5FU encapsulated polyglycerol sebacate nanoparticles as anti-cancer drug carriers (2024)

Diagrammatic flowchart for the synthesis of polymeric PGS and preparation of 5FU-loaded PGS nanoparticles.

2.1. Materials and methods

The chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. Sebacic acid (cat. no. 283258) and glycerol (cat. no. G5516) were purchased from Sigma-Aldrich Chemical Co. 5-Fluorouracil (5FU), Tween 80, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), rhodamine B and other chemicals used for this study were obtained from Sisco Research Laboratories Pvt. Ltd. NMR spectra were recorded using a Bruker ADVANCE III 500 MHz spectrometer with DMSO-d₆ as the solvent. FTIR spectra were recorded using a Jasco FTIR-4100 instrument equipped with an ATR accessory. UV-visible spectra were collected using a PerkinElmer spectrophotometer. Thermogravimetric analysis (TGA) was performed under a nitrogen atmosphere with a heating rate of 10 °C min⁻¹ using a TGA Q-50 thermogravimetric analyzer. Dynamic light scattering (DLS) was performed using a Malvern Instruments Zetasizer nano ZS90. Powder X-ray diffraction (XRD) was performed using a D8 Advance (Bruker). Scanning electron microscopy (SEM) was used to take images of the nanoparticles and the SEM instrument was bought from Thermo Fisher Scientific (Apreo S). The colorimetric caspase-3 assay was purchased from Takara Bio. Annexin V-FITC and propidium iodide were obtained from BD Biosciences. The primary and secondary-HRP conjugated antibodies used for western blot analysis were obtained from Cell Signaling Technology.

2.2. Maintenance of cell lines

Human non-small-cell lung cancer cell line A549 and human breast cancer cell line MDA-MB-231 were procured from the National Centre for Cell Sciences (NCCS), Pune, Maharashtra, India. They were cultured in DMEM (Gibco, Grand Island, U.S.A.) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, U.S.A.) with 1× penicillin–streptomycin (Gibco, Grand Island, U.S.A.) with 5% CO₂ at 37 °C in a humified incubator.

2.3. Synthesis of the polyglycerol sebacate pre-polymer

The polyglycerol sebacate polymer was synthesized using a previously reported procedure.²⁵ Equimolar amounts of glycerol and sebacic acid were taken and kept in an oil bath at 120 °C for 24 h under a nitrogen atmosphere with constant magnetic stirring. After 24 h, a sticky-gel polymer formed, which was dissolved in ethanol and purified using distilled water and a 10 000 Da dialysis bag. This solution was freeze-dried and stored at room temperature. FTIR (PGS) (cm⁻¹): 3443, 2926, 2854, 1733, 1410, 1180, 936; ¹H NMR (PGS) (500 MHz) δ/ppm: 1.24 (s, –CH₂–), 1.55 (d, –CH₂CH₂O(CO)–), 2.27 (m, –CH₂O(CO)–), 3.6–4.1 (m, OHCH₂CHO–), 4.2–4.35 (m, –OCH₂CHO–), 4.95 (2-acylglyceride), 5.09 (1,2-diacylglyceride), 5.26 (1,2,3-triacylglyceride).

2.4. Preparation of PGS nanoparticles

The nanoparticles were generated using the solvent displacement method. Different amounts of PGS were taken and dissolved in 1 mL of ethanol to obtain different concentrations of the nanoparticles (5 mg mL⁻¹, 10 mg mL⁻¹, 20 mg mL⁻¹, and 25 mg mL⁻¹).²² This solution was added dropwise to 10 mL of distilled water slowly and 100 μL of Tween 80 was also added. The solution was kept under constant stirring with sonication at 60 °C. It was then centrifuged at a high speed of 12 000g for 10 min and the supernatant was discarded. The pellet was washed with phosphate buffer saline (PBS) twice at high speed for 5 min. This pellet was then freeze-dried and weighed for further experiments.

2.5. Encapsulation of 5FU within the nanoparticles

The 5FU drug and PGS nanoparticles were taken in different ratios (1 : 1, 1 : 2, 1 : 2.5, 1 : 4, and 1 : 5) to optimize the loading efficiency of the 5FU loaded nanoparticles. 5FU was dissolved in 1 mL of DMSO and this solution was added to 9 mL of distilled water (1 : 10) containing 250 mg of PGS (25 mg mL⁻¹).²⁶ This mixture was left stirring overnight. Then, the solution was centrifuged at a high speed of 12 000g for 10 min and the supernatant was discarded. The pellet was washed with phosphate buffer saline (PBS) at high speed for 5 min and the supernatant was taken for UV-vis spectral measurement at 508 nm to calculate the amount of encapsulated 5FU. After washing with PBS, the pellet was lyophilized and weighed. The loading efficiency²⁷ was calculated using the following formula:

2.6. Characterization of 5FU loaded PGS nanoparticles

2.7. Drug release study

The dialysis method was used to determine the concentration of 5FU in PBS and, thus, the drug release pattern of the nanoparticles.³⁰ PGS-5FU NPs were dispersed in PBS and put in a dialysis tube in a beaker with 200 mL of PBS. Every hour, 3 mL of PBS was replaced with fresh PBS in the beaker with stirring at 50 rpm and this was done for 24 h. The drug release rate was obtained by taking readings of the 3 mL of extracted PBS using UV/visible spectroscopy. DD Solver software was used to simulate the drug release kinetics.

2.8. Cellular uptake of the NPs

The nanoparticles were loaded with rhodamine B (0.5 mg mg⁻¹ w/w)³¹ and the uptake of the NPs was observed using a fluorescence microscope. Around 25 000 cells (MDA-MB-231 and A549) were seeded in separate 60 mm dishes then treated with the rhodamine B loaded nanoparticles. After 12 h of incubation, the cells were washed with PBS and the nucleus was stained with 2 μL of live Hoescht (25 mg mL⁻¹) for 15 min. Images were then taken using a fluorescence microscope (Ti Eclipse Nikon, U.S.A.).

2.9. In vitro cytotoxicity study

Cells were seeded in a 96 well plate (5000 cells per well) and, 24 h later, the free drug, PGS nanoparticles and the drug loaded nanoparticles were added to the wells in triplicate at different concentrations; untreated cells were also used in the experiment.³² Readings were taken at different time points such as 12 h, 24 h, 36 h and 48 h. An MTT stock solution was prepared (5 mg mL⁻¹) and 10 μL was added to each well along with blanks at each time point. 2 h later, the stop solution (DMSO) was added to solubilize the crystals formed. The reading of the samples was performed at 560 nm using an ELISA plate reader (PerkinElmer). The cell viability percentage³³ was calculated using the following formula:

2.10. Apoptosis analysis using flow cytometry

The cells were treated for 24 h with free 5FU, PGS-NP, and PGS-5FU and 2 × 10⁵ cells were taken along with the untreated control cells. 50 μL of binding buffer was added to each tube and then annexin V-FITC (5 μL) was added with 10 μL of propidium iodide (PI).³⁴ After 10 min of incubation at 37 °C, the cells were run through a FACS Calibur instrument to calculate the number of cells undergoing apoptosis. The gating was done using unstained control cells.

2.11. Evaluation of apoptosis

The intrinsic apoptosis pathway is initiated by cleavage of caspase-9, and the cleaved caspase-9 cleaves caspase-3. This cleaved caspase-3 then translocates into the nucleus to induce apoptosis. To understand the mechanism of the NP induced apoptosis, a caspase-3 colorimetric assay kit was used.³⁵ The protocol given by Takara Bio was followed whereby 2 × 10⁶ treated and control cells were centrifuged at 400g for 5 min. Then, 50 μL of cold lysis buffer was added and the solution was kept in ice for 10 min. The tubes were then centrifuged at 12 000g for 10 min at 4 °C and the supernatant was transferred to new tubes. One set of positive control cells was treated with 1 μL of caspase-3 inhibitor and 50 μL of reaction buffer containing DTT. The rest of the samples were treated only with reaction buffer having DTT and the solution was incubated for 30 min in ice. Then, 5 μL of the 1 mM caspase-3 substrate was added and kept for 1 h in a water bath at 37 °C. A standard curve was plotted using pNA provided in the kit dissolved in DMSO at different concentrations ranging from 0 to 200 μM.

2.12. Expression levels of apoptosis proteins

MDA-MB-231 cells and A549 cells were treated with 5FU, PGS nanoparticles and PGS-5FU, the cells were trypsinized, and the pellets were washed with PBS. The supernatant was discarded, and the total protein was extracted using a radioimmunoprecipitation buffer (RIPA) with a 1× protein inhibition co*cktail (PIC).³⁶ 30 μg of protein of each sample was run using SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane after activation. This membrane was blocked with 5% skimmed milk and then incubated overnight at 4 °C with primary antibodies against procaspase-3, procaspase-9, and vinculin. The membrane was washed with TBS—T thrice and incubated with the respective alkaline phosphatase-conjugated secondary antibody. The blots were developed using an enhanced chemiluminescence (ECL) buffer, imaged using a BioRad ChemiDoc system and analysed by normalizing with an endogenous control vinculin protein.

2.13. Statistical analysis

The statistical data analysis was performed using Student's t-test to compare the different samples where p < 0.05 was taken to be significant. All the experiments were done in triplicate, the average was noted, and the standard error was plotted using Graphpad Prism 8. Origin Pro 8.5 software was used to plot graphs of FTIR, XRD, and TGA data. Flowjo v8 was used to plot the dot plot of flow cytometry experiments. Image Lab and Fiji ImageJ software were used to analyse western blot and fluorescence microscope images, respectively. To calculate the loading efficiency and the drug release rate, the supply of 5FU was measured over a range of different concentrations and λ_max was determined by UV-vis spectroscopy.

3.1. Synthesis and properties of 5FU-NPs

The synthesis of the polyglycerol polymer was done according to a previously reported protocol by taking equimolar amounts of glycerol and sebacic acid, as shown in Scheme 1. The characterization of the monomers and the pure polymer was performed using ¹H NMR spectroscopy and data were analyzed using MestReNova software (Fig. S1^†). The proton peaks at δ 1.30, 1.60, and 2.3 ppm in the polymer data corresponded to sebacic acid and the peaks at 3.43–3.96, 4.08–4.35, and 5.09, 5.26 ppm are related to glycerol. These data showed a linear structure of the PGS polymer with the molar ratio of monomers of 1. Additionally, this result was corroborated by FTIR analysis (Fig. 1A), which exhibited a broad peak at 3443 cm⁻¹ attributed to the stretch vibration absorption of O–H end groups in the molecules while their bending gave a peak at 1410 cm⁻¹. The sharp peaks at 1180 cm⁻¹ and 1733 cm⁻¹ are related to C–O whereby the 1733 cm⁻¹ peak indicated an ester carbonyl linkage. CO bonds and methylene groups were indicated by peaks at 2854 cm⁻¹ and 2926 cm⁻¹, respectively. The peak at 1691 cm⁻¹ indicates unreacted sebacic acid and this peak was absent in the spectrum of the polymer owing to the purification step. These data demonstrate the successful generation of the PGS polymer and the active groups on the surface can be functionalized further to attach compounds onto the surface of the nanoparticles. XRD analysis (Fig. 1B) showed a sharp peak in the pattern of the PGS-5FU nanoparticles at 30 2θ and a peak at 20 2θ present in the pattern of PGS-NP that had almost completely disappeared in the pattern of PGS-5FU, showing the encapsulation of 5FU within PGS-5FU. These data confirm the crystalline nature of the nanoparticles as compared to the semi-crystalline nature of the PGS gel and, therefore, the increased stability of PGS-5FU. The thermostability of PGS-NP and PGS-5FU was assessed using a thermogravimetric analyzer (TGA-Q50) and a representative thermogram is plotted in Fig. S2.^† The results showed a first weight loss of about 30% at 250 °C for the PGS nanoparticles and a second thermal decomposition at 400 °C of 40% weight loss. The first weight loss is due to absorbed solvent evaporation and the second one is due to polymer degradation. In the case of the 5FU loaded PGS nanoparticles, there was a weight loss of up to 90% in the range from 300 °C to 400 °C, indicating an increase in stability with the encapsulation of 5FU. The heat flow through the PGS prepolymer, PGS-NP and PGS-5FU was measured by differential scanning calorimetry (DSC Q200, TA Instruments) (Fig. S3^†). The results showed a glass transition temperature (T_g) of the PGS prepolymer of −7.52 °C and that of PGS-NP of 104.87 °C. The shift to positive temperature is due to the increased crosslinking within the nanoparticle. Additionally, the melting temperature (T_m) was 5.56 °C for the PGS prepolymer, which increased to 105.33 °C for PGS-NP, indicating that PGS is miscible with the sol–gel composite and that the nanoparticle has partial crystallinity. Interestingly, after loading 5FU into the nanoparticle, it became completely amorphous, and this suggests the successful entrapment of the drug within the nanoparticle matrix. Furthermore, GPC analysis (GPC, Waters, U.S.A.) showed a PDI of the PGS polymer of 1.0054, a calculated M_n of 93.9 kDa and a M_w of 94.4 kDa using polystyrene standards for generating the calibration curve.

3.2. Morphological characteristics of the nanoparticle

The size and zeta potential of the nanoparticles were assessed using a Malvern Instruments Zetasizer nano ZS90. The size distribution histogram (Fig. S5^†) showed varied sizes for different concentrations of PGS used to make the nanoparticles. Interestingly, the size and PDI data for the 25 mg mL⁻¹ concentration of PGS measured 30 days post synthesis showed hom*ogenous particles in the range of 200–350 nm and having long shelf-life stability (Fig. 1C). The observed zeta potential was between −35 mV and −20 mV. The zeta potential magnitude of PGS-NPs indicates high colloidal stability. These nanoparticles are also small-sized, within which the drug can be easily loaded. To corroborate the results, scanning electron microscopy (SEM) (Fig. 1D) was used, which exhibited PGS nanoparticles having a spherical shape.

3.3. In vitro 5FU release kinetics

The loading efficiency of different nanoparticle to drug ratios was calculated and, out of these, the 1 : 2.5 ratio of PGS-NP : 5FU showed a 90.39% encapsulation efficiency. Additionally, the release of 5FU via diffusion recorded over 15 h showed a controlled drug release (Fig. 2A). A graph was plotted of the drug release rate and, at 13 h, almost all of the loaded drug was released into the medium. With the help of mathematical modelling using DD Solver software, the kinetics of 5FU-NP was determined to be Hopfenberg with R square calculated as 0.985.

3.4. Cytotoxic effect of PGS-5FU

Stock solutions of different concentrations of PGS were prepared in distilled water and then working concentrations were diluted in biological media. At different time points, the absorbance was recorded after solubilizing crystals formed due to the addition of MTT, and the cell viability percentage was calculated (Fig. 2B and C). Interestingly, even at higher concentrations of PGS-NPs, there was no significant decrease in cell viability up to 48 h. This could illustrate the biocompatibility of the polymeric PGS nanoparticles. The 50% inhibitory concentration (IC₅₀) of 5FU was calculated as 10.86 ± 0.34 μg mL⁻¹ for A549 cells and 11.18 ± 0.42 μg mL⁻¹ for MDA-MB-231 cells. Furthermore, PGS-5FU had IC₅₀ values in similar ranges, calculated as 10.49 + 0.22 μg mL⁻¹ (A549) and 10.88 + 0.17 μg mL⁻¹ (MDA-MB-231). But, after 24 h, 36 h, and 48 h, the viability% increased when cells were treated with the drug alone, demonstrating that the cells became resistant and survived. Interestingly, the cell viability decreased with time upon treatment with PGS-5FU nanoparticles. This clearly shows the improved efficiency of PGS-5FU nanoparticles in the controlled release of 5FU as compared to 5FU alone. Therefore, this drug delivery system can enhance the competence of existing conventional chemotherapy and help avoid resistance development and metastasis of the disease.

3.5. Cellular uptake of PGS-5FU nanoparticles

The fluorescent dye rhodamine B was encapsulated in the PGS-NPs (Rho-NPs) to understand the efficiency of uptake of the nanoparticles by cancer cells. There was a gradual increase in the fluorescence with time (Fig. 3). At 6 h, almost all the cells showed the intracellular uptake of Rho-NPs. The images showed that the NPs were transported efficiently into the cells. One of the key requisites of a drug delivery system is the ability of the nanoparticle to penetrate the cell membrane; here, it can be seen that these PGS-NPs loaded with rhodamine were easily taken up by the cancer cells. Within a few hours, the nanoparticles were internalized in the cancer cells and, therefore, PGS-NPs could deliver the anti-cancer drugs enclosed within them inside the cells.

3.6. Apoptosis assay using flow cytometry

To estimate the induction of apoptosis upon treatment with the drug loaded nanoparticles, Annexin V-FITC assay was performed wherein the treated cells along with control cells were stained with propidium iodide and Annexin V-FITC dyes. Calibration gating was performed using unstained cells, and results were obtained using flow cytometry (Fig. 4). The dot plots validated that PGS-5FU treatment induced apoptosis in the cancer cells as there was a shift seen towards the Q2 and Q4 quadrants, as shown in Fig. 4A and B, indicating late and early apoptosis, respectively. A549, as well as MDA-MB-231 cells treated with PGS-NPs had lower populations in these quadrants, which confirms the previous toxicity findings. These outcomes provide insight into the molecular mechanism of PGS-5FU nanoparticles and confirm that the cell death is caused by apoptosis induction.

3.7. Immunoprecipitation analysis of apoptotic proteins

The western blot analysis (Fig. 5) also indicated cell death via apoptosis for the cells treated with PGS-5FU nanoparticles. To prove the same, the expression levels of inactive caspase-3 and caspase-9 proteins were recorded, and they remained almost the same when normalized with vinculin protein in the case of PGS-NP and 5FU treatment in A549 cells. This means that neither the PGS nanoparticles nor 5FU alone could initiate apoptosis in the cancer cells. However, there was a significant decrease in the protease levels upon treatment with PGS-5FU, evidently showing initiation. In MDA-MB-231 cells as well, a similar pattern of expression levels was observed. The significant downregulation of procaspases in both cell lines when treated with the drug loaded nanoparticles proves the augmented ability of PGS-5FU nanoparticles in initiating apoptosis in cancer cells.

3.8. Caspase-3 assay

When initiating apoptosis, caspase proteases play a very crucial role. The activity of caspase-3 indicates the process of apoptosis and, therefore, to assess the effect of PGS-5FU, colorimetric caspase-3 assay was performed. The protease levels of the A549 lung cancer and MDA-MB-231 breast cancer cells treated with PGS-5FU at the IC₅₀ concentration were different (Fig. 6). The breast cancer cells showed a 4.15-fold change in caspase-3 levels and the A549 cells showed a 4-fold change in levels, but the 5FU treated MDA-MB-231 cells, and A549 cells showed 2.9- and 3.29-fold changes of caspase-3 levels, respectively. This is due to the different cellular uptake rates of PGS-5FU within these two cell lines in the given period and also indicates that breast cancer cells are more sensitive to PGS-5FU treatment. As mentioned earlier, PGS-NPs did not initiate apoptosis as the caspase-3 levels were significantly low as compared to the untreated control cells.

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