Wortmannin

hTERT-molecular targeted therapy of ovarian cancer cells via folate-functionalized PLGA nanoparticles co-loaded with MNPs/siRNA/ wortmannin

Somayyeh Ghareghomi a, Shahin Ahmadian a,*, Nosratollah Zarghami b, Salar Hemmati c
a Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
b Department of Clinical Biochemistry and Laboratory Medicine, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran
c Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

A B S T R A C T

Effective telomerase-molecular targeted cancer therapy might be a promising approach for the efficient treat- ment of ovarian cancer. Therefore, folate-functionalized PLGA nanoparticles (NPs) were co-loaded with hTERT siRNA, Wortmannin (Wtmn), as a potent PI3K inhibitor, and magnetic nanoparticle (MNPs) as a theranostic agent to gain a multifunctional NPs for targeted drug delivery as well as molecular targeted therapy. 1HNMR, FTIR, DLS, FE-SEM and TEM were applied to characterize the synthesized NPs. In vitro discharge pattern for siRNA and Wtmn from the dual drug-loaded NPs showed an early fast release followed by a constant release up to 200 h. According to the MRI analysis, by increasing the concentration of Fe3O4 in NPs, the weaker T2 signal intensity was enhanced, and a considerable contrast was detected in the MRI images. MTT assay and median- effect analysis showed that the Wtmn/siRNA-loaded MNPs-PLGA-F2 NPs display the most synergistic cytotoX- icity on the SKOV-3 ovarian cancer cells. Moreover, the Wtmn/siRNA-loaded MNPs-PLGA-FA NPs could significantly reduce the expression of hTERT, AKT, and p-AKT than the single drug-encapsulated NPs (P < 0.05). Taken together, the findings showed that the multifunctional NPs relying on combinatorial therapy might have considerable potential for effective telomerase-molecular targeted therapy of ovarian cancer. Keywords: hTERT siRNA Wortmannin Magnetic nanoparticles PLGA Molecular targeted therapy Ovarian cancer 1. Introduction Cancer is the second most common cause of mortality worldwide, accounting for nearly 10 million deaths in 2020 [1]. Telomerase is a large ribonucleoprotein complex responsible for the progressive syn- thesis of telomeric DNA repeats (TTAGGG) at the 3′ ends of linear chromosomes, thereby reversing the loss of DNA from each round of replication [2,3]. Telomerase enzyme reactivation in 85% of cancer cells has made it to be proposed as a hallmark [4,5]. This enzyme has the main RNA part (hTR) in eukaryote cells. It has a catalytic subunit known as telomerase reverse transcriptase (hTERT) that is crucial for the syn- thesis of telomeric repeats [6]. Numerous investigations indicated that the regulation of telomerase/TERT has correlations with some cell sig- nalling pathways such as NF-κB, AKT/mTOR, and Wnt/b-catenin [7,8]. The regulation of hTERT/telomerase activity occurs at various levels, including transcription, mRNA splicing, maturation and modifications of hTR and hTERT, transport and subcellular localization of each component, assembly of active telomerase ribonucleoprotein, and accessibility and function of the telomerase ribonucleoprotein on telo- meres [9,10]. According to previous studies, activated AKT (p-AKT) phosphorylates the TERT subunit at specific residues such as serine (227, 824)/threonine/tyrosine. This modification leads to the correct localization of TERT in the nucleus and participates in the structure of the telomerase holoenzyme [11,12]. Some studies state that telomerase activity in various cancer cell lines can effectively be suppressed through targeting the hTERT gene expression by RNA interference (RNAi) [13,14]. Small interfering RNAs (siRNAs), the most commonly used RNA interference (RNAi) tool for inducing short-term silencing of protein- coding genes, are artificially synthesized 19–23 nucleotide long double-stranded RNA [15,16]. The absence of appropriate delivery systems with cancer cells' targeting potency is a major limiting factor in effective targeted siRNA delivery [17]. Wtmn, which acts as a phos- phoinositide 3-kinase (PI3K) inhibitor, can down-regulate the AKT/ mTOR signalling pathway by inhibiting AKT activation. According to some studies, free Wtmn has intense hepatotoXicity and displays poor in vivo stability (Wipf and [18]). Several previous studies indicated that the therapeutic efficacy of Wtmn could be significantly rectified by NP de- livery via increasing its solubility and protecting its bio-active furan moiety from degradation by extracellular amino acids [19,20]. Poly (lactic-co-glycolic acid) or PLGA NPs have been investigated to deliver a wide range of therapeutic molecules, such as small hydrophobic and hydrophilic drugs, peptides, and biological macromolecules, through various routes of administration [21,22]. These NPs enter into cells quickly, are traced via the discharge of their therapeutic agent by passive diffusion and the slow destruction of PLGA [23]. Besides, cell surface receptors for folic acid are often overexpressed on various cancer cells especially ovarian cancer cells [24]. Therefore, in this work, to enhance NP cellular uptake, the PLGA NPs were functionalized with folic acid (PLGA-FA) and then loaded with MNPs, as contrast agents, for thera- nostic imaging through magnetic resonance imaging (MRI). Thera- nostics, which includes the combination of therapy and diagnostic imaging into a single system, is one of the novel aspects of nanotech- nology [25]. Besides, for robust molecular targeting and inhibiting hTERT in SKOV-3 ovarian cancer cells, Wtmn and hTERT siRNA were co-loaded into the MNPs-PLGA-FA NPs. 2. Materials and methods 2.1. Cell line and chemicals SKOV-3 ovarian cancer cell lines (Code: 209) provided from the Cell Bank of Pasteur Institute of Iran were cultured in RPMI-1640 medium (Invitrogen, UK) contain 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Invitrogen, UK) with streptomycin (Merck Co, Germany) and penicillin G (Serva Co, Germany), Wortmannin, folic acid, and hTERT siRNA were obtained from Santa Cruz Biotechnology; MTT [(3,4,5- Dimethyl thiazol-2-yl)-2,5 diphenyl tetrazolium bromide], DMSO (dimethyl sulfoXide), DCM (dichloromethane), methylene chloride, and PVA (Polyvinyl alcohol) were purchased from Sigma-Aldrich (St. Louis, MO, USA); and PLGA (MW: 38000–54,000) was obtained from Evonik (Germany). All the antibodies were obtained from SantaCruz and Elabscience (USA). All other materials were acquired from Sigma- Aldrich. 2.2. PLGA functionalized with folic acid by 1,3 diaminopropane linker For PLGA functionalization with folic acid, we used 1,3 dia- minopropane as a linker according to Wang et al.'s method with some modifications [26]. Based on the findings of the mentioned work, the efficiency of this method is much higher than the old methods (about 46.7%). This synthesis was performed in four steps as follows: 2.2.1. Activation of PLGA In a 100 ml round-bottom three-necked flask equipped with a funnel and N2 atmosphere, 25 ml of dry DCM (dichloromethane) was placed, and 1.5 g (0.0325 mmol, MW 38000–54,000) of PLGA was dissolved in DCM, and the miXture was stirred for 15 min; subsequently, 13.0 mg (0.83 mmol) of EDC was dissolved in DCM (5 ml) and added to the re- action, and the miXture was stirred for 15 min. After adding 90 mg (0.78 mmol) of NHS in 5 ml of DCM, the miXture was stirred for 24 h. The miXture reaction was filtered for removing any insoluble materials. After the filtration, the product was precipitated by shedding into 50 ml of cold diethyl ether: methanol (1:2), pursued by centrifugation and washed with the same solvents. Then, the product was separated and dried in a vacuum oven (40 ◦C) to give 1.2 g of activated PLGA (com- pound.4), a yield of 80% was identified by FT-IR and CHN analysis. 2.2.2. PLGA-CONH(CH2)3 NH2 synthesis In a 50 ml round-bottom three-necked flask equipped with an N2 inlet and a funnel, 1.1 g (0.024 mmol) of activated PLGA was dissolved in dry CHCl3 (15 ml). Then, 2 ml (0.024 mol or 24 mmol) of 1,3dia- minopropane (in 3 ml of CHCl3) was added to the reaction blend and stirred for 12 h at room temperature. The solvent was evaporated by a vacuum rotary evaporator, and the PLGA-CH2CH2CH2NH2 was then precipitated by dropping into 60 ml of cold dimethyl ether-methanol (1:2) and centrifuged (4000 rpm, 5 min). The supernatant was dec- anted, and the polymer was washed further (twice) with diethyl ether and dried under vacuum to yield 0.85 g of product 6. This product was characterized by FT-IR, CHN analysis and Ninhydrin Test for primary amines. 2.2.3. Activation of folic acid A miXture of folic acid (1 g, 2.27 mmol), EDC (0.435 g, 2.24 mmol), and NHS (0.27 g, 2.34 mmol) were prepared in 30 ml of DMSO in a round-bottom flask covered by aluminum foil and stirred for 2 h in the dark. 2.2.4. PLGA-CONH(CH2)3NH-CO-folic conjugate In a 100 ml round-bottom flask covered by aluminum foil, 0.5 g of PLGA-1,3 diamino propane conjugate (compound 6) was dissolved in 5 ml of DMSO; then, the previously prepared solution of activated folic acid (Step 3) was slowly added to the reaction miXture and stirred for 24 h in the dark at room temperature. The solution was concentrated by an efficient vacuum (2–5 mm Hg) at a temperature lower than 50 ◦C for removing the DMSO. Then, the residue was precipitated in cold methanol (or acetone). The precipitates were centrifuged and washed with acetone to obtain PLGA-folic acid conjugated product (PLGA-FA). The final product was freeze-dried and fully characterized by 1HNMR,13CNMR, FT-IR, and CHN analysis. All the steps were summarized in Fig. 1. 2.3. Preparation of MNPs-PLGA-FA NPs The MNPs-PLGA-FA was prepared by applying a double emulsion (W1/O/W2) process with some alterations [27]. To produce the blank MNPs-PLGA-FA, 100 mg of MNPs was dispersed in 2 ml of DI water and sonicated by a Microtip Sonicator at 80 W of energy output in an ice bath for 30 s. Then, using a syringe with a narrow needle, the MNP suspension was added dropwise to 4 ml of FA-PLGA solution with a concentration of 25 mg/ml in DCM/DMSO (75:25). The blend was sonicated twice to obtain the primary water in the oil emulsion. In the next step, using a dropper, 8 ml of 5% PVA (w/v) was added dropwise to the blend and sonicated in an ice bath for 60 s to stabilize the emulsion. Next, the resultant double emulsion (water/oil/water) was diluted with 60 ml of 1% PVA (w/v) and then stirred 3–4 h at room temperature until the organic solvent was disappeared. Finally, the synthesized MNPs-PLGA- FA NPs were collected by ultracentrifugation at 72,000 rpm for 10 min, washed twice with N2-saturated DI water, lyophilized, and stored at 20 ◦C. 2.4. Preparation of Wortmannin/hTERT siRNA-loaded MNPs-PLGA-FA NPs For the preparation of Wtmn-loaded MNPs-PLGA-FA NPs, a certain amount of Wtmn (1:25 drug/polymer) was added to the FA-PLGA and dissolved in DCM/DMSO (75:25) through the emulsification procedure to formulate the hydrophobic drug-loaded MNPs-PLGA-FA. For the incorporation of the hydrophilic agent, before continuing toward the primary emulsion,100 μl of siRNA complex (20 μM) was added to the MNPs' dispersion in water. For the dual drug design of hydrophilic and hydrophobic drugs, Wtmn hTERT siRNA, an equal ratio of the two drugs (1:1) was added in the mentioned steps. To determine the effi- ciency of Wtmn/siRNA encapsulation, after the preparation of drug- loaded NPs, the supernatant of the tube was separated, and the amount of non-entrapped drugs was evaluated by an ultraviolet spec- trophotometer at the siRNA and Wtmn absorbance peak (260 and 254 Particle size, ζ potential and polydispersity (PDI) of the prepared NPs dispersed in PBS (50 mM, pH 7.4) Were determined via Dynamic Light Scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, Malvern, UK). A transmission electron microscope (TEM, Hitachi H-800, Japan) at an accelerating voltage of 10 kV was used to characterize the interior structure of NPs. Besides, the feasible chemical interactions of FA and polymers in the FA-PLGA preparations were approved by FT-IR and NMR spectra. 2.6. In vitro drug release assay The release of siRNA and Wtmn molecules from Wtmn/siRNA-loaded MNPs-PLGA-FA NPs was investigated at 7.4 pH for 168 and 48 h, respectively. In brief, 5 mg of NPs were suspended in PBS (5 ml, pH 7.4), added into a dialysis tubing (MW cut off:3000), and incubated under stirring at 37 ◦C. At various time intervals, the suspension of NPs was centrifuged at 12,500 rpm for 10 min. Then, the supernatants were taken for determining the Wtmn and siRNA amounts, and the same volume of PBS was added for sustained checking of discharge. Detection of Wtmn and siRNA was carried out by UV/visible spectrophotometry at the λmax of Wtmn and siRNA (254 and 260 nm, respectively). 2.7. In vitro MRI study MNPs-PLGA-FA NPs were diluted with PBS to different concentra- tions (0, 0.3, 0.5, 0.7,0.9, and 1.2 mM of Fe) and then were dumped into glass test tubes. Clinical 1.5 T whole-body magnetic resonance system (MAGNETOM Avanto, Siemens, Germany) was applied for T2-weighted MRI analysis of the MNPs-PLGA-FA NPs. For obtained multislice T2- weighted images were as follows: multispin-echo, numerous echo times (TE) from 20 ms to 352 ms, field of view (FOV) 256 190 mm2, repetition tim 3500 ms, section thickness 10 mm, flip angle 180◦, and point resolution 256 256 mm2. Then, the obtained images were processed, and the Sante DICOM Viewer Pro (version 11.8.0) was used to extract the magnitude of signal attenuation through manually drawn regions of interest (ROIs). Relaxivity rates R2 (= 1/T2) were measured for each concentration by the following formula (3) (S0 and S1 stand for constant and signal intensity, respectively): S1 = S0 × ep ( — R2 × TE) (3) 2.8. MTT assay and cytotoxicity determination The cytotoXicity of the siRNA-NPs, Wtmn-NPs and Wtmn/siRNA-NPs on SKOV-3 cells were studied using MTT assay. Briefly, 200 μl of the medium, including a density of 2 10 4 SKOV-3 ovarian cancer cells as folate-receptor positive cancer cells, was seeded into 96-well plates and incubated overnight in a humidified atmosphere containing 5% CO2 to allow the cell attachment before the treatment. Next, the cells were treated with different concentrations of NPs. After 48 h of incubation, the cell culture medium of all wells was removed and substituted with 200 μl of the PBS containing 0.5 mg/ml of MTT and incubated for 4 h at 37 ◦C. Then, All content of the wells was replaced with 200 μl of DMSO and incubated for 20 min. Finally, the absorbance of each well was After 48 h exposure to the drug-loaded NPs, Total cellular RNA isolation was carried out with TRIzol reagent (Invitrogen, Carlsbad, CA) based on the manufacturer's protocol. Then, Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA) was used to synthesize cDNA through 1 μg of total RNA. Quantitative PCR (qPCR) was carried out in a Mic qPCR Cycler (BioMolecular Systems, Australia) using specific primers of hTERT, SYBR green master miX (Roche Di- agnostics, Mannheim, Germany), and cDNA. The real-time PCR reaction included the following steps: initial denaturation phase was set at 95 ◦C for 10 min, followed by cycles of denaturation at 95 ◦C for 15 s, annealing phase at 60 ◦C for 30 s, and extension phase at 72 ◦C for 30 s. The qPCR data were calculated using ∆∆Ct formula. The β-actin gene was applied as a housekeeping control, and all tests were carried out in triplicate. 2.10. Western blot analysis Western blotting was carried out to evaluate the protein levels of hTERT, p-AKT and AKT. After treatment with the NPs for 48 h, the SKOV-3 cells were washed with ice-cold Tris-buffered saline (TBS) twice and lysed by lysis buffer (150 mM NaCl, 1 mM sodium orthovanadate, 0.1% sodium dodecyl sulphate, 50 mM Tris-HCl, 1% NP-40, 0.5% so- dium deoXycholate, and protease inhibitor cocktail). Bradford protein assay was utilized to assess protein concentration in the supernatants of the centrifuged lysates. SDS-PAGE electrophoresis was applied to sepa- rate the proteins in each sample, and then the proteins were electro- blotted to polyvinylidene difluoride (PVDF) membranes. Subse- quently, the membranes were blocked in PBS containing 5 mg/ml of BSA for 40 min at room temperature. Immunoblotting of the membrane was performed at 4 ◦C for 24 h via the following primary antibodies: hTERT (Elabscience, USA) with a dilution of 1:2000, p-AKT (SantaCruz, USA) with a dilution of 1:1000, AKT (Elabscience, USA) with a dilution of 1:2000 and anti-β-actin (SantaCruz, USA). Then, the membrane was washed trice with TBS-T (150 mM NaCl, 20 mM Tris-Cl, 1 g/l Tween20, pH 7.5) and incubated with secondary antibodies. The chem- iluminescence signals were visualized applying an enhanced chem- iluminescence (ECL) western blotting detection kit (Pierce Biotechnology, Rockford, IL). 2.11. Statistical analysis Graph Pad Prism software (version 8.0.2) was used for the statistical investigation of data. The significant difference in the values was determined using ANOVA at the significance level of P 0.05. All the experiments were conducted in three replicates. 3. Results and discussion 3.1. Characterization of FA-PLGA FT-IR, CHN analysis was performed for the characterization of PLGA- NH-CH2CH2CH2-NH2 (Conjugates). In FT-IR analysis (Fig. 2), over- lapping spectra of PLGA (starting material) with the product (PLGA- CONH-CH2CH2CH2-NH2) showed a disappearance in the peak of the carboXylic acid group of PLGA at 1760 cm—1 (CO stretching of end acid group). The appearance of a strong new peak at 1652 cm—1 attributed to CO stretching of amide indicate the creation of an AMIDE bond among PLGA and H2NCH2CH2CH2NH2. Also, the weak peak of OH of carboxylic acid at 3518 cm—1 completely disappeared in PLGA-NH-CH2CH2CH2- NH2, and strong peaks of NH stretching of the end amine group appeared at 3359 and 3285 cm—1. The conjugation of folic acid to PLGA-CONHCH2CH2CH2NH2 (PLGA- linker) was performed by activating folic acid with EDC/NHS (as a coupling reagent), and IR and NMR analysis specified the conjugates to compare starting materials with the conjugate product. The overlapping spectra of starting materials (FA, PLGA-CONH-CH2CH2CH2-NH2) and the conjugate product showed the appearance of vibration frequencies at 1652 cm—1 (amide C–O) and 1563 cm—1 (N–H bond) and the disappearance of the carboXyl group of FA (1701 cm—1) that were absent in starting materials (Fig. 2). The structure of the conjugate product was completely elucidated by 1H and 13C NMR (Fig. 3). The peak at 5.8 ppm protons belongs to lactide, and the peak at 4.5 ppm indicates the gly- colide of PLGA and -CH2-N of folic moiety in the conjugate product. Moreover, peaks at 4.27 and 4.20 ppm indicated the protons of CH in the folic moiety. As shown in the NMR spectrum, FA-linker-PLGA gave rise to a series of peaks at 5.8, 4.48, 4.27, 4.19, 3.95, 3.81, and 1.52 ppm, corresponding to protons linked to the C36, C11, C24, C40, C32, C30, and C38 (methyl group) carbon atoms in the PLGA-linker, respectively. In the spectrum, the signals at 8.6, 7.60, and 6.8 ppm regions were related to the aromatic protons of folic acid (C8, C15, 17, and C14, 18, respectively). The top at 2.4 ppm was associated with the carbon next to amide (C26) protons, thereby confirming the successful conjugation of folic acid to PLGA-linker. 3.2. NP characterization Drug-loaded NPs must be small enough to avoid the reconnaissance and demolition by the immune system and have a prolonged circulation lifespan in vivo [29]. Therefore, measuring the size and diameter dis- tribution of the synthesized NPs was performed through a zeta potential penetrate to cancer cells. On the other hand, NPs with a high enough size are required to escape from macrophage arresting and guarantee effi- cient endocytosis into the cancer cells [32]. Therefore, according to the desired physicochemical properties obtained, the dual drug-loaded MNPs-PLGA-FA NPs can be considered hopeful candidates for internalization into the cancer cells and exerting effective antitumor effects. Further investigations on the size and morphological features of NPs analyzer with the DLS method (Fig. 4A–C and Table 1). As displayed in Table 1, the drug-free NPs exhibited an average particle size of 63.94 nm with a PDI of 0.328 and a zeta potential of 8.93 mV. Nevertheless, the average diameter of Wtmn-loaded MNPs-PLGA-FA NPs, siRNA-loaded MNPs-PLGA-FA NPs, and dual drug-loaded MNPs-PLGA-FA NPs was higher than the drug-free NPs, demonstrating that the encapsulation of therapeutic molecules into the NPs could cause larger particles with the well-knit structures. Zeta potential is a crucial factor for assessing the stability of a colloidal dispersion. The high surface potential of the suspension de- creases the cohesion between the particles, and the particles are ex- pected to be electrochemically stable under the investigated condition after preparation [30]. According to the data, an average surface charge of 8.93 to 3.46 mV was identified for the produced NPs. The exis- tence of PLGA and folic acid on the outer surface creates a negative charge and consequently negative values of zeta potential for NPs. In comparison with larger NPs, smaller average size of NPs have a higher velocity of migration in a known applied electric field and hence have higher values of zeta potential, which can guarantee the stability of the particles in colloidal dispersions after preparation [31]. It has been described that NPs with sizes less than 400 nm can were performed applying FE-SEM and TEM analyses (Fig. 4D and E). The representative FE-SEM image showed that Wtmn/siRNA-loaded MNPs- PLGA-FA NPs were spherical, dispersed uniformly with an average thickness of approXimately 86.5 nm. Besides, the image gained from TEM indicated the presence of MNPs in the PLGA-FA NPs and also confirmed the size and morphology of the NPs described by the FE-SEM. It was detected that the encapsulation efficiencies of Wtmn and siRNA in the formulated dual drug-loaded NPs were 80.5 and 74.28%, with a loading capacity of 12.97 1.8 and 9.71% 2.3, respectively. Wtmn is a low-molecular fungal metabolite with a hydrophobic nature, and this can be the reason for its high encapsulation efficacy compared to the siRNA molecule. 3.3. In vitro drug release The dialysis membrane technique was applied to determine the release pattern of Wtmn and siRNA from the Wtmn/siRNA-loaded MNPs-PLGA-FA NPs at pH 7.4. As displayed in Fig. 5 for the first 6 h, an early fast release was observed for Wtmn, which was continued by a constant release up to 48 h. Besides, for the first 12 h, an early rapid discharge was detected for siRNA, which was continued by a constant release up to 200 h. ApproXimately 75% of Wtmn was released after 48 h while about 58% of siRNA was discharged from the NPs within 8 days. The difference in the released amounts of Wtmn and siRNA from Wtmn/ siRNA-loaded NPs may be related to the discrepancy in the levels of their hydrophobicity. It was confirmed that fast primary discharge is associ- ated with the portion of the molecules loosely connected to the high surface area of the NPs rather than the molecules entrapped within the polymeric NPs [30]. 3.4. MRI analysis Imaging modalities that have been utilized to track the fate of NPs in vivo and gain functional, molecular and structural data concerning the tumour area include fluorescence imaging and positron emission to- mography (PET), computed tomography (CT), magnetic resonance im- aging (MRI), which differ in detection sensitivity, penetration depths, and image resolution [33]. MRI provides supreme soft-tissue contrast for high spatial resolution imaging of structures deep within the body among these imaging devices. MRI contrast agents often administered to increase the contrast between diseased and normal tissues, are divided into two groups: T1-weighted (positive) contrast agents, which produce brighter images by shortening the longitudinal relaxation time of pro- tons; and T2-weighted (negative) contrast agents, which cause darker MRI images through shortening the protons' transverse relaxation time [34]. By lowering the amount of transverse relaxation time T2 and dephasing transverse magnetization, MNPs are identified as a reputable T2-weighted negative contrast agent. Therefore, to determine the MRI efficiency and relaxation time of MNPs-PLGA-FA at numerous concen- trations of Fe, the 1.5 T MRI technique was adopted. According to the findings, by increasing the concentration of Fe in MNPs-PLGA-FA, the weaker T2 signal intensity was enhanced, and a more excellent contrast was detected in MRI images (Fig. 6A). The plot of the relaxation rate, R2 (=1/T2), of MNPs-PLGA-FA as a function of Fe concentration is illus- trated in Fig. 6B. It can be observed that the R2 was linearly commen- surate to the Fe concentrations. Simultaneously, with increasing Fe concentration, the R2 value increased. These results proved that the MNPs in MNPs-PLGA-FA conserved their superparamagnetic properties, suggesting the MNPs-PLGA-FA as a notable T2-weighted contrast agent in MRI. It is in great accordance with the results gained from Fe3O4/ DOX/PLGA-PEG NPs, which exhibited an enhanced T2 contrast mag- netic resonance for the early detection of cancer and accurate drug de- livery via dynamic monitoring using MRI [35]. 3.5. Evaluation of SKOV3 shape and viability The impact of the single and dual drug-loaded NPs treatments was observable, looking at the morphological feature of cells after 48 h of treatment (Fig. 7A). Indeed, cells subjected to Wtmn-NPs, siRNA-NPs and Wtmn/siRNA-NPs seem to lose cell-to-cell junctions as well as cell- to-surface adhesion. Moreover, cells were floating in the culture me- dium. After 48 h exposure time, the cells had detached and shrunk with different debris formations compared to the untreated cells. Besides, the combined cytotoXic efficacy of Wtmn and hTRET siRNA co-liberated from the dual drug-loaded MNPs-PLGA-FA was studied on SKOV-3 ovarian cells by MTT assay. Preliminary data showed the drug-free MNPs-PLGA-FA on SKOV-3 cells have no toXicity at the used concen- trations (data not shown), confirming that the composite nanocarrier was cytocompatible. As shown in Fig. 7B, single- and dual-drug loaded MNP-FA-PLGA NPs presented dose-dependent cytotoXicity on SKOV-3 cells. The IC50 values for Wtmn-loaded MNPs-PLGA-FA NPs and siRNA-loaded MNPs-PLGA-FA NPs were 15.20 μM and 509.4 pМ, respectively, after 48 h of treatment (Table. 2). Also, it was found that the nano-co-delivery of Wtmn and siRNA led to a drastic reduction in IC50 relative to the treatments with single drug-loaded NPs. This strong cytotoXicity can be mainly ascribed to the enhanced cellular uptake of Wtmn/siRNA-loaded MNPs-PLGA-FA NPs through folate receptor- mediated endocytosis, increasing the intracellular concentrations of drugs and lead to an effective combinatorial growth inhibitory effect of Wtmn and siRNA on SKOV-3 ovarian cancer cells. To study the inter- action between hTERT siRNA and Wtmn, a combination effect analysis was done based on Chou and Talalay's method. CompuSyn software was applied to determine the combination index in which CI < 1 indicates synergism, CI = 1 shows additive, and CI > 1 signifies antagonism in drug combinations [36]. As presented in Table. 2, the CI50 value was found to be <1 for Wtmn/siRNA-loaded MNPs-PLGA-FA NPs, indicating their synergistic inhibitory effects on the viability of SKOV-3 cancer cells (Fig. 7C). Collectively, the findings proved that co-loading Wtmn and hTERT siRNA in a multifunctional magnetic-polymeric system might be a promising combination therapeutic strategy for increased antitumor therapy. In consistent with our results, some reports have clarified that using Wtmn via other chemotherapy drugs is much more effective in cancer cell targeting [19,37]. Au et al. engineered sub-50 nm diameter di-block copolymer NPs that can sequentially discharge Wtmn and docetaxel (DTX, genotoXic anticancer agent) to cancer cells to maximize therapeutic efficacy [19]. It was showed that Wtmn enhances the ther- apeutic efficacy of DTX and increases the efficiency of radiotherapy in PC3 prostate cells and H460 lung cancer. The relevant in vivo data showed that the DTX/Wtmn co-loaded NPs are more effective than each single drug-loaded NPs or combination of both single drug-loaded NPs in chemoradiotherapy. Besides, PLGA/PEG-based NP co-delivery of cisplatin (CP) and Wtmn synergistically increases chemoradiotherapy and reverses CP resistance in platinum-resistant ovarian cancer (PROC) [38]. EXposure of platinum-sensitive ovarian cancer (PSOC) and PROC murine models with these Wtmn/CP-encapsulated NPs considerably decreased tumour burden versus treatment with combinations of free drugs or single-drug loaded NPs. 3.6. Expression of hTERT, AKT, and p-AKT in SKOV-3 cells treated with Wtmn/siRNA-NPs Growth evidence has confirmed that blocking telomerase, especially hTERT, is a highly promising approach for cancer treatment by genetic, antisense RNAi as already proved in numerous cancer cells. Zhang et al. transfected a plasmid encoding hTERT-specific shRNAs into human hepatocellular carcinoma cells and detected that they could firmly inhibit hTERT expression, which caused the suppression of cell prolif- eration and an attenuated tumorigenic potency [14]. Dong et al. reported that transfection of encoding hTERT-specific siRNAs into human breast cancer cells could suppress cell proliferation and cause cell apoptosis induction [39]. In recent years, numerous works also confirmed that the knock down of the hTERT through using siRNA efficiently suppressed cell proliferation and telomerase activity and arrested the cell cycle of cancer cells in vitro [40–42]. Shi et al. revealed that siRNA targeting hTERT could efficiently knock down hTERT expression, significantly inhibit cell proliferation, migration and inva- sion, and telomerase activity as well as caused apoptosis induction in cervical cancer cells in vitro [43]. Besides, siRNA targeting hTERT dramatically inhibited tumorigenesis in nude mice and significantly suppressed the constitutive phosphorylation of Akt, PI3K, which might imply that a decrease of hTERT suppressed tumour growth through the PI3K/Akt signalling pathway to some extent. Due to the correlation of PI3K/AKT signalling pathway to post- translational modulation of TERT, Wtmn as a potent PI3K inhibitor can be used in combination with hTERT siRNA for effective telomerase targeting in different levels (Fig. 8). Therefore, following the evaluation of cytotoXicity, the expression of target genes for Wtmn and siRNA, including hTERT, AKT, and p-AKT, were analyzed in SKOV-3 cells treated with Wtmn/siRNA-NPs. Based on the qPCR analysis, after treatment with Wtmn/siRNA-NPs, the mRNA expression of the hTERT gene significantly decreased during a period of 48 h. The findings dis- played that the dual drug-loaded NPs lead to more effective inhibition of the hTERT gene than the single drug-loaded NPs so that the reduction of the hTERT gene was 0.51, 0.8, and 0.1-fold for siRNA-NPs, Wtmn-NPs, and Wtmn/siRNA-NPs, respectively (Fig. 9A). The results displayed that dual drug-loaded NPs have a more significant impact on hTERT gene expression than single drug-loaded ones. Besides, the Western blot method was applied to determine the AKT, p-AKT, and hTERT proteins' levels of SKOV-3 cells treated with Wtmn/ siRNA-loaded NPs (Fig. 9B–E). According to the results, AKT protein level showed no significant change relative to the control group. The expression levels of p-AKT and hTERT in the cells treated with Wtmn/ siRNA co-loaded NPs were more significant than in the control group (P < 0.05). Besides, the Wtmn-NPs diminished the expression of hTERT and p-AKT, where the reduction of p-AKT protein was found to be greater than the hTERT protein levels. Also, siRNA-NPs decreased the expression of hTERT. Also, the Wtmn/siRNA NPs significantly sup- pressed hTERT and p-AKT expression, and the rate of protein reduction was more significant than single drug-loaded NPs (P < 0.05). These results demonstrated that the active targeting and co-delivery of Wtmn and hTERT siRNA through the folate-decorated polymeric NPs leads to an effective targeted molecular inhibition of hTERT synergis- tically in SKOV3 ovarian cancer cells. The current study's findings are in accordance with previous reports, offering that FA-conjugated polymeric NPs exert potent antitumor effect both in vitro and in vivo through selective and efficient delivery of various anticancer agents to tumour cells. It was described that FA- conjugated doXorubicin (DOX)-loaded PLA-PEG-based polymeric NPs a strong boosting effect on cancer cells. Therefore, the molecular mechanisms that amplify each other during tumorigenesis and their targeting by different agents as a novel molecular perspective in cancer therapy merit attention. Further experiments on the efficacy of the platform for inducing apoptosis as well as in vivo investigations can open a new horizon for ovarian cancer treatment. 4. Conclusion In the present study, targeting of hTERT as an important hallmark of cancer was investigated via co-loading of hTERT siRNA and Wtmn into MNFs-PLGA-FA NPs for effectively active targeting and growth inhibi- tion of SKOV3 cancer cells. The results confirmed that combination therapy considering the molecular mechanisms of different drugs exerts and translocates TERT from the nucleus in mouse and human prostate cancer cells via the deactivation of Akt and PKCα, ovarian cancer cells in vitro [44]. 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