A potent formula against Triple-negative Breast Cancer - Sorafenib-Carbon Nanotubes- Folic Acid: Targeting, Apoptosis triggering, and bioavailability enhancing

doi:10.21203/rs.3.rs-3013795/v1

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A potent formula against Triple-negative Breast Cancer - Sorafenib-Carbon Nanotubes- Folic Acid: Targeting, Apoptosis triggering, and bioavailability enhancing

https://doi.org/10.21203/rs.3.rs-3013795/v1

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Triple-negative breast cancer (TNBC) has short survival rates, a high recurrence rate, aggressive clinical behavior, a high risk of metastasis and it is difficult to be targeted by ordinary medicines. This study aimed to (i) prepare a novel formula of sorafenib, carbon nanotubes, and folic acid to be tested as a drug delivery system targeting TNBC with higher safety and efficacy compared with free sorafenib and to (ii) evaluate the formula stability, in vitro pharmacodynamic, and in vivo pharmacokinetic properties. The formula preparation was done by the synthesis of polyethylene glycol bis amine linker, carbon nanotube pegylation, folic acid attachment, and sorafenib loading. The prepared formula has been characterized using XRD, FTIR, ¹HNMR, UV, HR-TEM, FESEM, Zeta sizer and Zeta potential. In vitro studies included drug release determination, MTT assay, flow cytometry to determine the apoptotic stage with percent, cell cycle analysis, and apoptotic marker assays for caspase-3, 8, 9, cytochrome c, and BCL-2. The in vivo study was performed to determine bioavailability and half-life in rats. The in vitro MTT anti-proliferative assay revealed that the formula was 3-fold more cytotoxic towards TNBC cells than free sorafenib, and the flow cytometry showed a significant increase in apoptosis and necrosis. The formula has a greater inhibitory effect on BCL-2 and a lessening effect on cytochrome c and caspases 3, 8, and 9 than free sorafenib. Moreover, in vivo experiments proved that our novel formula was superior to free sorafenib by increasing bioavailability by eight times and prolonging the half-life by three times. These results confirmed the successful preparation of the desired formula with better pharmacodynamic and pharmacokinetic properties. These promising results may show a novel therapeutic strategy for TNBC patients.

carbon nanotubes CNTs

sorafenib

polyethylene glycol PEG

folic acid

triple negative breast cancer TNBC

Female breast cancer (BC) has now surpassed lung cancer as the leading cause of global cancer incidence, with an estimated 2.3 million new cases, representing 11.7% of all new cancer cases in 2020.¹ It is the fifth-leading cause of cancer mortality among women worldwide, with 685,000 deaths in 2020. Breast cancer accounts for 1 in 4 cancer cases and for 1 in 6 cancer deaths, ranking first for incidence in the vast majority of countries.¹ Breast cancer's main treatment strategies are surgery; radiation therapy; hormonal therapy by targeting the hormonal receptor; and chemotherapy by blocking the proliferation in many different ways according to the type of drug used and the types of receptors or enzymes of the type of cancer.² Unfortunately, all have a number of side effects such as lymphedema, neuropathic pain, fatigue, hair loss, muscle pain, bone pain, nausea, vomiting, and increased chance of infection due to low white blood cell counts.^3–6

Breast cancer has four subtypes: luminal A, which has estrogen receptors and a lot of progesterone receptors; luminal B, which has estrogen receptors and may have a few progesterone receptors or not; HER2-overexpression, which has not any estrogen or progesterone receptors but has HER2 receptors; and basal-like (triple-negative) breast cancer (TNBC), which does not have any of these receptors that could be blocked by an ordinary antagonist, and that is why it`s difficult to be targeted by hormonal drugs. TNBC accounts for about 15% of all BC cases, with short survival rates (most deaths occur within the first 5 years) and a high recurrence rate (in just 3 years), as well as aggressive clinical behavior and a high risk of metastasis. Because of these reasons, it has been urgent to find a way to treat TNBC. But unfortunately, TNBC is difficult to be targeted by hormonal drugs, also because of the side effects and difficulties of radiation and surgical therapy, chemotherapy has been the logical way to be used in the treatment of TNBC.⁷

Sorafenib is an FDA-approved drug for the treatment of hepatocellular cancer, so it has a degree of efficacy and safety. Sorafenib works both outside and inside the cells by (i) blocking VEGFR and reducing the vascular supply permeability and cell survival and (ii) FGFR1, PDGFRP, KIT, RET, and FLT3 in the cancer cells themselves, and (iii) working against RAF1 and BRAF enzymes to block cell proliferation, differentiation, migration, invasion, and metastasis (prevent angiogenesis and induce apoptosis).^8–11 It has been proven that sorafenib is a multikinase inhibitor and could improve the survival of breast cancer patients by inhibiting their invasive and metastatic properties because it has some cytotoxic effect and anti-proliferative activity on breast cancer cells by inhibiting the invasion and migration of breast cancer cells in vitro. Sorafenib increased mitochondrial superoxide production while suppressing breast cancer stem cell self-renewal, inhibiting epithelial-mesenchymal transition, and inhibiting ERK signalling.¹² Thus, sorafenib has some activity against breast cancer, especially against TNBC MDA-MB-231 cell lines.^13–15 Unfortunately, sorafenib still has poor pharmacokinetic qualities and some side effects like leukopenia, neutropenia, anaemia, diarrhoea, nausea, pain, sensory neuropathy, hand-foot skin reaction, and skin rash or acne.⁸ Furthermore, the efficacy of sorafenib used alone is limited not only by their systemic toxicity and narrow therapeutic window but also by drug resistance and limited cellular penetration. Thus, the development of efficient delivery systems with the ability to enhance cellular uptake of existing potent drugs is needed.

Nanoscale formulas have been developed for drug delivery applications to increase absorbability and biological activity due to their size distribution and large surface area.¹⁶ The utilization of nanoparticles as drug delivery vehicles has a lot of advantages, such as improving the pharmacokinetic profile, increasing absorption, utilization, and stability, helping medicine target, having a higher circulation time in comparison with the administration of free drugs, and having lower toxicity as a consequence of the lower dosages employed in the nanovehicles.^17,18

Over the last three decades, there has been an enormous advantage of using biotechnology and nanotechnology within the medical fields in several ways. Among these techniques is the use of the carbon nanotubes (CNTs) which proved its efficacy in enhancing the kinetic properties by increasing absorption and half-life time. Further, CNTs can easily cross the cytoplasmic membrane and nuclear membrane due to a needle-like shape that facilitates transmembrane penetration and intracellular accumulation.^19,20 An anticancer drug transported by CNTs will be liberated in situ with intact concentration, and consequently, its action in the tumor cell will be higher than that administered by traditional therapy alone. So, CNTs proved its efficacy in enhancing the dynamic properties by increasing intracellulization with the drug-loaded. It has been known that functionalized CNTs seem to have a high propensity to cross cell membranes. The high aspect ratio of CNTs offers great advantages over the existing delivery vectors because the high surface area provides multiple attachment sites for drugs and the chemistry of CNTs offers the possibility of introducing more than one function on the same tube, so that targeting molecules and drugs can be attached at the same time.²¹

Poly ethylene glycol (PEG) is preferred as a promising candidate for the surface decoration of CNTs due to its assured safety in humans, good biocompatibility, and aqueous solubility, yet also because of its exceptional ability to connect a large range of biologically active agents. Also, it has been proved that PEG improves biodistribution and adjusting the clearance from the body by adjusting the formula size to avoid renal filtration.^22,23 The studies showed that the PEG increases hydrodynamic size of nanoparticles NPs, which in turn inhibits the clearance by liver and kidney, thereby prolonging the circulation time.²⁴ PEG enhances the circulatory half-life with no protein binding.²⁵ Moreover, PEG helps in drug deliver.²⁶ On the other hand, folate receptors are overexpressed by BC and TNBC to help the cells get folic acid, which is necessary for their proliferation. So, it has been helpful to use different formulations containing folic acid moieties to target BC with fewer side effects on normal cells.^27–29

Herein the objectives of this work are as follows: 1. the preparation of a formula of CNTs connected to polyethylene glycol PEG to improve kinetic properties and also connected to folic acid to target TNBC; and the drug sorafenib is physically loaded on this formula, which could be used as a drug delivery system targeting TNBC with greater safety and efficacy than standard drugs. 2. Evaluate the effect of the formula from the point of view of its (i) stability properties, (ii) pharmacokinetic properties (bioavailability and half-life), and (iii) pharmacodynamics in vitro.

2.1. Materials

Sorafenib, 1-(3-dimethyl amino propyl)-3-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma Aldrich. Carboxylated multiwall carbon nanotubes (MWCNTs-COOH) was obtained from PSAS, Beni-Suef University. PEG, chloroacetyl chloride (CCA), ethylenediamine, folic acid, dimethyl sulfoxide (DMSO), hexane, dichloromethane, ethanol, methanol, acetonitrile, KH₂PO₄, and diethyl ether were bought from Vikaash Chemicals, India. Acetonitrile, ethyl acetate, methanol, potassium dihydrogen orthophosphate, and dialysis bag MWCO 12KD–14KD were purchased from LOBA CHEMIE in India. Phosphate buffered saline (PBS) at PH 7.4 was prepared according to the published protocol.³⁰ All used chemicals and solvents were of analytical grade and high purity. We used nitrogen gas to achieve N₂ atmosphere. A rotary-evaporator, a heating magnetic stirrer, an ultrasonicator, centrifugate, a paddle-type dissolution test apparatus and YL9100 HPLC system (Republic of Korea) have also been used. Mammalian cell lines: MDA-MB-231cells (epithelial, human breast cancer cell line) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Fetal Bovine serum, MTT, and trypan blue dye were purchased from Sigma (St. Louis, Mo., USA). DMEM, HEPES buffer solution, L-glutamine, gentamycin, and 0.25% Trypsin-EDTA were purchased from Lonza.

2.2. Synthesis of the formula

2.2.1. Synthesis of PEG bis amine linker

To a dissolved PEG (5 g, 1 equiv.) in 110 ml of hexane/dichloromethane (1:1) under a nitrogen atmosphere, chloroacetyl chloride (0.4 ml, 6 equiv.) was added gradually and the reaction mixture was stirred for 24 hours at room temperature. Then, 14 ml ethanol was added and the system was kept under reflux for 1 hour. Finally, the reaction mixture was concentrated under vacuum to 20 ml and about 0.2 ml was taken from the crude mixture to the ¹HNMR to measure the structures of ClCH₂-CO-PEG-CO-CH₂Cl (the intermediate compound in PEG bis amine linker synthesis). Ethylenediamine (0.6 ml, 10 equivalents) was added to the crude reaction mixture at room temperature and stirred for 48 hours. The final product was purified by precipitation with diethyl ether (250 ml), followed by slight vacuum filtration and a small amount was taken to the ¹HNMR to measure the structure of NH₂-PEG-NH₂ (PEG bis amine linker).³¹ Scheme 1 shows the synthesis equations for PEG bis amine linker.

2.2.2. Pegylation of CNTs

MWCNTs-COOH (500 mg) were activated by EDC (500 mg) and NHS (300 mg) in DMSO (20 ml) for 3 hours under a nitrogen atmosphere with magnetic stirring to fully activate the carboxyl groups on the MWCNTs. A DMSO-PEG-NH₂ solution (500 mg in 10 ml) was added dropwise to the above mixture. After 3 days under a nitrogen atmosphere, the reaction mixture was dialyzed against phosphate-buffered saline (PBS) at PH 7.4 (3 times, 2 l) and deionized water (6 times, 2 l) for 3 days with a dialysis membrane (MWCO 12–14 KD) and left to dry in the air to acquire MWCNT-PEG-NH₂.²⁹

2.2.3. Attaching of folic acid

Covalent immobilization of FA onto MWCNT-PEG via the formation of an amid bond is therefore desirable. In brief, FA (400 mg) was activated by EDC (200 mg) and NHS (120 mg) in DMSO (10 ml), and the reaction was kept under stirring for 3 hours under a nitrogen atmosphere. The activated FA was then added to MWCNT-PEG-NH2 (500 mg) suspended (with sonication) in DMSO (5 ml) under nitrogen atmosphere, and the reaction was kept under stirring for 5 days, Then the reaction mixture was dialyzed against PBS at PH 7.4 (3 times, 2 l) and deionized water (6 times, 2 l) for 3 days with a dialysis membrane (MWCO 12–14 KD) and left to dry in the air to acquire MWCNT-PEG-FA.²⁹

2.2.4. Drug Loading on The PEGylated CNTs

Sorafenib loading on the functionalized MWCNTs was achieved via physical adsorption. In brief, 410 mg of MWCNT-PEG-FA in 15 ml ethanol were left in ultrasound for 30 minutes before adding 30 mg of sorafenib in 5 ml ethanol and stirring for 1 week. The product was collected by repeated centrifugation (at 15000 rpm for 30 min) until the supernatant became colorless. The precipitated product was obtained, and the supernatant was measured by UV to determine the amount absorbed.²²

2.3. Characterization techniques

Using CuK_α radiation at 40 kV and 30 mA, x-ray diffraction (PANalytical Empyrean, Netherlands) was used to analyze the crystalline phase of the SFN, MWCNT, and MWCNT-SFN in the 5–50° range. Flourier-transform infrared (FT-IR) analysis was carried out using a Bruker (Vertex 70 FTIR, Germany). The scanning resolution was adjusted at 1 cm^− 1, and the average of spectra were taken after three scans. Proton magnetic resonance spectroscopy (¹HNMR) was measured for ClCH₂-CO-PEG-CO-CH₂Cl (the intermediate compound in PEG bis amine linker synthesis), and NH₂-PEG-NH₂ (PEG bis amine linker) using the ¹HNMR instrument Bruker (Ascend 400 MHZ with Avance III HD console). A UV-Vis spectrophotometer (Shimazu 3600, Japan) was used to measure the UV-Vis spectra of the final formula and the intermediates (MWCNT-COOH, MWCNT-PEG-NH2, FA, MWCNT-PEG-FA, sorafenib, and MWCNT-FA-sorafenib). The size distribution of the prepared nanoformula was estimated using Malvern Instruments Ltd., (Malvern, UK) in a 0.05 mg/ml sample concentration suspended in PBS (pH 7.4). In order to detect the influence of various chemical modifications on the MWCNTs' surface charge, the zeta potential was estimated using the same instrument.

The microstructural details, morphological characteristics, and size distribution of the MWCNT-PEG-FA-sorafenib, were investigated using a high-resolution transmission electron microscope (HRTEM) (JEOL, JEM 2100, Japan). While the topographical features, fracture surface details, elemental analysis (EDAX), and elemental distribution (mapping) of the same formula were investigated by using field emission scanning electron microscopy (FESEM) (Zeiss Sigma 500-VP, Germany).

2.4. Determination the drug loading content and entrapment efficiency EE%

The formulated SFN-MWCNT-FA was centrifuged at 15,000 rpm for 30 min. The amount of unbound drug present in the supernatant was measured to determine the amount of absorbed SFN. The absorbance of the diluted supernatant solution was measured at λ_max 265 nm using a single beam UV spectrophotometer as mentioned, the blank was methanol solvent. Drug content was calculated by subtracting the amount of free SFN from the total amount of SFN used and dividing by the total amount of SFN used in the synthesis (the starting drug loaded).

$$\text{E}\text{E}\text{%}=\frac{\text{T}\text{o}\text{t}\text{a}\text{l} \text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \text{o}\text{f} \text{d}\text{r}\text{u}\text{g} \text{a}\text{d}\text{d}\text{e}\text{d} – \text{T}\text{h}\text{e} \text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \text{o}\text{f} \text{f}\text{r}\text{e}\text{e} \text{d}\text{r}\text{u}\text{g} \text{N}\text{o}\text{n} \text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{e}\text{d}}{\text{T}\text{o}\text{t}\text{a}\text{l} \text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \text{o}\text{f} \text{d}\text{r}\text{u}\text{g} \text{a}\text{d}\text{d}\text{e}\text{d}}x100 Eq.1$$

2.5. Determine the drug release

In vitro release of SFN itself and SFN from the formulated formula through a dialysis bag was performed at room temperature with a paddle-type dissolution test apparatus as described in the literature. In brief, 500 ml of simulated pH 7.4 was added, and the experiment was run for 12 hours, with a three-ml aliquot aspirated and filtered every 1-hour interval to measure absorbance against a corresponding blank.

2.6. Bio-study

2.6.1. In vitro measurements

The MTT assay was used to assess the anti-proliferative activity of the free SFN and MWCNTs- PEG-FA- SFN formula against MDA-MB-231cells (human breast cancer cell line).

Cell line Propagation: The cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer, and 50 µg/ml gentamycin. All cells were maintained at 37 ^oC in a humidified atmosphere with 5% CO₂ and were sub-cultured twice a week. Cytotoxicity evaluation using the viability assay: For antitumor assays, the tumour cell lines were suspended in a medium at a concentration of 5x 104 cells per well in Corning® 96-well tissue culture plates, then incubated for 24 hours. The tested compounds were then added to 96-well plates (three replicates) to achieve twelve concentrations for each compound. Six vehicle controls with media or 0.5 percent DMSO were run for each 96-well plate as a control.

After incubating for 48 hours, the numbers of viable cells were determined by the MTT test. Briefly, the media was removed from the 96-well plate and replaced with 100 µl of fresh culture on RPMI 1640 medium without phenol red, then 10 µl of the 12 mM MTT stock solution (5 mg of MTT in 1 ml of PBS at PH 7.4) was added to each well, including the untreated controls. The 96-well plates were then incubated at 37°C with 5% CO2 for 4 hours. An 85 µl aliquot of the media was removed from the wells, and 50 µl of DMSO was added to each well and mixed thoroughly with the pipette. The wells were incubated at 37°C for 10 minutes. Then, the optical density was measured at 590 nm with the microplate reader (SunRise, TECAN, Inc., USA) to determine the number of viable cells, and the percentage of viability was calculated as [(ODt/ODc)]x100%, where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The relation between surviving cells and drug concentration is plotted to get the survival curve of each tumour cell line after treatment with the specified compound. The 50% inhibitory concentration (IC₅₀), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose-response curve for each concentration using GraphPad Prism software (San Diego, CA, USA).³²

2.6.2. Apoptotic stage and percent assay by flow cytometry

2.6.2.1. Apoptosis analysis (Annexin V-FITC assay)

Apoptotic cells were further analyzed by Annexin V-FITC assay. Control and treated cells were collected on day 2. Briefly, MCF-7 (breast carcinoma) cells were cultured to a confluent monolayer and then treated with the tested samples at the concentrations described earlier (500, 200, and 100 µg/ml). After treatment for 48 hours, the cells were then harvested and rinsed twice in ice-cold phosphate buffered saline (PBS) (20 min. each). The cells were centrifuged at 1500 g for 5 min and resuspended in 500 ml of binding buffer (BD, Franklin Lakes, NJ). Moreover, MCF-7 cells (treated or untreated) were re-suspended in 100 µl of kit binding buffer with the addition of 1 µl of FITC-Annexin V (Becton Dickinson BD PharmingenTM, Heidelberg, Germany), followed by a 40-min incubation at 4°C. Cells were then washed and re-suspended in 150 µl of binding buffer with the addition of 1 µl of DAPI (1 µg/ml in PBS) (Invitrogen, Life Technologies, Darmstadt, Germany). Then, the cells were analyzed using the flow cytometer BD FACS Calibur (BD Biosciences, San Jose, CA).³³

2.6.3. Cell cycle analysis by flow cytometry

To determine the effect of the tested active samples on the cell cycle distribution of the MCF-7 cell line, cell cycle analysis was performed using the CycleTESTTM PLUS DNA Reagent Kit (Becton Dickinson Immunocytometry Systems, San Jose, CA). The MCF-7 cells (treated with the tested samples or untreated) were stained with propodium iodide stain following the procedure provided by the kit and then run on the cytometer. Cell-cycle distribution was calculated using Cell Quest software (Becton Dickinson Immunocytometry Systems, San Jose, CA).³³

2.6.4. Determine the pathway of apoptosis via cytochrome c, BCL-2, caspase 3,8&9 enzyme assay

2.6.4.1. ELISA immunoassay:

Caspase activation is a common apoptotic mechanism by which anticancer agents induce apoptosis. Thus, the level of caspase activation was determined to elucidate the mode of action of untreated cells (normal control), sorafenib-treated cells, and the formula (sorafenib-loaded MWCNT-PEG-FA) nanocarrier. Briefly, MCF-7 cells were cultured as a monolayer and then treated with compounds at the IC₅₀ concentration as described earlier. Cells were then harvested through trypsinization and rinsed twice in PBS. The analysis of the apoptotic markers caspase-3, caspase-8, caspase-9, and cytochrome C levels as well as the anti-apoptotic marker BCL-2 levels were assessed using ELISA colorimetric kits according to the manufacturer's instructions.³³ The difference in caspases activities during apoptosis was determined by comparing the results of sorafenib-treated cells and the formula (Sorafenib-loaded MWCNT-PEG-FA) nanocarrier treated cells to the level of untreated cells (normal control).

2.7. Pharmacokinetics in vivo

To determine the formula's bioavailability and half-life, the pharmacokinetic behaviour of the drug-loaded MWCNTs was evaluated using ten healthy male Wistar rats (weighing 160 ± 15 g). The animals were randomly divided into two groups (5 rats each) and allowed to acclimatize for one week under regular environmental conditions (temperature, 22 ± 2°C; humidity, 50 ± 5%; night/day cycle, 12 hours) with free access to an ordinary rodent diet and tap water ad libitum. Subsequent to a 12-hour fasting period, animals in the first group were given Sorafenib (1.5 mg/kg, per os), while animals in the second group were given MWCNTs-FA-SFN (1.5 mg/kg SFN-equivalent, per os). After oral administrations, blood samples were collected from each animal via the retro orbital plexus at predetermined time intervals.

These blood samples were centrifuged at 10,000 rpm for 15 minutes, and plasma was isolated and kept frozen at -20°C until analysis. The plasma SFN concentration in each sample was quantified using a YL9100 HPLC system (Republic of Korea) consisting of a quaternary gradient pump, a UV detector, a G137PA automatic degasser, a standard auto-sampler, a column oven, a chromatography workstation, and a Kromasil C18 column (250 mm × 4.6 mm length × internal diameter, 5 µm particle size). The used mobile phase consisted of potassium dihydrogen phosphate (20 mM) and acetonitrile (35:65, v/v) (pH 6.3) with a flow rate of 1 ml/min. According to these chromatographic conditions, the SFN concentration in the collected plasma samples was determined at 265 nm after sample handling according to a previously described processing method.

Briefly, 50 µl of each plasma sample was added to 50 µl acetonitrile in an Eppendorf tube to precipitate proteins. Subsequent to the 1-minute vortex, each tube was centrifuged at 10,000 rpm for 10 minutes, and the obtained supernatant was transferred into a glass tube containing 250 µl deionized, purified water. Then, SFN was extracted by adding 1 ml of ethyl acetate. After mechanical shaking for 5 min, each tube was centrifuged at 4000 rpm for 10 min, and the obtained supernatant was transferred into a 5 ml glass tube to be evaporated until dryness in a 25°C water bath under a nitrogen stream. The obtained residue was dissolved in 100 µl methanol before being injected into the chromatographic system in a 20 µl aliquot. Using PKSolver 2.0, a non-compartmental analysis of plasma data after extravascular input using a linear trapezoidal method was done to estimate the pharmacokinetic parameters.

3.1. The rationale design of the formula

As sorafenib alone have poor pharmacokinetic properties and a lot of side effects,⁸ the nanotechnology is a new trend used to enhance the traditional medicines’ kinetic and dynamic effects.¹¹ The goal of this formula is to synthesis a PEG bis amine linker and use it to connect MWCNT to folic acid, and the drug (Sorafenib) is physically loaded on this formula, which could be used as a drug delivery system targeting TNBC with greater safety and efficacy than traditional drugs.

The role of each component is briefly discussed in the following sentences. (i) MWCNTs have efficacy in enhancing the pharmacokinetic properties by increasing absorption and half-life and also enhancing pharmacodynamic properties by increasing intracellulization with the drug-loaded thereon due to a needle-like shape that facilitates transmembrane penetration and intracellular accumulation.^19,20 (ii) PEG works as a decorating agent for MWCNTs due to its assured safety in humans, good biocompatibility and aqueous solubility. Also, it has been proved that PEG improves biodistribution and adjusting the clearance from the body by adjusting the formula size to avoid renal filtration.^22,23 In addition PEG works as a linker because of its exceptional ability to connect a large range of biologically active agents.^22,23 (iii) PEG bis amine linker has amine groups at the two ends of the PEG polymer and here we could use it as a linker by using the amine groups of one side of PEG bis amine linker to react with the carboxylic group of the carboxylated MWCNTs and the amine group of the other side to react with the carboxylic group of the folic acid. (iv) Folic acid helps in targeting the folate receptors in breast cancer cells with a fewer side effects on normal cells.^28,29

3.2. X-ray diffraction

The X-ray diffraction patterns of SFN, MWCNT, and SFN-MWCNT are shown in Fig. 1. Figure 1(a) represents the XRD pattern of free SFN, which shows a sharp reflection at 2θ = 11°, 15°, 21°, 22°, and 22° and several other reflections, suggesting their crystalline nature. In addition, MWCNT reveals its crystalline nature as XRD patterns of MWCNT show a sharp reflection at 2θ = 25° (Fig. 1(b)). Fig. Figure 1(c) shows a slight decrease in the crystalline nature of the SFN -loaded MWCNTs. A few sharp reflections at 2° of 19° and 23° matched with SNF with slight right shifting when compared with free SNF reflection patterns, confirming SNF loading onto the MWCNT. For the SFN-MWCNT, the crystalline reflections of SNF were observed to be embedded in the crystalline MWCNT phase.

3.3. FTIR spectra analysis

The structure of the final product and synthesis intermediates were measured by FT-IR measurement as presented in Fig. 2. The FT-IR spectra of MWCNTs-COOH (Fig. 2(a)), shows a characteristic peak at 1709, 1573 and 1466.59 cm^− 1, related to C-O, C = O and C = C functional groups of the carboxylic group, respectively. The wide peak in the region of 3374.93 cm^− 1 is assigned to the -OH (carboxylic acid) group, and the two peaks at 2916.9 and 2852.8 cm^− 1 are the absorption peaks of C-H stretching. The FT-IR spectra of MWCNT-PEG-CONH-C₂H₂-NH (Fig. 2(b)) shows a new peak at Fig. 09 cm^− 1 related to the C-N functional group. The wide peak in the region of 3396.9 cm^− 1 is assigned to the -NH (secondary amine) group and the -OH of the remaining COOH groups on the MWCNT. and the peak at 2878.2 cm^− 1 is the absorption peak of C-H aliphatic bond stretching of the MWCNT and PEG. The FT-IR spectra of folic acid shown in Fig. 2(C), the broad peak at 3542 cm^− 1 is assigned to the -OH (carboxylic acid) group, and the two peaks at 3414 and 3324 cm^− 1 are the absorption peaks of NH₂ primary amine functional groups. The peak at 1695 cm^− 1 related to the C = O of the carboxylic group, and the peak at 1608 cm^− 1 related to the C = O of the amide group. The FT-IR spectra of MWCNT-PEG-FA presented in Fig. 2(d) shows a peak at 1096 cm^− 1 related to the two C-N groups from the binding amide sites on the PEG. The peak at 1596 cm^− 1 related to the C = O of the carboxylic group, the peak at 1473 cm^− 1 related to the C = O of the amide group, the peak at 2876 cm^− 1 is the absorption peak of C-H aliphatic bonds stretching in the MWCNT and PEG, and the wide peak in the region of 3416 cm^− 1 is assigned to the -OH (carboxylic acid) group. The FT-IR spectra of the SFN (Fig. 2(e)) shows peaks from 1124 to 1318 cm^− 1 related to the C = O functional group of (-NH-CO-NH-). The two different peaks at 3080 and 3288cm^− 1 are the absorption peaks of the –NH functional group of (-CO-NH-phenolic) and (-CO-NH-methyl) due to the two different chemical environments around the NH. The FT-IR spectrum of MWCNTs-PEG-FA-SFN illustrated in Fig. 2(f) shows all of the characteristic peaks in all of the individual FA and SFN compounds.

3.4. ¹HNMR

The structures of ClCH₂-CO-PEG-CO-CH₂Cl (the intermediate compound in PEG bis amine linker synthesis) and NH₂-PEG-NH₂ (PEG bis amine linker) were measured by ¹HNMR measurement. As clear in Fig. 3(a), which represents the intermediate product, the Peak of CH2Cl of CCA part and of CH2-CH2 of PEG part appears at 4.08 PPM and 3.61 PPM. While in Fig. 3(b), which represents the final product of this synthesis step, we could observe the disappearance of the peak of CH2Cl of CCA part at 4.08 PPM but still have the peak of CH2-CH2 of PEG part at 3.61 PPM.

The absence of the peak at 4.08 PPM assigned to CH2Cl groups of CCA part could be interpreted as these protons might appear overlapped by the signal of (CH₂–CH₂) groups of PEG due to the release of chloride group after the amination process, which suggests the success of the amination process.³¹

3.5. Surface morphology and elemental analysis

The HR-TEM image of the prepared formula (sorafenib-loaded MWCNT-PEG-FA) was shown in Fig. 4(a), indicating the longitudinal axis of the MWCNTs with a diameter of 20 nm. The FESEM images in Fig. 4 (b &c) depict the prepared formula's surface details, topographical features, and tangled MWCNTs. Further, the MWCNTs-PEG surface appeared rough and irregular due to SFN adsorption. Figure 5(a) illustrated the EDX spectrum of the prepared formula. The spectrum showed the content of C, O, N, f, and Cl elements with different weightings in percent. The presented elements confirmed the entities in the formula. Figure 5(b) demonstrates the spatial distribution of each element in the formula matrix.

3.6. Zetasizer and zeta potential measurement

The main concern in the drug delivery application of nanoscale formula is preventing the elimination of the nanoformula by the reticuloendothelial system and prolonging its circulation time in the blood to enhance the bioavailability at the target. In this regard, the clearance rate of the nano formula changes with increasing size.^16,34 Using DLS, the mean hydrodynamic diameter size distribution (Z-average (d. nm.)) of SFN was determined to be 3094 nm, with a PdI of 1.00 and an Intercept of 0.461. The polydispersity index (PDI value of 1.00) predicted a high width, thus reflecting a heterogeneous size distribution, whereas the intercept value reflected a poor signal-to-noise ratio.

The measured Z-average (d. nm.) of the prepared formula was 197 nm, Fig. 6 with a PdI of 0.483 and an intercept of 0.942. It is important to highlight that CNT needs two dimensions to be measured: length and diameter, and will have two characteristic diffusion times: one along the tube’s axis and one perpendicular to it. However, the concept of using DLS to measure the size distribution is based on the assumption that the measured particles have a sphere shape. So, the Zetasizer assumed the formula was a sphere, and that is not the diameter of CNT.

The size of the formula was within an acceptable nanometer range, as this size is an appropriate size to deliver the drug into the cells. Other studies showed NPs could internalize at small sizes in the range of 15–30 nm, 30–50 nm, and 70–240 nm, while larger sizes could be internalized by phagocytosis.³⁴ Also the size of the formula was within an acceptable nanometer range to prolong the half-life by reducing the renal clearance and protect the NPs from being eliminated by the reticuloendothelial system and to prolong its circulation time in the blood, thus enhancing the bioavailability at the target.³⁴

To detect the influence of various chemical modifications on the MWCNTs' surface charge, the zeta potential, one of the benchmarks of stability of the colloidal system, was determined for all MWCNT dispersions. The oxidized, aminated PEG, PEG-loaded folic acid and SFN-loaded MWCNTs were negatively charged in the aqueous solution. As in Table 1, The oxidized MWCNTs showed a negative zeta potential (-23.9 mV) most likely due to the insufficient ionization reaction of carboxylic acids in water. The aminated PEGylated MWCNTs showed a negative zeta potential (-41.1 mV), which is lower than that of the oxidized MWCNTs. A possible explanation for this phenomenon could be the resonance effect of amide. Specifically, aminated PEG functionalization shifted the value of the zeta potential to negative. The negative zeta potential of aminated PEGylated MWCNTs has been postulated to be attributed, in part, to the long hydrocarbon chain of PEG. The additional increase in the negative value of zeta potential in the case of MWCNTs-FA (-45.3 mV) could be due to the presence of folic acid. The additional change in the value of zeta potential in the case of the formula MWCNTs-FA-SFN (-28.1 mV) could be due to the presence of the adsorbed SFN tosyl group. High positive or negative charges increase the repulsive interaction, increasing particle stability by preventing aggregation.³⁵ So the resulting formula value of -28.1 mV seems good enough to maintain the dispersion of the formula.

Table 1

Zeta potential results of: oxidized CNTs, CNT-PEG-NH2, CNT-PEG-FA and CNTs-FA-SFN
Compound	Values of zeta potential
oxidized CNTs	-23.9 mV
CNT-PEG-NH2	-41.1 mV
CNT-PEG-FA	-45.3 mV
CNTs-FA-SFN	-28.1 mV

3.7. UV-Analysis

As shown in Fig. 7, the characteristic peak of the standard SFN was observed in the UV–vis spectra at λ = 265 nm, as well as the remnant SFN in the supernatant remaining after its absorption into the formula. According to Beer–Lambert law:

$$A=\epsilon lc eq.2$$

where A is the absorbance, ε is the molar attenuation coefficient or absorptivity of the attenuating species, $l$ is the optical path length in cm, and c is the concentration of the attenuating species. The absorbances of the standard SFN and the supernatant were 0.95 and 0.86 dil mol^− 1 cm^− 1, respectively. Given that the standard SFN concentration was 1 mg/ml and $l$ was 1 cm, so the $\epsilon$ was calculated to be 0.95 dil mol^− 1 cm^− 1. According to Beer-Lambert law, the calculated concentration of the supernatant by the following equation $0.86 =0.95x1c$, so the C (supernatant) = 0.91 mg/ml.

Calculating the absorbed concentration was done by using the following equation:

$${C}_{\left(\text{a}\text{b}\text{s}\text{o}\text{r}\text{b}\text{e}\text{d}\right)}={C}_{0} – {C}_{\left(\text{s}\text{u}\text{p}\text{e}\text{r}\text{n}\text{a}\text{t}\text{a}\text{n}\text{t}\right)} eq.3$$

knowing that C₀ = 30 mg SFN in 20 ml ethanol = 1.5 mg/ml and C(supernatant) = 0.91 mg/ml.

So, the C(absorbed) = C₀ – C(supernatant) = 1.5–0.91 = 0.59 mg/ml

Calculating the amount of the SFN absorbed into the formula was done by knowing that for the initial concentration of 1.5 mg/ml, there is an absorption of 0.59 mg/ml and by knowing that the start loading amount was 30 mg, so the absorbed amount was found to equal 11.84 mg of SFN absorbed on 410 MWCNT (in 422 mg of total formula). This means that there is 11.84 mg of sorafenib in every 422 mg of the formula.

The EE% was 39.47% according to, indicating that SFN could be trapped within the formula.

3.8. SFN release profile

As in Fig. 8, The in vitro release profile of SFN from the formula was assessed at pH 7.4. Under these alkaline conditions, which mimic those in the blood (pH 7.4), there was a relatively slower release of the loaded drug from the formula than that of the drug itself, allowing efficient completion within 12 hours in the formula instead of 7 hours in SFN alone. SFN appears to be released from the new formula in two stages: a relatively rapid burst release followed by a characteristic slow-release rate lasting up to 12 hours, indicating a sustained release pattern. After 7 hours, the percentage of free SFN cumulative release was approximately 100 percent, whereas after this 7 hours the percentage of SFN formula showed a sustained release pattern of approximately 85 percent and take a total 12 hours to reach approximately 100 percent. This delayed release of SFN from the formula is most likely due to the adsorption of the SFN on the MWCNTs, achieving the sustained release pattern.

3.9. In vitro measurements

3.9.1. MTT assay

The cytotoxic activity of the developed formula against MDA-MB-231 cells was assessed using the MTT assay and was compared with that of the conventional SFN alone, which served as the positive control. As in Fig. 9, The obtained results revealed that the viability of the MDA-MB-231 cells was significantly reduced after 48 hours of incubation with both the free drug and MWCNTs-FA-SFN, with corresponding IC50 values of 2.91 µg/ml and 0.86 µg/ml, respectively. This apparent triple increase (338.37% ) in the anti-proliferative activity of SFN towards TNBC cells after being loaded onto the functionalized MWCNTs indicates enhanced bioavailability, which could be due to (i) the feasible delivery of the nanosized MWCNTs-SFN inside the cells with a subsequent more efficient and enhanced pharmacodynamics by faster intracellular release and accumulation of SFN from the formula than that of the free SFN alone and (ii) also could be due to endocytosis by the affinity of the folic acid moieties to the folate receptor on TNBC.²⁷

These results are in agreement with previous studies that reported the improved bioavailability of anticancer agents other than SFN via their binding to MWCNTs.^36,37 Additionally, our results are consistent with other recent investigations that reported increased cellular uptake and, consequently, enhanced anticancer activity of SFN via its loading onto nanosystems other than CNTs.³⁸ Of note, the previously reported limited anti-proliferative activity of CNTs-PEG towards cancer cells proves to a large extent that the developed cytotoxicity of the novel formula is attributed only to the loaded SFN.²² These findings are in accordance with a recently published work in which PEG-functionalized CNTs were described as non-cytotoxic.³⁹ These data collectively revealed that the PEGylated MWCNTs could be used as drug carriers and that the SFN-loaded formula could be a promising potent system for in vivo TNBC treatment.

3.9.2. Flow cytometry measurements

As shown in Fig. 10, the apoptotic analysis using flow cytometry shows a significant increase in early apoptosis, late apoptosis, and necrosis in the cells treated with the formula (sorafenib-loaded MWCNT-PEG-FA) at IC50 after 48 hours of treatment (Fig. 10(a)) by a percent of 15.05, 4.41, and 2.49%, respectively, and in the cells treated with free SFN at IC50 after 48 hours of treatment (Fig. 10(b)) by a percent of 22.63, 19.67, and 4.72%, respectively, when compared to the control cells (Fig. 10(c)) which were 0.43, 0.17, and 1.01%, respectively.

On the other hand, Fig. 11 shows the spectrum of the DNA cell cycle analysis. The high point of the peak of the spectra reflects the intense propidium iodide (PI) fluorescence emission, reflecting the DNA content, while the width of the peak indicates the size and complexity of the DNA content. The results revealed a significant decrease in G0/G1 phase and S phase and a significant increase in the G2/M phase in the cells treated with the formula (Fig. 11(a)) and in the cells treated with SFN (Fig. 11(b)) when compared to the control cells (Fig. 11(c)). In total, the spectra revealed that both free SFN and the formula arrested cell growth at the G2/M cell cycle phase.

3.9.3. Apoptotic marker measurements (caspase 3,8,9, cytochrome c & BCL-2)

The anticancer agents mediate the apoptotic pathway through caspase activation.⁴⁰ As shown in Fig. 12(a), the activated caspase-3, 8, and 9 in SFN-treated cells and the formula SFN-loaded MWCNT-PEG-FA nanocarrier-treated cells were significantly increased; this reflected that SFN and the formula have activity against the proliferation of the cells. As observed, the measured caspases 8 revealed a significant difference among the SFN and the formula and a significant pattern in caspase 9, which reflects the intrinsic pathway of apoptosis. Furthermore, as shown in Fig. 12(b and c), the activities of the apoptotic marker’s cytochrome c and BCL-2 were significantly different in the SFN-treated cells and the formula-treated cells. Both SFN and the formula induce apoptosis by increasing the levels of cytochrome C and inhibiting BCL-2. As observed the formula has more inhibition effect on BCL2, which represent the intrinsic pathway of apoptosis than Sorafenib and less effect on cytochrome c and caspase 3,8&9,⁴¹ which represent the extrinsic pathway of apoptosis than sorafenib and that may be due to the ability of the formula to get the drug into the cells. Moreover, sorafenib could inhibit BCL2.⁴² also this result could be supported by the published reports that proved DNA damage and the effect of gene expression of the cancer cells by sorafenib nanoformula.¹⁵ To sum up, SFN markedly suppressed the proliferation of MCF-7 cells and induced cytotoxicity, which favors the induction of apoptosis. However, the targeted drug delivery of the formula to tumor cells enhanced the apoptosis-induced cell death compared to the control.

3.10. Pharmacokinetics in-vivo study

According to the obtained results, the used HPLC method was found to be satisfactory in terms of its sensitivity and specificity, with no interference with SFN determination from the endogenous substances in plasma under the selected chromatographic conditions. The obtained chromatograms and the average plasma SFN concentration-time profiles following administrations of the various formulations are shown in Fig. 13, and the corresponding pharmacokinetic parameters are listed in Table 2 and in Fig. 14.

Table 2

Mean Pharmacokinetic Parameters for SFN in Rat Plasma Following Administrations of the Oral Solution of SFN and the formula
Parameter (Unit)	Value of SFN	Value of Formula
C_max (µg/ml)	15.39	27.31
T_max (h)	4.00	12.00
K_e (1/h)	0.10	0.03
t_1/2 (h)	6.72	21.71
AUC_0 − t (µg/ml*h)	134.00	710.32
AUC_{0 − inf_obs} (µg/ml*h)	136.03	1107.73
MRT_{0 − inf_obs} (h)	8.73	35.29
F_rel	--------	814.32%
Vz/F_obs (mg/kg)/(µg/ml)	0.11	0.04
Cl/F_obs (mg/kg)/(µg/ml)/h	0.011	0.001

The C_max in rat plasma following administrations of an oral suspension of SFN and the formula were 15.36 and 27.31 µg/ml, respectively, revealing an approximately 1.77-fold increase. The mean residence time (MRT) for the formula was 4.04-fold higher than that of the SFN. And AUC_{0 − inf} of SFN and the formula were (136.03 and 1107.73) µg/ml*hour, respectively, nearly eight-times higher in the formula when compared with the oral administration of the SFN, the F_rel (relative bioavailability) of the SFN from the formula was about 814.31%. This superior bioavailability of the formula could be accredited to the augmented permeability as a result of (i) the influence of CNTs as a penetration enhancer due to a needle-like shape that facilitates transmembrane penetration, increasing absorption, which is consistent with the published reports. (ii) The use of CNT which proved its efficacy in enhancing the kinetic properties as well as intracellular accumulation into the cancer cells. (iii) Also, PEG is known to reduce the formula clearance from the body.^19,20,22,23

As manifested by the higher T_max value, the formula achieved slow absorption. The T_max of the SFN and the formula were 4 and 12 hours, respectively, as the recognized sustained in vitro release pattern. Additionally, the half-life (T_1/2) of SFN was three times prolonged after its administration in this new formula in comparison with conventional SFN alone (21.71 versus 6.72 hours, respectively). This could be an effect of (i) the drug wrapping into the formula, which avoids the metabolic action of CYP enzymes, allowing the drug to stay longer in the systemic circulation. (ii) As with several published papers, the use of CNT proved its efficacy in enhancing the kinetic properties by increasing the half-life and absorption by intracellular accumulation. (iii) Also, PEG is known to reduce the formula clearance from the body.^20,22,23 Overall, the formula system provided more sustained release and exhibited a prolonged residence time, superior bioavailability, and a higher half-life as compared with the SFN alone.

In conclusion, our results have confirmed the successful preparation of the desired formula (SFN-loaded on MWCNTs-PEG-FA), the pharmacodynamic studies proved the distinctive cancer-suppressive nature of the newly designed formula, which was (3-times) more effective against TNBC cells in vitro, and the pharmacokinetic studies disclosed a markedly increased bioavailability (8-times) besides the prolonged half-life (3-times), also increasing T_max, C_max, and MRT of the optimized formula as compared with SFN alone. These promising results may shed light on a novel therapeutic strategy for efficient delivery of SFN to TNBC. This is in agreement with several published reports that confirm that the nano formula loaded with the drugs is an effective potential candidate in comparison with free drugs. The following are the study's outcomes: 1. Improving TNBC treatment. 2. enforcing the concept of drug delivery administration. 3. Demonstrating the impact of biotechnology and nanotechnology in the medical field. Nevertheless, further preclinical investigations are still needed to ascertain that the formula could targets the cancer cells with fewer side effects and superiority and efficiency of the formula over oral SFN forms. Moreover, delivery of anti-cancer drugs with the use of nanobiotechnology techniques could be the world's next step to further enhance the therapeutic efficacy to treat the cancer cells and reduce the side effects.

*Authors' contributions

The authors confirm their contribution to the paper as follows: A. G. conceived the idea. A. G., A. Z., W. R., and H. N. participated in the study design. W. R. and H. N. prepared the materials. A. G. and H. N. analyzed and interpreted the data. A. G. and H. N. wrote the manuscript. All the authors revised and approved the final version of the manuscript to be published and agreed to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

*Funding

The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript. The standing research paper was maintained through individual funding.

*Availability of data and material

The authors emphasize the availability of data and materials

*Statements and Declarations

*Ethics approval and consent to participate

The authors followed the ethics of research, approved, and consented to participate in this study

*Consent for publication

The authors approved this version of the manuscript for publication

*Competing interests

The authors declared no competing interests

This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue.

*Data Sharing Statement

The authors emphasize the availability of data and materials

Sung H et al (2021) Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 71:209–249
Yildizhan H et al (2018) Treatment strategies in cancer from past to present. in Drug Targeting and Stimuli Sensitive Drug Delivery Systems 1–37 doi:10.1016/B978-0-12-813689-8.00001-X
Collins KK et al (2011) Effects of breast cancer surgery and surgical side effects on body image over time. Breast Cancer Res Treat 126:167–176
Pérez M, Schootman M, Hall LE, Jeffe DB (2017) Accelerated partial breast irradiation compared with whole breast radiation therapy: a breast cancer cohort study measuring change in radiation side-effects severity and quality of life. Breast Cancer Res Treat 162:329–342
Berkowitz MJ et al (2021) How patients experience endocrine therapy for breast cancer: an online survey of side effects, adherence, and medical team support. J Cancer Surviv 15:29–39
Kayl AE, Meyers CA (2006) Side-effects of chemotherapy and quality of life in ovarian and breast cancer patients. Curr Opin Obstet Gynecol 18:24–28
Gautam P (2017) Chemical systems biology studies of triple negative breast cancer cell lines.
Abdelgalil AA, Alkahtani HM, Al-Jenoobi FI (2019) Sorafenib. Profiles of Drug Substances, Excipients and Related Methodology, vol 44. Academic Press Inc., pp 239–266
Bronte G et al (2017) Sorafenib for the treatment of breast cancer. Expert Opin Pharmacother 18:621–630
Gong L et al (2017) PharmGKB summary: Sorafenib pathways. Pharmacogenet Genomics 27:240–246
Sheng X et al (2017) Preparation, pharmacokinetics, tissue distribution and antitumor effect of sorafenib-incorporating nanoparticles in vivo. Oncol Lett 14:6163–6169
Dattachoudhury S, Sharma R, Kumar A, Jaganathan BG (2020) Sorafenib Inhibits Proliferation, Migration and Invasion of Breast Cancer Cells. Oncol 98:478–486
Nagaria TS et al (2017) Combined targeting of Raf and Mek synergistically inhibits tumorigenesis in triple negative breast cancer model systems. Oncotarget 8:80804–80819
Zafrakas M, Papasozomenou P, Emmanouilides C (2016) Sorafenib in breast cancer treatment: A systematic review and overview of clinical trials. World J Clin Oncol 7:331–336
Abdellatif AAH et al (2022) Formulation of polymeric nanoparticles loaded sorafenib; Evaluation of cytotoxicity, molecular evaluation, and gene expression studies in lung and breast cancer cell lines. Nanotechnol Rev 11:987–1004
Bhatia S (2016) Natural polymer drug delivery systems: Nanoparticles, plants, and algae. Nat Polym Drug Delivery Systems: Nanopart Plants Algae. 10.1007/978-3-319-41129-3
Baeza A, Ruiz-Molina D, Vallet-Regí M (2017) Recent advances in porous nanoparticles for drug delivery in antitumoral applications: inorganic nanoparticles and nanoscale metal-organic frameworks. Expert Opin Drug Deliv 14:783–796
Sui J et al (2017) Synergistic chemotherapeutic effect of sorafenib-loaded pullulan-Dox conjugate nanoparticles against murine breast carcinoma. Nanoscale 9:2755–2767
Fabbro C et al (2012) Targeting carbon nanotubes against cancer. Chem Commun 48:3911–3926
Sanginario A, Miccoli B, Demarchi D (2017) Carbon Nanotubes as an Effective Opportunity for Cancer Diagnosis and Treatment. Biosensors vol. 7 at https://doi.org/10.3390/bios7010009
He H et al (2013) Carbon nanotubes: Applications in pharmacy and medicine. Biomed Res. Int. (2013)
Elsayed MaMA et al (2019) Design and characterisation of novel sorafenib-loaded carbon nanotubes with distinct tumour-suppressive activity in hepatocellular carcinoma. Int J Nanomedicine 14:8445–8467
Suk JS, Xu Q, Kim N, Hanes J, Ensign LM (2016) PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Advanced Drug Delivery Reviews vol. 99 28–51 at https://doi.org/10.1016/j.addr.2015.09.012
Jokerst JV, Lobovkina T, Zare RN, Gambhir SS (2011) Nanoparticle PEGylation for imaging and therapy. Nanomedicine 6:715–728
Dos Santos N et al (2007) Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: Relating plasma circulation lifetimes to protein binding. Biochim Biophys Acta - Biomembr 1768:1367–1377
Verhoef JJF, Anchordoquy TJ (2013) Questioning the use of PEGylation for drug delivery. Drug Deliv Transl Res 3:499–503
Low PS, Henne WA, Doorneweerd DD (2008) Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Acc Chem Res 41:120–129
Vinothini K, Rajendran NK, Ramu A, Elumalai N, Rajan M (2019) Folate receptor targeted delivery of paclitaxel to breast cancer cells via folic acid conjugated graphene oxide grafted methyl acrylate nanocarrier. Biomed Pharmacother 110:906–917
Yan Y et al (2018) Stacking of doxorubicin on folic acid-targeted multiwalled carbon nanotubes for in vivo chemotherapy of tumors. Drug Deliv 25:1607–1616
Chazotte B (2012) Labeling Golgi with fluorescent ceramides. Cold Spring Harb Protoc 7:913–915
Icart LP et al (2016) PLA-b-PEG/magnetite hyperthermic agent prepared by Ugi four component condensation. Express Polym Lett 10:188–203
Mosmann T (1983) Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J oflmmunologicalMethods vol. 65
Eldehna WM et al (2018) Novel [(3-indolylmethylene)hydrazono]indolin-2-ones as apoptotic anti-proliferative agents: design, synthesis and in vitro biological evaluation. J Enzyme Inhib Med Chem 33:686–700
Foroozandeh P, Aziz AA (2018) Insight into Cellular Uptake and Intracellular Trafficking of Nanoparticles. Nanoscale Res Lett 13,
Mahalingam M, Krishnamoorthy K (2015) Fabrication, physicochemical characterization and evaluation of in vitro anticancer efficacy of a novel pH sensitive polymeric nanoparticles for efficient delivery of hydrophobic drug against colon cancer. J Appl Pharm Sci 5:135–145
Sajid MI et al (2016) Carbon nanotubes from synthesis to in vivo biomedical applications. Int J Pharm 501:278–299
Zakaria AB et al (2015) Nanovectorization of TRAIL with single wall carbon nanotubes enhances tumor cell killing. Nano Lett 15:891–895
Tang S, Li Y (2019) Sorafenib-Loaded Ligand-Functionalized Polymer-Lipid Hybrid Nanoparticles for Enhanced Therapeutic Effect Against Liver Cancer. J Nanosci Nanotechnol 19:6866–6871
Habibizadeh M, Rostamizadeh K, Dalali N, Ramazani A (2017) Preparation and characterization of PEGylated multiwall carbon nanotubes as covalently conjugated and non-covalent drug carrier: A comparative study. Mater Sci Eng C 74:1–9
Fulda S, Debatin KM (2006) Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene vol. 25 4798–4811 at https://doi.org/10.1038/sj.onc.1209608
Cao L, Quan XB, Zeng WJ, Yang XO, Wang MJ (2016) Mechanism of hepatocyte apoptosis. J Cell Death 9:19–29
Younis NS, Ghanim AMH, Saber S (2019) Mebendazole augments sensitivity to sorafenib by targeting MAPK and BCL-2 signalling in n-nitrosodiethylamine-induced murine hepatocellular carcinoma. Sci Rep 9,

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A potent formula against Triple-negative Breast Cancer - Sorafenib-Carbon Nanotubes- Folic Acid: Targeting, Apoptosis triggering, and bioavailability enhancing

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Synthesis of the formula

2.2.1. Synthesis of PEG bis amine linker

2.2.2. Pegylation of CNTs

2.2.3. Attaching of folic acid

2.2.4. Drug Loading on The PEGylated CNTs

2.3. Characterization techniques

2.4. Determination the drug loading content and entrapment efficiency EE%

2.5. Determine the drug release

2.6. Bio-study

2.6.1. In vitro measurements

2.6.2. Apoptotic stage and percent assay by flow cytometry

2.6.2.1. Apoptosis analysis (Annexin V-FITC assay)

2.6.3. Cell cycle analysis by flow cytometry

2.6.4. Determine the pathway of apoptosis via cytochrome c, BCL-2, caspase 3,8&9 enzyme assay

2.6.4.1. ELISA immunoassay:

2.7. Pharmacokinetics in vivo

3. Results and discussion

3.1. The rationale design of the formula

3.2. X-ray diffraction

3.3. FTIR spectra analysis

3.4. 1HNMR

3.5. Surface morphology and elemental analysis

3.6. Zetasizer and zeta potential measurement

3.7. UV-Analysis

3.8. SFN release profile

3.9. In vitro measurements

3.9.1. MTT assay

3.9.2. Flow cytometry measurements

3.9.3. Apoptotic marker measurements (caspase 3,8,9, cytochrome c & BCL-2)

3.10. Pharmacokinetics in-vivo study

4. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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3.4. ¹HNMR