oblates, pollen type vesiculate, bisaccate. Cedrus libani pollens are monad, heteropolar and bilateral symmetry. The pollen shape is an equatorially appearance oblate, pollen type vesiculate, bisaccate [2].
To the best of our knowledge, no study has been reported on the C. libani and P. nigra sporopollenin production and its utilization as a controlled-drug vehicle for Oxaliplatin and the effect of this slow release on cell death. Besides the conventional release measurement assay (simulated pH systems), the first-time real-time cell analyzer model (xCELLigence) has been employed to determine the slow release from sporopollenin in real time. Here, the sporopollenin production and drug loading were confirmed by using analytical tools such as light microscopy, SEM, FT-IR, and TGA. The release assays were conducted both in simulated pH solutions and cell culture (Caco-2 and Vero) using a real-time cell analyzer (xCELLigence). The effects of sporopollenin-mediated slow release of oxaliplatin over apoptosis was investigated. The cell apoptosis was analyzed using two reference genes with main roles in cell apoptosis known as FOXO-3 and c-MYC.

Pollen collection
In the current study, the pollens of Cedrus libani and Pinus nigra were collected in the year 2015 from the garden of Education Faculty and Vocational School of Kastamonu University, respectively. Male cones collected with vintage scissors were placed in clean paper bags and brought to the laboratory. It was then spread over clean papers and covered with paper to prevent contamination and allowed to dry at room temperature for 3 days. Drying male cones were shaken to shed pollen. The obtained pollens were sieved (having a pore diameter of 10 µm) to be free from dust and other unwanted particles. To obtain pure pollens, the remaining pollen was again passed through a sieve having a pore diameter of 100 µm to remove large particles. The purified pollen samples were stored in sealed falcon tubes at − 20° for further experiments.

Sporopollenin isolation
To obtain sporopollenin, the obtained pollens were treated with acid, base, and chloroform/methanol/water solution for demineralization, deproteinization and depigmentation. Briefly, for demineralization, 10 g of C. libani and P. nigra pollen were treated with 4 M HCl solution at 50° C for 2 h. Then, the samples were filtered by a Whatman filter paper with a pore size of 110 µm following by an extensive wash with distilled water until neutral pH. For deproteinization, the samples were incubated with 4M NaOH solution at 150° C for 6 h. Then, the treated samples were filtered using Whatman filter paper and extensively washed with distilled water until neutral pH. The demineralization and deproteinization treatments were repeated 4 times to ensure the complete removal of genetic and cellulosic materials inside the pollen. The acid and base-treated pollen samples were then allowed at room temperature for 1 h in chloroform/methanol/water solution (4: 2: 1/v: v: v). Finally, the samples of sporopollenin were thoroughly washed with distilled water and dried in the oven at 60° C for 48 h [24].

TGA
All samples were heated from 30 to 650 °C with a temperature change of 10 °C/ min and their thermal stability, water, and ash content were determined. All the Polymer Bulletin (2022) 79:519-540 thermograms were recorded with an EXSTAR S11 7300 (USA) instrument under the nitrogen atmosphere.

SEM
The drug, pollen, and sporopollenin samples were placed on aluminum stab with double side adhesive type, and it was covered with gold by Cressington, Sputter Coater 108 Auto Au-Pd Coating Machine at Kastamonu University Central Research Laboratory, under 40 mA for 30 s. SEM photos were taken with FEI-Quanta FEG 250 model scanning electron microscope.

Light microscopy
The sporopollenin production and drug loading were also confirmed by analyzing the samples under a LEICA DFC425 C light microscope. The samples were analyzed in ambient conditions using glass slid.

Loading of Oxaliplatin to sporopollenin via passive loading technique
Oxaliplatin was loaded onto C. libani and P. nigra sporopollenin by the passive loading technique used in our previous study [29]. Briefly, in the passive loading technique, 100 mg of Oxaliplatin was dissolved in 4 mL of distilled water with the aid of sonification for 10 min. Then, 200 mg of C. libani and P. nigra sporopollenin were added to this solution separately and the suspension was vortexed for 15 min. Each sporopollenin-Oxaliplatin solution was then incubated at 350 rpm using a thermo-shaker for 4 h and at 4 °C. The samples were then filtered with filter paper having a pore size of 110 µm and washed twice with 3 mL of distilled water. The drug-loaded sporopollenin samples were incubated at − 80° C for 30 min. Finally, Oxaliplatin loaded sporopollenin were dried at room temperature for 24 h and then stored at − 18° C for further experiments.

Encapsulation efficiency
Quantitative determination of Oxaliplatin loaded on C. libani and P. nigra sporopollenin was carried out by following previously reported methods with minor modifications [25]. Oxaliplatin was dissolved in different concentrations i.e., 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL and 25 μg/mL to obtain the calibration curve. The absorbance of these solutions was measured at 255 nm using a UV-vis spectrophotometer. To evaluate the drug loading efficiency of both sporopollenin samples, 10 mg of Oxaliplatin loaded C. libani and P. nigra samples were vortexed for 10 min by adding 3 mL of PBS. Then, the samples were filtered through a Whatman filter paper with a pore size of 110 µm. The clear solution was collected and measured at 255 nm using UV-spectrophotometer. The following equations are used to calculate the loading efficiency:

In vitro release studies with Oxaliplatin loaded microcages
In vitro, drug release studies of Oxaliplatin loaded sporopollenin obtained using passive loading technique were performed using PBS (pH 7.4) buffer as simulated intestinal fluid. Briefly, 10 mg of drug-loaded sporopollenin were added separately to 5 mL of PBS buffer solution. Each solution was then transferred to dialysis bags and placed in 50 mL of PBS solution. The samples were then shaken in a water bath at 37 °C at 100 rpm for 120 h. Then, to calculate the amount of Oxaliplatin released from the sporopollenin contained in the dialysis bag into the PBS solution, 2 mL of PBS was drawn at 5th min, 15th min, 30th min, 1st, 2nd, 6th, 24th, 48th, 72nd, and 120th h time intervals. The same amount of fresh PBS buffer solution was added back to the medium for maintaining the volume. The amount of Oxaliplatin released into the medium was measured using a UV-VIS spectrophotometer at 255 nm. Besides, to control the release of free drug, 10 mg Oxaliplatin was added to the PBS solution and in vitro release studies were performed by following the above procedure.
The following equations were used to calculate the cumulative percent release of Oxaliplatin: Pt denotes the percentage release at time t, P (t − 1) is the percentage release previous to t.  (Lonza, Verviers, Belgium), at 37 °C in a humidified atmosphere containing 5% CO 2 . Vero, non-cancerous cell line, was cultured in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% Fetal bovine serum (Lonza, Verviers, Belgium), 1% penicillin/streptomycin (Gibco, Grand Island, NY, USA) in a 5% CO 2 humidified incubator at 37 °C.

Antiproliferative effect by sporopollenin-based control release (xCELLigence, RTCA)
The cell proliferation was also continuously monitored using the xCELLigence RTCA Instrument (xCELLigence RTCA, Roche, Germany) according to the manufacturer's instructions. CaCo-2 and Vero cells seeded at a density of 5 × 10 3 cells and 7 × 10 3 per well in 200 μl media containing 10% FBS in of 16 well E-plate (Roche Applied Science, Germany), respectively. After 24 h, drug-loaded sporopollenin of P. nigra, C. libani and pure Oxaliplatin (control) were added at different concentrations (at the range of 5-20 mg/ml). The E-Plates were continuously monitored on an RTCA system for 120 h at 37 °C with 5% CO 2 . The proliferation of examined cells was monitored every 30 min and a time-dependent cell index (CI) graph was produced by the device using the RTCA software program of the manufacturer. The baseline cell index for molecules treated cells compared to cancer and control cells was calculated for at least two measurements from three independent experiments.

Total RNA isolation and cDNA synthesis
The cells were plated at a density of 5 × 10 5 cells in a 6-well plate with a 2-mL culture medium. After keeping the drug-loaded C. libani and P. nigra sporopollenin for 24 h. in the cell culture of CaCo-2 and Vero, the cells were washed twice with PBS, and total RNA was isolated with Trizol Reagent. 1 µg RNA was reverse transcribed by oligo(dT) primers with Transcriptor High Fidelity cDNA Synthesis Kit (EUX, Germany) according to the manufacturer's instructions.

Quantitative real-time RT-PCR
Quantitative real-time RT-PCR analysis was performed using a Lightycycler 480 (Roche, Germany). Complementary DNA (10 ng), 2.1 μL of PCR-grade water, 5 μL of SYBR Green real-time PCR master mix, and 1.5 μL of primer pairs were mixed in a PCR tube. PCR was performed with an initial denaturation at 95 °C for 10 s, followed by amplification for 40 cycles, each cycle consisting of denaturation at 95 °C for 5 s, annealing at 65 °C for 60 s. The primers are shown in Table 1. The housekeeping gene, GAPDH, was used for normalization. The qRT-PCR experiments were repeated three times. The 2 −ΔΔCT method was used to calculate the fold change of mRNA expression level. To validate the size of amplified fragments, PCR products were separated by electrophoresis through 2.0% agarose gels and visualized with ethidium bromide.   pollens were recorded as the broad peaks between the 1100-1028 cm −1 for CD and PN.

FT-IR
Although the certain chemical structure of sporopollenin has not been explained until today, some research groups have been identified unsaturated fatty acids, olefinic, and aromatic functional groups found in the sporopollenin [6]. The authors determined that sporopollenin which consists of C, H, and O is an organic polymer and is nitrogen-free [8,44]. The chemical structure of sporopollenin can be different by species [44]. In the present study, the FT-IR spectra of CD-SP and PN-SP showed that the chemical structures of the extracted sporopollenin's are different from CD and PN. When compared the CD spectrum with the FT-IR spectrum of the CD-SP, the new peaks were observed at 1575.6 and 1538.9 cm −1 due to the sporopollenin polysaccharide structures. In the PN-SP spectrum, the O-H bonding peak was shifted to 3325.2 cm −1 and the intensity of the peak decreased. However, a sharp peak appeared at 1713.4 cm −1 due to the olefinic acid structures. In the CD-SP-OX spectrum, it was seen that the C-H stretching of CD-SP shifted to higher wavenumber due to the effect of the C-H stretching of drug molecules. N-H bending vibrations of Oxaliplatin was observed at 1603.6 cm −1 . Alkane C-H bending of OX and C-H bending of CD-SP were overlapped and intensified at 1378.3 cm −1 . In the PN-SP-OX spectrum, the O-H band was becoming broader due to the effect of the N-H bending of OX. The peaks in the region of 1245-1500 cm −1 were intensified because of the drug molecules. These results were showed that the drug was successfully loaded into CD-SP and PN-SP.

Scanning electron microscopy (SEM) and light microscopy analysis
SEM analysis was carried out to evaluate the pollen and sporopollenin structures and to better understand the drug loading mechanism. C. libani and P. nigra have monad pollens consisting of two pollen saccus which are connected to the main body called Corpus at the distal pole called Corpus. The main body of the pollen is surrounded by two wall layers, intin and exin. The pollen saccus is alveolar vesicles, which consist of exine (Figs. 2c, g and 3b, f). As is known that sporopollenin isolation consists of two steps, i.e., acid hydrolysis and base hydrolysis for the removal of internal proteins and pigment structures. The same procedure was followed for the production of CD-SP and PN-SP. SEM micrographs confirmed the successful isolation of sporopollenin. Compared to the untreated pollens, the porous surface becomes visible after the removal of organic compounds through acid and base treatments. SEM micrographs revealed that acid treatment leads to the structural disintegration of sporopollenin. Following the acid hydrolysis treatment, CD preserves its structural integrity to a large extent (85-90%), and transformed into sporopollenin capsules (cages) only with a small cleavage on the leptoma (Figs. 2d and 3g). In the case of PN, the acid hydrolysis resulted in a complete structural disintegration (90-95%) and form sporopollenin sheets like structures or plaques. Only a small percentage (5-10%) of the pollens retained their original shape (cages), leaving only a cleavage on the leptoma (Figs. 2h and 3c). Although the acid hydrolysis resulted in the structural deformation of PN-SP, the sporopollenin was porous which could be help trap or adsorb the Oxaliplatin particles on its surface (Fig. 2d, h). In the current study, a commonly known anticancer drug Oxaliplatin was applied in the form of a water solution. Considering the cumulative control release and drug encapsulation results showing, it can be assumed that the drug has been encapsulated into CD-SP by adsorption or direct penetration through porous walls (Figs. 2e, f and 3h). PN, which were highly decomposed into sporopollenin plaques, form big granules together with Oxaliplatin solution (Figs. 2i, j and 3d).
In the release tests, PN-SP-OX has initially released 35-40% of the drug while CD-SP-OX revealed a 15-20% release in the first 4 h. As is clear from SEM images that acid hydrolysis of CD retained its structural integrity to a large extent (85-90%) compared to PN. SEM images of CD-SP-OX have revealed very little superficial Oxaliplatin residues after the encapsulation. This confirmed that the loaded drug to CD-SP has been encapsulated inside or adsorbed on the surface of the cages. On the other hand, as evident from SEM micrographs, following the acid treatment, the pollens of P. nigra lost its structural integrity (90-95%) and form sporopollenin plaques. The drug loaded to these sporopollenin plaques was adsorbed on the surface and some drug particles remain on the surface and acted as adhesive for other plaques leading to the formation of granule like structures. The drug particles trapped between the sporopollenin plaques and those present superficially released quickly into the media followed by slow release from sporopollenin cages and adsorbed on the walls. These phenomena also explained the initial release in 4 h by CD-SP-OX and PN-SP-OX.

Thermogravimetric analysis (TGA)
TGA was carried out to evaluate the thermal stabilities of the pollens and drugloaded sporopollenin. The obtained thermograms of PN and CD, PN-SP, CD-SP, OX, PN-SP-OX, and CD-SP-OX are given in Fig. 4. The TG curves revealed that Oxaliplatin has been degraded at 270-330 °C (Maximum degradation 283 °C). Oxaliplatin has been reported to be thermally degraded at approximately 300° C in previous studies [45,49]. During this degradation, elements like C, H, O, N were completely degraded, leaving only platin residues. The metal residue for Oxaliplatin after TGA degradation was recorded as 46.74% (Fig. 4a). When the percent platin content of Oxaliplatin was calculated, it was recorded as approximately 49.10% and this value was close to the platin residue recorded during thermal degradation. Five different decomposition stages were recorded for the PN and PN-SP (Figs. 4b, 5a). The initial mass loss in the thermograms of all the tested samples is attributed to the evaporation of adsorbed structural water molecules [37]. The second, third, and fourth mass loss can be attributed to the degradation of pollen intine (structural materials such as cellulose, genetic material, lipid, etc.) [30]. Degradation of PN-SP-OX was recorded in four stages (Fig. 5c). The first decomposition up to 100° C can be attributed to the evaporation of the bound water, while the second decomposition, maximum degradation around 345° C is due to the degradation of the carbohydrates remaining in the sporopollenin structure after the removal of genetic material and pectin from pollen (P. nigra) structure. The higher ash content in Oxaliplatin loaded sporopollenin (24.3%) is due to the platin residue, a structural component of Oxaliplatin. The thermograms of CD revealed the thermal degradation in four steps (Fig. 4c). The first mass loss around 53.1 °C, can be ascribed to the evaporation of structural water molecules. The second and third mass losses have occurred at 250 and 380 °C displaying a big difference. This mass loss is probably caused by the degradation of pectin and cellulose [46]. The maximum thermal decomposition temperatures for pectin and cellulose are 262.8° C and 378.9° C, respectively. The sharp decomposition at 432.5 °C may be due to partial depolymerization and disintegration of the sporopollenin wall. For CD-SP, three separate degradations were observed (Fig. 5b). After the addition of Oxaliplatin (CD-SP-OX), the mass loss in the second degradation increased from 37.8 to 41.5% (Fig. 5d). The maximum degradation of sporopollenin was achieved at approximately 440 °C (Table 2). It is thought that the mass loss occurring around 400-650 °C is caused by the deterioration of the wall (exine) of sporopollenin. According to these results, sporopollenin has high thermal stability. Furthermore, it shows that the normal thermal properties of Oxaliplatin change after incorporation Oxaliplatin into sporopollenin samples and that Oxaliplatin has high decomposition temperatures. The higher the thermal decomposition temperature, the higher the thermal stability has been noted in previous studies [22,23,50]. Mujtaba et al. [24] reported that P. orientalis sporopollenin has higher thermal stability than biopolymers such as cellulose and chitin [24]. Compared to the results of the current study, it was observed that P. nigra and C. libani sporopollenin have high dTGmax temperature and this sporopollenin has more stable structures. In this study, the TGA results of P. nigra and C. libani species did not show a significant difference in terms of thermal stability.

Encapsulation efficiency
The encapsulation efficiency (%) of CD and PN was recorded as 10.06% and 38.62% respectively. An important difference was observed among the encapsulation % of the two pollens. Here in the current study, a passive drug loading technique was adopted for the Oxaliplatin encapsulation into CD and PN. The variance in the encapsulation percentages can be attributed to the structural modifications during the isolation. As discussed in detail in the SEM section, acid treatment of PN leads to major structural disintegration resulting in sporopollenin plaques formation providing a larger surface area for the drug to interact and penetrate. Due to the enhanced surface area, a large amount (38.63%) of Oxaliplatin has been adsorbed into the sporopollenin porous surface. On the other hand, the CD preserved its structural integrity up to a large extent during the isolation, thus resulting in sporopollenin cages. As passive loading techniques do not involve any active external forces [25] for loading the drug inside the sporopollenin cages, the loading efficiency of CD was recorded lower as compared to PN. It is known that the morphologies of pollen grains vary according to the species. The sporopollenen, generally has a large internal space, pore, reticulated or flat surface, uniform size and is highly biocompatible. The uniformity in size is specific specie property and shows significant variation from specie to specie. Different surface morphologies may also vary in different usage areas of sporopollens. In a study, it has been shown that P. orientalis sporopollenen has a porous and reticulated surface and this surface has a good drug encapsulation ability. In addition, it was determined that not only the surface but also the drug loading technique had an effect on the encapsulation efficiency [24]. The sporopollenin of Phoenix dactylifera L. sporopolenes have also been reported to increase the encapsulation efficiency due to macroporous surface morphology [3]. In the same regard in current study, sporopollenin from different species have different morphologies in terms of pore size, number of pores and overall size. These difference size and pores significantly effected the encapsulation and release of encapsulated drug.

Drug release measured in simulated pH solutions
In vitro release studies of the Oxaliplatin (OX) and Oxaliplatin loaded P. nigra (PN-SP-OX) and C. libani sporopollenin (CD-SP-OX) were performed in PBS (pH 7.4) and results are presented in Fig. 6. The release profile of the OX revealed an initial fast release during the first 4 h and then dramatically decreased. Overall drug-loaded sporopollenin has shown a slower release rate than the pure drug. The reason behind this slow release can be due to the successful entrapment of the drug inside of the sporopollenin microcapsules and sporopollenin plaques of P. nigra. Oxaliplatin was completely dissolute in PBS for 4 h, whereas in the same time frame, the cumulative releases of Oxaliplatin from CD-SP-OX and PN-SP-OX were recorded as 15.72% and 28.97% respectively. This fluctuation in the initial release values is attributed to the structural changes (sporopollenin plaques formation) that occurred during the P. nigra sporopollenin extraction process (Better explained in SEM section). The

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results were also found in agreement with the cell studies conducted for checking the release effect in real time using a real-time cell analyzer (xCELLigence). Also, chitosan nanoparticles [42] and immune-hybrid nanoparticles [39] were used as drug delivery systems for Oxaliplatin in the literature. The Oxaliplatin release from chitosan nanoparticles was found to be under 20% for the first 8 h in the Vivek et al. study [42]. Tummala et al. [39] reported that the Oxaliplatin release from immunehybrid nanoparticles showed a biphasic pattern of drug release in pH = 7.4. The authors explained that the cause of the initial burst release was unentrapped drug molecules. However, the initial burst release did not observe in the present study. The reason for this result can be due to the entrapment of the drug molecules into the sporopollenin cages and plaques. Consequently, the results of the present in vitro release study demonstrated that the PN-SP and CD-SP could be used as a drug carrier.

Drug release measured via the xCELLigence RTCA system
In the current study, xCELLigence a real-time cell analyzer (RTCA) system was used to determine the slow release activity of P. nigra and C. libani sporopollenin in CaCo-2 and Vero cells in real time (Fig. 7). Although traditional drug release and cytotoxic assays are performing well, it is time to support them through advancing technologies of the field. RTCA system monitors cell growth by measuring electrical impedance, which is represented as a cell index. xCELLigence detects and measures the phenotypic changes occurring in the cells as a result of the released drug from the sporopollenin through gold microelectrodes mounted at the bottom of the system-specific plates. The prolonged cell proliferation activity reflects the slow  to Vero cells for over 120 h incubation time (Fig. 6a-h). A prolonged-release pattern was observed for CD-SP-OX and PN-SP-OX compared to free Oxaliplatin drug, with an IC 50 value of 5 and 14.62 mg/ml after 120 h incubation time. Oxaliplatin displayed an antiproliferative effect in CaCo-2 cells at approximately 5 h at all concentrations, while PN-SP-OX revealed a long-delivery effect on CaCo-2 cancer cells for about 41 h. PN-SP-OX showed about an 8 times longer release effect than the control drug (free Oxaliplatin). Interestingly, PN-SP-OX applied at the concentration of 10 mg/ml displayed a lower antiproliferative effect on Vero normal cells providing a clue that the slow release of the therapeutics also minimizes the harmful effects on the normal healthy cells (Fig. 7a-d). In the case of CD-SP-OX, at the concentration of 5 mg/ml, a prolonged proliferation effect on CaCo-2 cell was observed, which lasts for 45 h. At the same concentration, the free Oxaliplatin showed a 5 h antiproliferation activity. Besides, slow release activity from CD-SP-OX was observed in Vero cells (Fig. 7e-h).
Considering the overall results, it can be stated that sporopollenin-mediated slow release revealed a prolonged and slow release of Oxaliplatin in both CaCo-2 and Vero cells. However, the slow release in healthy cells decreases the antiproliferative effect as a result of the loaded therapeutic i.e., Oxaliplatin. The use of RTCA gives an idea about the beneficial effect of slow release of Oxaliplatin on healthy cells.

Expression of FOXO3 and MYS
To better understand the apoptosis effects of sporopollenin-mediated slow release of Oxaliplatin on cancer (CaCo-2) and healthy cells (Vero), the expression profiles of two reference genes for apoptosis were investigated using a qRT-PCR. FOXO3 and MYS genes were selected for this purpose as they are thought to be responsible for controlling key pathways during cell apoptosis (detailed functions of both genes have been described in the "Introduction" section).
As shown in Fig. 8, a 20-fold increase was recorded for the FOXO3 mRNA expression in the CaCo-2 cell line when treated with PN-SP-OX (p < 0.05) (Fig. 7a). Interestingly, in the case of Vero cells, no significant increase was recorded in the expression of FOXO3 after treating it with PN-SP-OX (Fig. 7b) (p < 0.05). The level of MYC gene expression decreased approximately twofold on the CaCo-2 cell compared to the control (Fig. 7c). In Vero normal cell, the expression level of the MYC gene decreased about 8 times compared to control (p < 0.05) (Fig. 7d). In the case of CD-SP-OX, the level of FOXO3 gene expression on the CaCo-2 cell increased by about 36-fold compared to the control but a decrease of twofold was detected in the Vero cell (p ˂ 0.05) (Fig. 7e, f). MYC gene expression of CD-SP-OX was not statistically significant compared to the control (OX) but increased gene expression compared to the control of CaCo-2 cells (Fig. 7g). The same material showed a threefold decrease in MYC gene expression in Vero cell (p < 0.05) (Fig. 7h). In summary, the expression of MYC and FOXO-3 genes was increased in CaCo2 cells and decreased non-cancerous Vero cells. The results demonstrated that the increased expression level of these gene-induced apoptosis. The slow release of anticancer drugs in tumor cells may lead to the development of important and novel strategies based on slow release in the treatment of cancer. However, considering the current clue given by this study a more detailed study is needed to investigate the apoptosis linkage with the slow release on the protein level.

Conclusion
Sporopollenin exine capsules (SECs) extracted from plant pollens have successfully established its position in the category of biomaterials for control release applications, thanks to its robust structural and morphological features making it an ideal vehicle for encapsulation and control release of drugs. Considering the results of the current study, it can be stated that the sporopollenin of C. libani and P. nigra  (40-45 h). The sporopollenin-mediated extended-release of Oxaliplatin can overcome the negative effect of the routine used cancer drugs. Both the in vitro release assay in PBS and CaCo-2 and Vero cell cultures assay in real-time cell analyzer (xCELLigenece) had confirmed the slow release by sporopollenin of C. libani and P. nigra. Apoptosis plays a critical role in tumorigenesis, in this study, we have provided an understanding of the linkage of sporopollenin aided slow release and apoptosis mechanisms by checking the expression levels of MYC and FOXO-3 genes. However, further investigation of other genes related apoptosis pathway is required to advance the results of the present study.