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1. Introduction
Cancer is a complex disease that arises and progresses via the accumulation of genetic mutations and epigenetic alterations in somatic cells. Dysregulation of epigenetic mechanisms leads to changes in gene transcription, which are associated with tumor progression, therapy resistance, and poor survival rates [1]. Histone deacetylases (HDACs) are epigenetic modifiers that silence transcription by removing acetyl groups from the lysine residues of histone tails [2]. This increases the positive charge on the histone proteins, enhancing their interaction with DNA and leading to chromatin condensation, which blocks the access of the transcriptional machinery [2, 3]. HDACs are involved in regulating key cellular functions including cell cycles, DNA repair, differentiation, and tumor suppression [2, 3]. Thus far, 18 HDACs have been identified in humans and were grouped into four classes (I, IIa/IIb, III, and IV). The overexpression of class I isoforms (HDAC1, HADC2, and HDAC3) has been frequently associated with advanced disease stages and poor patient outcomes across various cancers [4]. Hence, they have gained considerable attention as promising targets to disrupt the oncogenic regulations.
HDAC inhibitors are emerging as promising anticancer agents due to their ability to reverse epigenetic abnormalities that regulate cell cycle, proliferation, angiogenesis, and apoptosis [3, 5]. MS-275, also known as entinostat, is an orally administered benzamide HDAC inhibitor characterized by the presence of lipophilic aromatic rings, which contribute to its moderate lipophilicity [6]. MS-275 exhibits high selectivity towards HDAC1, HDAC2, and HDAC3 and has demonstrated potent anticancer activity against various hematological and solid malignancies [7, 8]. In vitro analysis has demonstrated the major advantage of MS-275 in inhibiting the growth of chemotherapy-resistant cells, which is devoted to its ability to induce simultaneous actions on multiple regulations including cell cycle, apoptosis, and tumorigenic pathways [9], whereas the in vivo analysis showed a significant reduction in tumor size and a considerable improvement in many solid and liquid tumors including lung, breast, ovarian, gastric, pancreatic, and colon tumors [8, 10]. Studies have shown that treating cancer cells with low concentrations of MS-275 can act as an antiproliferative agent by increasing the expression of cyclin-dependent kinase inhibitor p21 (also known as CDKN1A), a growth-arresting protein [2, 11]. The increased p21 level then leads to hypophosphorylation of the retinoblastoma tumor suppressor protein (pRB), which in turn leads to the downregulation of the cell cycle-related proteins including cyclin D1 [2, 9, 10], whereas higher concentrations of MS-275 have been reported to prompt an early increase in reactive oxygen species (ROS), which eventually triggers apoptosis [9, 12]. MS-275 has also been reported to induce tumor cell death by increasing the expression of death receptor 5 (TNFRSF10B) and BCL-2 proapoptotic factors. Consequently, this stimulates apoptosis by downregulating the inhibitor of apoptosis proteins (IAPs) and inducing the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [2, 11, 12].
Clinical data revealed the remarkable specificity of MS-275 to malignant cells with minimal effects on normal cells, which qualifies MS-275 as an alternative or adjuvant to chemotherapies [13]. MS-275 has been evaluated in several phase I and II clinical trials to treat solid and hematologic malignancies. Phase I clinical studies have demonstrated the high efficiency of MS-275 when used alone or in combination with other anticancer treatments. However, dose-limiting toxicities were observed at the first dose level [8, 14]. The phase II trial revealed considerably higher overall survival rates and progression-free survival in the MS-275-treated cohort [15]. A separate study reported that a 5 mg weekly dose of MS-275 caused severe adverse effects in 6% of patients with grade 3 cancers, which led to study discontinuation for that group [16]. Currently, MS-275 is in phase III clinical trials to validate its preclinical and clinical application further and improve the treatment outcomes of solid tumors [13, 17].
According to the existing clinical evidence, the adverse effects of MS-275 are dose-dependent and can cause mild to moderate cytotoxicity, and hematologic and metabolic dysfunctions [8, 13, 15, 16, 18]. The clinical application of MS-275 in central nervous system diseases is also limited by its low bioavailability due to the restricted permeability of the blood-brain barrier (BBB) [19]. These adverse effects warrant a better design of efficient drug delivery to reduce systemic toxicity and enhance the delivery into tumor tissues. Current clinical findings suggest a sustained delivery of low MS-275 doses to reduce the dose-limiting toxicity, systemic toxicity, and off-target effects [8, 16, 20, 21]. Thus, enhancing the MS-275 anticancer effect and decreasing the systemic free drug are novel therapeutic approaches to minimize the impediment associated with MS-275 administration.
Previously, MS-275 was encapsulated in silver nanoparticles (AgNPs) to address the therapeutic challenges of aggressive lung cancer [22]. The findings have shown that encapsulation enhanced its anticancer activity at a low half maximal inhibitory concentration (IC50) and induced higher death in the A549 lung cell line compared with either AgNPs or MS-275 alone [22]. The study also demonstrated that the encapsulation preserved the MS-275 mechanism of action, which is mediated by apoptosis and ROS induction [22]. These findings indicate that the MS-275 off-target effects can be overcome by using a suitable nanoparticle carrier, which could improve the drug’s therapeutic index. Although the encapsulation has improved the anticancer activity of MS-275, AgNPs have been reported to induce genotoxic effects on normal cells [23, 24]. Therefore, developing more effective and safer encapsulation strategies is essential to provide a high therapeutic value.
In this context, TPGS, a highly stable, water-soluble form of vitamin E, has been approved by the U.S. Food and Drug Administration as a safe nanocarrier for drug delivery [25]. TPGS has been widely used in various chemotherapy drug delivery systems to enhance the bioavailability and therapeutic efficacy of orally administered agents, as it retains an anticancer activity [25, 26]. It has also shown great potential to cross the BBB [27]. Moreover, TPGS has been reported to inhibit ATP-binding cassette subfamily B member 1 (ABCB1) transporter and cytochrome P450 detoxifying enzyme. Accordingly, it may help to overcome multidrug resistance (MDR) and drug inactivation mechanisms [25].
Hence, to overcome the undesired side effects of using high concentrations of MS-275 and to improve its anticancer activity, we hypothesized that MS-275 encapsulated in TPGS micelles would be more effective against cancer growth than a single agent. In this regard, the main objective of this study is to encapsulate MS-275 in TPGS nanoparticles and to examine the physicochemical characteristics and anticancer activity of TPGS loaded with MS-275. The successful development of nanoformulation and encapsulation could minimize the adverse effects of MS-275 and potentially improve the drug delivery to challenging targets, such as brain tumors and those overexpressing ABCB1.
2. Materials and Methods
2.1. Preparation of TPGS Formulations
In this study, medicated and nonmedicated TPGS (Sigma-Aldrich, St. Louis, MI, USA) micelles nanoformulations were prepared. In brief, TPGS (2% w/v) and drug (0.1% w/v) were dissolved in 20 mL of ethanol (Sigma-Aldrich, St. Louis, MI, USA) under gentle agitation over a magnetic stirrer. Distilled water was subsequently added, and the ethanol was completely evaporated on Buchi Rotavapor R-200 (BÜCHI Labortechnik AG, Flawil, Switzerland). The clear micellar solution obtained was kept at 4°C. MS-275 (AK Scientific) was dissolved in dimethyl sulfoxide (DMSO, Molequle-On) to prepare the stock solution.
2.2. Characterization of the Prepared TPGS Formulations
The size distribution of the prepared nanoformulation was assessed using the Malvern Zetasizer Nano ZSP instrument (Malvern Panalytical Ltd, Malvern, United Kingdom). Specifically, we employed the polydispersity index (PDI) to gain insight into the uniformity of the micellar system. In addition, the surface charge (zeta potential) was determined by electrophoretic mobility, which facilitated the evaluation of colloidal stability and interactions with biological components. All the measures were performed in triplicates.
The entrapment efficiency was performed by subjecting the formulations to centrifugation at 20,000 rpm (3K30 Sigma centrifuge) for 1 hour at 4°C to separate the drug-loaded nanocarriers from the unentrapped drug. Once centrifugation was complete, the studied samples were carefully removed, and the supernatant containing the unentrapped drug was separated and filtered through a 0.2 μm syringe filter (Acrodisc). The concentration of the unentrapped drug in the supernatant was determined spectrophotometrically at 235 nm using a Shimadzu UV‐2600 UV-VIS spectrophotometer (Shimadzu Corporation, Kyoto, Japan).
2.3. Studying the Cellular Uptake
The cellular uptake of the nanoformulation was indirectly assessed by comparing the MS-275 concentration in the culture media before and after cell treatment. The analysis was conducted by treating the cells with 10 μM of the MS-275-TPGS formulation. After 4 hours, the culture media was collected, and an equal volume of absolute ethanol (Fisher Chemical) was added to emulsify the TPGS nanoparticles. The mixture was vortex-mixed and centrifuged at
2.4. Assessing Cell Viability Using the MTT Assay
The HepG2 (human hepatocellular carcinoma), HCT116 (human colon carcinoma), and MCF7 (human breast adenocarcinoma) cell lines were kindly provided by Dr Muhammad Imran Khan, Faculty of Science, King Abdulaziz University. The cell lines were cultured in Dulbecco’s Modified Medium (DMEM, Molequle-On) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin antibiotic (Gibco). The cells were grown in a 37°C incubator with a humidified atmosphere of 5% CO2 air. The cells were harvested at 70–90% confluence using 0.25% trypsin (Gibco). Harvested cells were counted after staining with Trypan blue, and a cell concentration of 3.5 × 104/mL was used to seed 96-well plates (Nunclon), and the plates were incubated for 24 hours. The media of each well was replaced by media containing a spectrum of different concentrations (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, and 24 μM) of MS-275, TPGS-free, and TPGS-loaded (hereinafter known as, MS-275-TPGS). Each concentration was assessed in triplicate, and a control of cell treated with DMSO was included in all experiments. The plates were incubated in a 37°C incubator with a humidified atmosphere of 5% CO2 air for 72 hours. The MTT assay was used to measure cell viability by replacing the treatment media with 100 μL of 0.5 mg/mL MTT solution (AK Scientific), and the plates were incubated for 90 min at 37°C in the dark. The MTT solution was then replaced with 100 μL of DMSO, and the plates were agitated for 15 min in the dark. The absorbance was measured at 590 nm wavelength with a reference filter of 720 nm using a SpectraMax i3x (Molecular Devices) spectrophotometer plate reader.
2.5. Cell Cycle Assays
The cells were seeded at 1 × 105 cell/mL density in 6-well plates using complete DMEM culture media and incubated overnight at 37°C in 5% CO2 air. The cells were then treated with a single dose of MS-275, MS-275-TPGS, or TPGS at a concentration of 10 μM for 36 hours. Subsequently, the cells were harvested as described above, washed with PBS, fixed in 70% cold ethanol, and stored at −20°C for at least 24 hours. After two washes with ice-cold PBS, the cells were resuspended in 500 μL PBS supplemented with 50 μg/mL propidium iodide (PI, Sigma) and 0.5 mg/mL RNase A (Qiagen). The suspension was incubated at 37°C for 1 hour.
Flow cytometry analysis was performed using BD FACSCalibur (BD Biosciences) with customized acquisition settings for forward scatter (FSC), side scatter (SSC), and PI fluorescence. At least 20,000 singlet cells were gated for the analysis, and the percentages of the cells in the G1, S, and G2/M phases were estimated and plotted using FlowLogic software (version 8.6, Inivai Technologies).
2.6. Apoptosis Assays
The cells were seeded at 1 × 105 cell/mL density in 6-well plates using complete DMEM culture media and incubated overnight at 37°C in 5% CO2 air. The cells were treated with a single dose of MS-275, MS-275-TPGS, or TPGS at a concentration of 10 μM for 36 hours. Subsequently, the cells were harvested by collecting both floating and adherent cells as described above and maintained on ice. The apoptosis analysis was performed using APC Annexin V and 7-AAD (BD Pharmingen) according to the manufacturer’s recommendations.
Flow cytometry analysis was performed using a BD FACSCalibur (BD Biosciences) with customized acquisition settings for FSC, SSC, FL4 for APC, and FL3 for 7-AAD fluorescence. At least 10,000 singlet cells were gated for the analysis, and the percentages of viable, necrotic, and early/late apoptotic cells were determined using APC vs. 7-AAD plot by applying a quadrant gate using the FlowLogic software (version 8.6, Inivai Technologies).
2.7. Growing Human Cell Line Tumors on the CAM of Chick Embryos
White leghorn chicken (Gallus gallus domesticus) pathogen-free fertilized eggs were obtained from Ommat, Jeddah, Saudi Arabia. Eggs were incubated for 9 days at 37.5°C and 60% humidity with continuous rocking at an hour interval using a Brinsea OvaEasy 190 Advance Series II Cabinet Incubator (Bristol, UK). For embryo manipulation, the air space (blunt end) of the egg was windowed under aseptic conditions. A 500 μL of PBS was added, then the shell was pinched by a watchmaker forceps, and the vitelline membrane was carefully removed to expose the CAM. A sterile silicone O-ring (0.5 cm diameter) was gently placed on top of the CAM, after which the surface of the membrane inside the ring was scratched with a 20 μL sterile filter tip.
The eggs were divided into five groups (two as negative controls and three as treated groups). One of the negative controls was treated with PBS, and the second group was treated with the culture media. Tumors were induced in vivo by inoculating the CAM with 40 μL of either HepG2, HCT116, or MCF7 cancer cell lines at 5 × 105 cells xenografted into the center of silicone O-ring after which the eggs were sealed and reincubated for another 4 days. The xenograft formation was imaged as 2D images using a Zeiss V12 SteREO microscope equipped with Zeiss Axiocam 305 color camera (Carl Zeiss Microscopy GmbH, Germany). Induced tumors were initially treated with MS-275-TPGS at a concentration of 5, 10, 20, 40, 80, and 100 μM four times every 24 h to examine the efficacy of the MS-275-TPGS nanoformulation in inhibiting the tumors’ growth. At the end of the treatment course, the tumors were 2D-reimaged using the same magnification that was used prior to the MS-275-TPGS treatment. The 2D images were converted into 3D images using ZEN Blue image analysis software (version 3.3.8), and the images were used to evaluate the inhibition in tumor growth by measuring the reduction in the tumors’ size.
2.8. Statistical Analysis
Statistical analysis was conducted using Microsoft Excel 2021. Statistical significance was determined by unpaired Student’s T-test, and all data presented in this study met the statistical test’s assumptions. Quantitative data were presented as the mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). A
3. Results
3.1. Characterization of TPGS Micelles
Characterization of drug delivery systems is pivotal for evaluating their suitability for therapeutic applications. The physicochemical properties of the synthesized micelles including size, PDI, zeta potential, and entrapment efficiency were systematically evaluated (Table 1). The prepared medicated MS-275-TPGS and nonmedicated TPGS formulations exhibited a nanosize of 42.41 ± 0.51–38.35 ± 0.68 nm, respectively. The PDI values observed, ranging from 0.201 ± 0.021 to 0.206 ± 0.026 indicate a low polydispersity. The zeta potential provides information about the surface charge, which can influence the particles’ stability and interaction with the biological systems [28]. The negative values of the zeta potential, ranging from −0.397 ± 0.041 to −0.441 ± 0.039 mV, are close to neutrality. The result of drug entrapment efficiency revealed a high value of 98.77 ± 0.37%, indicating that the TPGS micelles can efficiently encapsulate drug compounds.
Table 1
Summary of the physicochemical characteristics of the prepared micelles formulations.
| Size (nm) | PDI | Zeta potential (mV) | Entrapment efficiency (%) | |
| MS-275-TPGS | 42.41 ± 0.51 | 0.201 ± 0.021 | −0.397 ± 0.041 | 98.77 ± 0.37 |
| Nonmedicated TPGS micelles | 38.35 ± 0.68 | 0.206 ± 0.026 | −0.441 ± 0.039 | — |
The results represent the average of three measurements ± standard deviation.
Moreover, the stability of the TPGS micelles was reassessed by remeasuring the size and zeta potential after three months of preparation in both physiological (pH 7.4) and tumor-mimicking (pH 6.5) environments. The results did not show significant differences in the size or zeta potential of the micelles in both conditions. This finding suggests that the MS-275-TPGS nanoformulation maintains its stability in physiological and tumor-mimicking pH conditions. These results support the robustness of the formulation and its potential for consistent performance in varying biological environments.
Furthermore, the cellular uptake of the MS-275-TPGS micelles was investigated by quantifying the concentration of MS-275 in the culture media before and after a 4-hour treatment using LC-MS. The mass spectrum of MS-275 revealed protonated daughter ions at m/z 149.2 and 359.3, which were subsequently monitored across all the samples. The results showed that the concentration of MS-275 in the collected media was lower than that in the original treatment media (Figure 1). The observed decrease could be attributed to the internalization of the micelles by the cells, suggesting the occurrence of cellular uptake.
[figure(s) omitted; refer to PDF]
3.2. MS-275 Nanoformulations Showed Enhanced Anticancer Effect against Various Cancer Cells
Three human cancer cell lines were used to study the anticancer efficacy of MS-275 encapsulation: HepG2, HCT116, and MCF7. The cells were treated with various concentrations of MS-275, MS-275-TPGS, and TPGS, and cell viability was measured using the MTT assay after 72 hours of exposure (Figure 2). The results displayed a lower sensitivity to MS-275 in a dose-dependent manner. The complete cell death of HepG2, HCT116, and MCF7 was observed at 24 μM concentration (Figures 2(a), 2(b), and 2(c)).
[figure(s) omitted; refer to PDF]
Interestingly, the MS-275-TPGS nanoformulation has shown a significant (
3.3. MS-275-TPGS Treatment Altered the Cell Cycle Dynamics and Increased Apoptosis
The impact of MS-275-TPGS treatment at a concentration of 10 μM on the cell cycle distribution closely paralleled that of MS-275 treatment. MS-275 and MS-275-TPGS induced a significant cell cycle arrest in the G1 and G2M phases by more than 15% after 36 hours of treatment compared to the untreated control cells (Figure 3). Concurrently, they led to a notable reduction of over 20% in the S-phase cell population relative to the control. Interestingly, the TPGS alone showed a significant accumulation in the G1 by more than 20%, accompanied by a reduction in the S-phase cells (Figure 3).
[figure(s) omitted; refer to PDF]
Furthermore, to investigate the effect of MS-275-TPGS on cell death, we treated the cells with 10 μM concentration for 36 hours. The Annexin V/7-AAD, flow cytometry analysis, revealed a significant (
[figure(s) omitted; refer to PDF]
3.4. MS-275-TPGS Showed Enhanced Anticancer Effects against Various Tumors Induced on CAM
To examine the efficacy of MS-275-TPGS nanoformulation on tumor inhibition, we used the CAM of the chick embryo to evaluate tumor growth inhibition. In this assay, HepG2, HCT116, and MCF7 cell lines were xenografted onto the CAM of 9-day embryos for 4 days. 2D images of the xenografted tumors were taken (Figures 5(e), 5(i), and 5(m)) and converted to 3D images (Figures 5(f), 5(j), and 5(n)) to measure the size of the tumors. Induced tumors were initially treated with MS-275-TPGS nanoformulation at a concentration of 5, 10, 20, 40, 80, and 100 μM. MS-275-TPGS at 100 μM was sufficient to inhibit the tumor growth. Interestingly, this concentration was equal to ten times the IC50 of the cell lines. At the end of the treatment course, 2D images of the tumors were taken again (Figures 5(g), 5(k), and 5(o)) and converted to 3D images (Figures 5(h), 5(l), and 5(p)) to assess the effect of the MS-275-TPGS. The results showed a prominent reduction in tumor size and growth in the MS-275-TPGS-treated tumors when compared to the baseline measurements (Figures 5(f), 5(j), and 5(n)). The MS-275-TPGS treatment resulted in a remarkable shrinkage in tumor size by approximately 80–90%. Treatment CAM with PBS (Figures 5(a) and 5(b)) or culture media (Figures 5(c) and 5(d)), which served as negative controls, showed no noticeable effects.
[figure(s) omitted; refer to PDF]
4. Discussion
MS-275, a selective HDAC1 and HDAC3 inhibitor, exhibits remarkable anticancer activity against various malignancies, including some chemoresistant tumors [1, 9, 10, 29]. However, the therapeutic application of MS-275 has been constrained by its dose-limiting toxicities, leading to the early termination of some clinical trials [16]. The recommendations presented from the clinical data suggested using a sustained delivery of low MS-275 doses to mitigate its toxicity. In this study, we aimed to lower the IC50 level of MS-275 by using a combination therapeutic approach through nanoencapsulation within TPGS drug delivery micelles.
In the current work, TPGS-encapsulated MS-275 was successfully developed. The low PDI values indicated a uniform size distribution, which is highly desirable for pharmaceutical applications. The zeta potential charge values were close to neutrality, an advantage that reduces the likelihood of nonspecific interactions with cell membranes or other components. The combination of nanosized dimensions and a slightly negative charge may also contribute to colloidal stability and enhanced cellular uptake by repelling similarly charged particles. The PDI and ZP results obtained in this study are similar to the previously published work, further validating the successful development of the nanoformulation [30, 31].
In addition, the high entrapment values demonstrated the effectiveness of encapsulation, suggesting TPGS micelles as a promising drug delivery system. Furthermore, the cellular uptake data validated the internalization of the micelles. These characterization data highlight the prepared micelles’ appropriate physicochemical properties and drug-loading capacity, making them suitable for effective drug delivery.
In this study, the MS-275-TPGS nanoformulation caused a dramatic inhibition of cell proliferation at a lower dose than MS-275 alone. In addition, it induced cell cycle arrest at G1 and G2M, accompanied by a reduction in the proportion of cells in the S phase. This cellular response is similar to the effect observed with MS-275 monotherapy, suggesting that the MS-275-TPGS conjugate exerted a cell cycle effect comparable to MS-275. This similarity implies that the combination did not significantly alter the cell cycle regulatory mechanism targeted by MS-275 alone. Moreover, the increased apoptosis level further supports the efficacy of the MS-275-TPGS formulation. In contrast, treating the cancer cells with TPGS micelles alone resulted in a significant accumulation in the G1 phase, concurrent with a decrease in the S phase and a reduction in cell viability.
These findings align with prior studies, demonstrating the ability of the TPGS micelles to arrest cell cycle progression and inhibit cell survival in diverse cancer cell lines [32–35]. TPGS has been reported to induce cell cycle arrest and apoptosis through diverse mechanisms. These include the activation of oxidative stress signaling pathways, which reduce the mitochondrial membrane potential, leading to electron leakage and cellular damage [33, 35–37]. In addition, TPGS has been reported to inhibit the ABCB1 activity, a drug efflux pump involved in MDR [38]. This inhibition enhances the intracellular accumulation of anticancer drugs, which augment their cytotoxicity.
The results of this study also presented the low sensitivity of HepG2, HCT116, and MCF7 cell lines to MS-275, as evidenced by their requirement for higher concentrations to achieve complete cell death. In line with these findings, other studies have also observed the reduced sensitivity of these cells to MS-275 treatment [7, 39–42]. Although the sensitivity of cancer cells to MS-275 has been extensively investigated, it is not completely known why certain tumors exhibit higher sensitivity to MS-275 than others. These differences have been attributed to the variations in HDAC isoform expression, the p53 wild-type/mutant status, and oncogenic regulatory mechanisms specific to each tumor type [7, 43]. The oncogenic impact of HDAC1 and HDAC3 varies widely among diverse types of tumors. For example, knocking down HDAC3 in HepG2 has been reported to stimulate cell growth [44], whereas the combined knockdown of HDAC1 and HDAC2 has been reported to reduce cell proliferation and induce cell death, as they work collectively to promote G1 to S phase progression.
Similarly, in HCT116 cells, the combined knockdown of HDAC1 and HDAC2 has been reported to induce apoptotic cell death [45]. MCF7 is a noninvasive breast cancer cell, overexpressing HDAC1, HDAC2, and HDAC3 when compared to the aggressive MDA-MB-231 cell line [46]. Interestingly, knocking down HDAC1 and HDAC3 in MCF7 has been reported to have no significant impact on cell growth [47]. These diverse responses suggest that HDACs may not be the primary drivers of oncogenesis, and MS-275 may act through different molecular regulations, which warrant further investigations.
The chick embryo in vivo CAM assay has been widely and effectively used in a considerable number of studies over many years to test the efficacy of anticancer drugs [48–52]. Chick embryos share at least 70% genomic similarity to humans, making them an excellent model to bridge the gap between the in vitro and in vivo systems [53, 54]. Therefore, we utilized the CAM assay to investigate the effectiveness of MS-275-TPGS in inhibiting tumor growth, and the results demonstrated the ability of MS-275-TPGS in inhibiting the growth of the induced tumors. To demonstrate this inhibition, we used the advantages of 2D and 3D imaging analyses to assess the tumor size before and after treatment. The current study presents a robust experimental drug testing model that combines an in vitro cancer cell line with an affordable in vivo CAM system to examine drug efficacy and sensitivity.
In conclusion, our data showed that the combination of MS-275 and TPGS has dramatically reduced tumor cell viability at low pharmacological doses, providing a dosage advantage. This combination has the potential to reduce the toxicity associated with MS-275 treatment while enhancing its anticancer effects against low-sensitivity cells. Notably, TPGS, as an effective blood-brain barrier nanocarrier, may facilitate MS-275 delivery to brain tumors. The MS-275-TPGS formulation holds promise in the treatment of various cancers, including those characterized by ABCB1 overexpression. However, comprehensive assessments of formulation stability, pharmacokinetics, and in vivo behavior are essential to establish this formulation as a novel cancer therapy.
Disclosure
The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Authors’ Contributions
A.S.A., T.A.A., and M.A.E. conceptualized the study. A.S.A., T.A.A., F.A., and M.A.E. developed the methodology. A.S.A., T.A.A., and M.A.E. validated the study. A.S.A., T.A.A., F.A., and M.A.E. performed the formal analysis. A.S.A. T.A.A., F.A., and M.A.E. investigated the study. A.M.O., A.S.A., T.A.A., F.A., and M.A.E. collected the resources. A.S.A., T.A.A., F.A., and M.A.E. curated the data. A.S.A., T.A.A., M.A.E., F.A., A.H., A.M.O., and K.M.E. wrote, reviewed, and edited the manuscript. A.S.A., M.A.E., and F.A. visualized the study. A.S.A. wrote and prepared the original draft, supervised the study, administered the project, and acquired the funding. All authors have read and agreed to the published version of the manuscript.
Acknowledgments
This research was funded by King Abdulaziz University Institutional Fund Projects under grant no. (IFPRC-170-141-2020). The authors gratefully acknowledge the technical and financial support provided by the Ministry of Education and King Abdulaziz University, Jeddah, Saudi Arabia.
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Abstract
MS-275, a histone deacetylase inhibitor, has proven anticancer activities against various malignancies. However, its clinical application has been constrained by dose-limiting toxicity, off-target effects, and variable clinical outcomes. Clinical data suggest that sustained low MS-275 doses could achieve a more selective and consistent effect. This study aimed at enhancing the anticancer activity of MS-275 by encapsulating it in D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) micelles. The produced nanoformulations were characterized by their low polydispersity (0.201), negative zeta potential (−0.397 mV), and high entrapment efficiency (98.8%). Experimental evaluation of the formulation showed a significant reduction in HepG2, HCT116, and MCF7 cells’ viability, associated with enhanced apoptosis at a lower IC50 compared to MS-275 alone. The formulation was further examined on cancer cells xenografted on the chorioallantoic membrane (CAM) of chick embryos. The results showed a substantial reduction in tumor size. TPGS micelles alone induced an accumulation in G1 and slightly reduced the cellular viability of the examined cell lines. Our results suggest that encapsulating MS-275 in TPGS micelles represents a promising strategy to enhance MS-275 therapeutic impact while minimizing its pharmacological dosage.
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Details
; Ahmed, Tarek A 2
; Ahmed, Farid 3
; Haque, Absarul 1
; El-Say, Khalid M 2
; Omar, Abdelsattar M 2
; Abu-Elmagd, Muhammad 4
1 Department of Medical Laboratory Sciences Faculty of Applied Medical Sciences King Abdulaziz University Jeddah Saudi Arabia; King Fahd Medical Research Center King Abdulaziz University Jeddah Saudi Arabia
2 Department of Pharmaceutics Faculty of Pharmacy King Abdulaziz University Jeddah Saudi Arabia
3 Department of Medical Laboratory Sciences Faculty of Applied Medical Sciences King Abdulaziz University Jeddah Saudi Arabia; Center of Excellence in Genomic Medicine Research King Abdulaziz University Jeddah Saudi Arabia
4 Center of Excellence in Genomic Medicine Research King Abdulaziz University Jeddah Saudi Arabia