Encapsulated Docetaxel in Synthesized PCL-PEG-PCL Nano-Carriers as a Strategy for Controlled Drug Delivery in Breast Cancer

doi:10.21203/rs.3.rs-3092787/v1

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Research Article

Encapsulated Docetaxel in Synthesized PCL-PEG-PCL Nano-Carriers as a Strategy for Controlled Drug Delivery in Breast Cancer

https://doi.org/10.21203/rs.3.rs-3092787/v1

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Background

Nano-drug delivery systems are rapidly evolving in the treatment of cancer due to reduced side effects and improved therapeutic properties of drugs through the drugs’ slow release and as a result, enhancing permeability and retention of drugs.

Methods and Results

Encapsulated docetaxel in PCL-PEG-PCL nano-carrier (P1) was prepared by the “modified nano-precipitation” method. TEM, AFM, and DLS were used for character evaluation. HPLC and dialysis bags were also used to evaluate the amount of drug release and drug encapsulation. The MCF-7, MDA-MB231, and fibroblast cell lines were treated with different concentrations of the synthesized nano-carriers loaded DTX and free DTX over 48 and 72 hours. The MTT assay was used for investigating cytotoxic effects. The Annexin-V/PI staining and Hoechst-33342 staining were performed for apoptosis assay. The characterization techniques showed the designed nano-carrier has suitable properties for carrying drugs. The percentages of encapsulation, drug loading, and drug releasing were obtained by the HPLC technique. The MTT-assay results showed that inhibition of MCF-7 and MDA-MB231 cell growth and proliferation by P1 during 72 hours is significantly higher than 48 hours. The apoptosis assays showed the cytotoxic effects of synthesized nano-carriers that cause apoptosis cell death.

Conclusion

The results showed that DTX is effectively encapsulated by PCL-PEG-PCL nano-carriers, which can increase the solubility and bioavailability of the drug by high penetration into the cell. The nano-carrier P1 significantly caused early apoptosis of MCF-7, and MDA-MB231 cells. Accordingly, the IC50 of P1 on MCF-7 and MDA-MB231 cells in 72 hours was reported higher than at 48 hours. Based on the results obtained, it can be concluded that controlled drug release from the designed nano-carriers has been achieved and induction of apoptotic cell death has occurred.

Breast Cancer

Controlled Drug Delivery

Docetaxel

Nano-Carrier

PCL-PEG-PCL

Currently, cancer is a severe disease for humans, and with the change in the lifestyle of society, more and more people get it every year. Chemotherapy is one of the most common treatments for cancer, but due to problems such as the gradual creation of multiple drug resistance (MDR), tumor metastasis, low bio-availability and the numerous and severe side effects of chemotherapy drugs, restrictions on its use exist [1–3]. The MDR provides the ability for cancer cells to minimize the effects of chemotherapy drugs by resisting the toxicity of drug agents during treatment [3]. Initially, to reduce the side effects of the pharmaceutical agents and to prevent drug resistance, combined therapies were suggested using two or more medications with different or similar classes of treatment (with lower doses) [1]. Although there have been successes in the combined use of several drugs, this method has not been able to introduce a definitive treatment for a wide range of cancers due to its inability to control the amount and proportion of each drug [1]. Today, depending on the type of cancer and its progress, the combined use of cancer treatment methods such as surgery, chemotherapy, radiation therapy, hormone therapy, etc. are being used [4]. The series of applications of nanobiotechnology in the targeted drug delivery base suggests that nano drugs may increase drug effects and potentially reduce drug side effects effectively. Targeted drug delivery is an efficient strategy for locally accumulating drug agents in tumor sites [5]. Challenges of conventional chemotherapy have led to the improvement of exact drug delivery systems based on nano-carriers, also known as smart drug delivery systems (SDDSs) [3]. Depending on the type, base material, and synthesis method, the nano-carrier can reside in the bloodstream for a prolonged period, so it has more chances to reach the target tissue, and also release the drug gradually [3]. The trapping of anticancer agents not only causes an efficient delivery of the drug to the target site but also decreases the side effects of the drug [4]. Therefore, the trapping of anticancer drugs in polymer nano-carriers can be an excellent way to improve the effectiveness of anticancer drugs [6]. Although nanoparticles (NPs) are the basis of nano-carriers, not all nanoparticles can be safe and effective carriers for drug agents [7]. Polymeric micelles are an absorbing platform for enhancing the bioavailability and solubility of insoluble / low-soluble medicines in water. Polymeric micelles can be used to target solid tumors by Enhancing permeability and retention (EPR). The structure of polymeric micelles is in the form of cores/shells, which are usually formed by the self-arrangement of amphiphilic copolymer blocks [6, 8]. Biocompatible and biodegradable two-block and three-block copolymers containing hydrophilic and hydrophobic parts are widely used in the medical industry, especially in targeted drug delivery. The Poly ε-caprolactone-poly ethylene glycol-poly ε-caprolactone (PCL-PEG-PCL) three-block copolymer, which includes one central hydrophilic (PEG) strand and two hydrophobic strings (PCL), can form micelles because of existing hydrophobic interactions [9, 10]. The PEG strands on the micelle form a hydrophilic shell, that due to the hydrophilicity of most biological environments in nature, can reduce the absorption of opsonin and clearance rates by a single-core phagocytic system and increase blood circulation time. The outstanding benefits of this copolymer include high stability, the formation of small-sized micelles, high drug loading, ease of surface modification, biocompatibility, and biodegradability [11, 12]. The nano-carriers use physiological differences between cancer cells and healthy cells (such as cell growth rate) to identify cancerous tissues. There are two main approaches to identifying target tissue: 1. Passive targeting, 2. Active targeting. Passive targeting takes place on the nano-carrier surface without the use of active receivers and is based solely on the EPR effect. In contrast, active targeting uses one or more active agents that can recognize and interfere with the target cell [3]. A targeted drug delivery system that operates actively can reach predetermined organs and cells and release the drug, using molecules attached to the surface, such as saccharides, peptides, antibodies, aptamers, and so on [5]. Docetaxel (DTX) with the brand name “Taxotere” is a powerful anti-mitotic chemotherapy drug that prevents the growth and spread of cancer cells in the other tissues by inhibiting micro polymerization of microtubules [4, 13, 14]. Docetaxel is used to control and treat advanced or metastatic breast cancer, prostate cancer, stomach cancer, lung cancer, and head and neck cancer of the cervix [5, 15]. The clinical form of DTX has side effects such as paresthesia, pain, and burning sensation, weakness, fluid retention, alopecia, nausea, diarrhea and vomiting, anemia, neutropenia, leukopenia, allergic reactions, etc [15, 16]. The use of Tween 80 and ethanol to facilitate the solubility of DTX in aqueous solutions has caused these side effects. Therefore, dexamethasone is used in patients treated with DTX [13]. Docetaxel has a hydroxyl functional group on carbon number ten, which increases its solubility in aqueous solutions compared to other taxane-based drugs such as paclitaxel. However, the solubility of DTX in aqueous solutions has been reported to be approximately 5–6 µg/ml with a low bioavailability [4, 15]. Numerous studies have been performed on DTX-induced apoptotic cell death [17, 18]. Apoptosis is a major cellular process that promotes the normal growth of tissue, which is essential for removing old and worn cells [19]. Apoptosis is also a way to remove cancer and malignant cells [20, 21]. The main features of this process are cell shrinkage, condensation the of nucleus and cytoplasm, and the porosity and fragmentation of the membrane that eventually leads to nuclear fragmentation and cell death [22]. Uncontrolled growth of breast tissue cells called breast cancer covers the milk glands and their canals and continues to grow by spreading across the body through blood and lymph nodes. Breast cancer is the most common malignancy type of invasive cancer in the world after liver cancer. One in eight women has been reported to have breast cancer in their lives [4, 23, 24]. The present study aimed to synthesize nano-carriers of PCL-PEG-PCL and load the DTX using the modified “modified nano-precipitation” method. In this study, particle size, particle scattering, zeta potential, drug loading percentage, encapsulation percentage, the drug release rate of nanoparticles synthesized and their cytotoxic effects, and also apoptosis induction on Michigan cancer foundation-7 (MCF-7), M.D. Anderson metastatic breast 231 (MDA-MB231), and fibroblast cell lines were examined.

Basic Materials

DTX, PCL-PEG-PCL, Hoechst-33342 dye, and Phosphate buffered saline (PBS) tablet were bought from Sigma-Aldrich. Methanol, acetone, water, acetonitrile (HPLC grade), and an Annexin-V/PI kit were purchased from MERK. Other chemical materials were purchased from other companies.

Synthesis of DTX-loaded Polymeric Micelles

The blank NPs [PCL-PEG-PCL/(P0)] and DTX-loaded NPs [DTX-PCL-PEG-PCL/(P1)] have been prepared by a modified nano-precipitation method [12, 25]. In this method, the acetone solvent is used as the main solvent. In summary, 16 mg of PCL-PEG-PCL copolymer and 4 mg of DTX were dissolved in 2 ml of acetone to prepare nano-carriers containing DTX. The organic phase was added by the syringe (G22) to 25 ml of distilled water under constant stirring. A magnetic stirrer stirred the resulting suspension until complete evaporation of the solvent and the formation of micelles. The suspension was filtered by a 0.45 µm filter membrane to remove the free drug. Finally, the loaded nano-carriers were frozen at a pressure of 14 Pa at -78°C and then stored at 4°C for later use. The blank NPs were synthesized in the same way without the use of DTX.

Characterization of Nano-carriers

The particle size, polydispersity index (PDI), and zeta potential of DTX-loaded NPs, and blank NPs were measured by dynamic light scattering (DLS). Atomic Force Microscopy (AFM) and transmission electron microscopy (TEM) were used for morphological observation. One drop of the blank NPs and one drop of the DTX-loaded NPs solution were placed on a substrate (1 cm²), followed by air drying.

Drug Loading Content (%DL) and Encapsulation Efficiency (%EE)

The percentage of drug loading and drug encapsulation was studied using the lyophilized powder dissolved in 10 ml of methanol. Then 40 µl of this solution was injected into HPLC. The DTX content in the drug-loaded NPs was determined using high-performance liquid chromatography (HPLC). The HPLC analysis such as the percentage of drug loading and drug encapsulation was carried out using a system equipped with a C-18 reversed-phase column (150×4.6 mm, five µm, C-18; KNAUER Technologies). Methanol was used for %EE and %DL due to being the mobile phase of HPLC for identifying DTX. First, three times, standard DTX diagrams were drawn using 3 different concentrations. The DTX detection wavelength was 227 nm, and the elution rate was 1.0 mL/min. The mobile phase was a mixture of water and acetonitrile, with a volume rate of 50:50. The encapsulation efficiency and drug loading percentage were measured by two methods. In the first method, the lyophilized powder was used, and in the second method, the total volume of the sample was used to measure the encapsulation efficiency and drug loading percentage. In fact, in the first method, the encapsulation efficiency and drug loading percentage were calculated directly, while in the second method, the amount of unloaded drug was calculated. Then the encapsulation efficiency and the drug loading percentage were calculated. The drug-loading content (%DL) and drug entrapment efficiency (%EE) were calculated based on the following formula;

$${\text{% DL = }}\left( {{\raise0.7ex\hbox{${{\text{weight of drug in micelles}}}$} \!\mathord{\left/ {\vphantom {{{\text{weight of drug in micelles}}} {{\text{weight of micelles}}}}}\right.\kern-0pt}\!\lower0.7ex\hbox{${{\text{weight of micelles}}}$}}} \right) \times 100$$

$${\text{EE% = }}\left( {{\raise0.7ex\hbox{${{\text{weight of drug in micelles}}}$} \!\mathord{\left/ {\vphantom {{{\text{weight of drug in micelles}}} {{\text{weight of initial drug}}}}}\right.\kern-0pt}\!\lower0.7ex\hbox{${{\text{weight of initial drug}}}$}}} \right) \times 100$$

In vitro Drug Release Studies

Drug release from nano-carriers synthesized by dialysis bag was investigated. Initially, 100 ml of PBS (PH 7.4), containing 2% (v/v) of tween 80 with a ratio of 1:10 (v/v) ethanol was prepared [26]. Then, 10 mg of dry powder was spread in 4 ml of PBS. The resulting suspension was immersed in 40 ml of PBS after being placed inside the dialysis bag (Mw KDa 12) and incubated at 37°C. At scheduled time intervals, 2 mL of samples were withdrawn and replaced by 2 ml PBS. The above experiment was repeated with pH = 5.5 to review the sensitivity of nano-carrier release to pH. The release of free drug (DTX) was studied in PBS with pH 7.4, similar to ECF (Extracellular fluid), and pH 5.5, similar to tumor microenvironment as a control. All the release studies were carried out in triplicate.

Cell Culture

The MCF-7, MDA-MB231, and human skin fibroblast cells (HSF) were grown in RPMI 1640 medium and enriched with 10% fetal bovine serum (FBS), 100 U/ml penicillin-streptomycin, at 37°C in a 95% air-humidified-5% CO2 incubator. The cells were passaged 2 to 3 times weekly and regularly checked for contamination and impurity. For research objectives, cells were cultured in 96-well plates. The appropriated cell concentration, as determined by the growth protocol of the cell line, was 5000 cells/well [19].

MTT-assay

The effect of DTX as a free drug, P0 as a drug-free nano-carriers, and P1 as nano-carriers containing DTX on living cells were investigated using the MTT assay. MCF-7, MDA-MB231, and fibroblast cell lines were plated in 96-well plates. Free drugs of DTX, P0, and P1 at different concentrations (25–200 µg/ml) were used to evaluate cytotoxicity. After 48h and 72h of incubation, MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide) solution was injected into each well (0.5 mg/ml). Then, the cells were incubated for 4 hours to assess the severity of cytotoxicity caused by free drugs and nano-carriers. The living cells that have active mitochondrial succinate dehydrogenase can transform MTT into formazan crystals. After aspirating the supernatants, 200 µl of dimethyl sulfoxide (DMSO) was added to each well. The plates were incubated in the dark for an additional 10 min, and the microplate reader read the absorbance values at 570 nm [19]. Also, half maximal inhibitory concentration (IC50) was calculated using GraphPad Prism. The number of viable cells was measured using the following formula.

$${\raise0.7ex\hbox{${mean{\text{ }}OD{\text{ }}of{\text{ }}treated{\text{ }}cells}$} \!\mathord{\left/ {\vphantom {{mean{\text{ }}OD{\text{ }}of{\text{ }}treated{\text{ }}cells} {mean{\text{ }}OD{\text{ }}of{\text{ }}control{\text{ }}cells}}}\right.\kern-0pt}\!\lower0.7ex\hbox{${mean{\text{ }}OD{\text{ }}of{\text{ }}control{\text{ }}cells}$}} \times 100$$

Detection of Apoptosis

Annexin V-FITC/Propidium Iodide Kit

Investigation of apoptosis was done using the Annexin-V/PI apoptosis detection kit. In summary, cells were cultured at a density of 400,000 cells/well in a 6-well plate with different concentrations of P0 and P1. Residual attached cells and floating dead cells were separated by a rinse with PBS three times and treated for 20 min at room temperature with Annexin-V-FITC and Propidium Iodide (PI). Then, cells were surveyed using the provided software with the flowcytometer device. Annexin-V binds to phosphatidylserine in the plasma membrane of cells under conditions of apoptosis. This adhesion makes it possible to distinguish between living cells (unstained with either fluorochrome) from early apoptotic cells (stained only with Annexin-V) and late apoptotic cells (stained with both Annexin-V and PI) [19].

Fluorescent Staining by Hoechst-33342

The morphological changes assay within the apoptotic process in the nuclear chromatin was performed by DNA-binding fluorochrome, Hoechst-33342 [19]. To investigate nuclear changes as the second feature of apoptotic death, cells were seeded in 6 wells plate for 24 hours and then incubated with different concentrations of P0 and P1 for a maximum of 72 hours. Then, the cells were washed thoroughly with phosphate-buffered saline (PBS) and fixed with an acetate-methanol combination for 15 min and then incubated with Hoechst-33342 solution (1 µg/ml in PBS) for 15 min in the dark at room temperature. In the last step, after 3 washes with PBS, the cells were investigated by an inverted fluorescence microscope.

Characteristics of Synthesized Nano-carriers

Docetaxel is encapsulated and self-assembled inside polymer filaments. The atomic force microscopy (AFM) was used to confirm the synthesized nano-carriers. As expected, the synthesized nano-carriers have a homogeneous spherical morphology (Fig. 1).

Moreover, the Transmission Electron Microscopy (TEM) analysis was carried out to study the internal structure of nano-carriers (Fig. 2). The morphology of the obtained micelles by TEM showed that the micelles were spherical in shape.

The size of the copolymer micelles measured by the AFM device was reported to be about 100 nm. Since the hydrodynamic diameter of nanoparticles is a more reliable indicator of particle size, the size of the synthesized nano-carriers was once again examined by the DLS device. The size of the Blank copolymer micelles measured by the DLS device was about 158.4 nm. Also, the size of the nanoparticles containing DTX measured by this device was reported to be about 177.5 nm. The scattering of particles and the potential of Zeta nano-carriers synthesized are shown in Table 1.

Table 1

Characteristics of synthesized nano-carriers
Nano-carriers	Particle size (nm)	Zeta potential (mV)	PDI	%DL	%EE
Blank NCs (P0)	158.4	-12.8	0.336	NA	NA
DTX/NCs (P1)	177.5	-10.21	0.225	16.5 ± 0.86	82.5 ± 4.3

Encapsulation Efficiency (EE) and Drug Loading (DL)

The encapsulation efficiency and loading ratio of DTX on the nano-carriers were 82.5 ± 4.6 and 16.05 ± 0.86, respectively. It should be noted that one of the factors affecting the %EE and %DL is the ratio of drug to polymer, which in the present study, this ratio is considered to be 0.25.

In vitro Drug Release

Drug release was investigated in vitro from nano-carriers synthesized in two saline phosphate buffer media with pHs of 5.5 and 7.4. A solution of DTX was prepared to evaluate the release of the drug and comparing with the encapsulated state. As shown in Fig. 3, the free drug was released in the medium at pH 5.5 faster than in the medium at pH 7.4. This condition also applies to the release of the drug from the synthesized nano-carriers, causing a more release at about 6%, in 336 hours with a pH of 5.5. The release of the synthesized nano-carriers was performed in two steps. In the first release phase, about 39% of DTX was released in the medium at pH 5.5 and about 34% at pH 7.4 within 96 hours. In the second stage, a slow and sustained release of the synthesized nano-carriers was observed, releasing only 28% of the drug in the medium at pH 5.5 and 21% of the drug in the medium at pH 7.4 within the next 240 hours. In general, it can be concluded that drug release from synthesized nano-carriers is associated with a rapid explosion in the early stages and will slow down over time.

Cell Viability Assay

The in vitro cell cytotoxicity of synthesized nano-carriers and the free drugs were assessed by MTT assay. The cell lines were exposed to different free DTX and P1 concentrations for 48 and 72 hours. The cell viability rate is shown in Fig. 4.

As shown in Fig. 4, the free DTX at a concentration of 200 µg/ml within 48 hours, caused survival of about 43.56%, 26.74%, and 51.92% of MCF-7, MDA-MB231, and fibroblast cells, respectively. However, in the same time interval of 48 hours and at the same concentration, P1 nano-carriers caused survival of about 38.86%, 24.35%, and 46.32% of MCF-7,. MDA-MB231, and fibroblast cells, respectively. As the concentration decreases, the survival rate of the cells increases, so the free DTX at a concentration of 25 µg/ml in 48 hours, caused survival of about 89.58%, 87.88%, and 93.26% of the MCF-7, MDA-MB231, and fibroblast cell, respectively. P1 nano-carriers in the same time interval of 48 hours and at the same concentration, caused survival of about 80.16%, 70.37%, and 86.87% of MCF-7, MDA-MB231, and fibroblast cells, respectively. These results indicated that P0 nano-carriers alone had little effect on cell death, so that synergistic effects of P0 were seen with docetaxel compared to the free form of drug. After 72 hours and at a concentration of 200 µg/ml, the free DTX caused survival of about 23.91%, 22.47%, and 33.09% of MCF-7, MDA-MB231, and fibroblast cells, respectively. In 72 hours, the same P1 nano-carriers caused survival of about 20.21%, 17.44%, and 28.02% of MCF-7, MDA-MB231, and fibroblast cells, respectively. By decreasing the concentration for example 25 µg/ml, the free DTX caused survival of about 69.61%, 74.85%, and 75.50% of MCF-7, MDA-MB231, and fibroblast cells, respectively, and P1 nano-carriers at the same concentration caused survival of about 67.92%, 68.40% and 73.85% of MCF-7, MDA-MB231 and fibroblast cells, respectively. In the same way, viability is shown in Fig. 4 in its entirety for concentrations of 50 and 100 µg/ml. The IC50 value of P1 was reported to be 29.60 µg/ml and 32.50 µg/ml, and 34.96 µg/ml for MCF-7, MDA-MB231, and fibroblast cells respectively.

Apoptosis Assessment

Flowcytometric Assay by Annexin-V/PI Labeling

For investigating and confirming that nano-carriers P1 inhibits cell growth through induction of apoptosis, the effects of nano-carriers P0 (as a control) and P1 were analyzed with two concentrations higher than IC50. The cells were exposed to the P0, 50, and 100 µg/ml concentrations P1 for 48h and 72h and assayed by flowcytometry using Annexin-V-FITC (FL1-H) and PI (FL2-H) double staining. Figures 5 and 6 show a significant increase in the rate of both early (Annexin-V positive, PI negative) and late (Annexin-V positive, PI positive) apoptosis in a concentration-dependent manner. The fibroblast cells as control were weakly positive for both Annexin-V–FITC and PI compared with MCF-7 and MDA-MB231 cells. Within 48 hours and at a concentration of 50 µg/ml, about 3.45%, and 4.36% of nano-carriers P1 caused early apoptosis of MCF-7, and MDA-MB231 cells, respectively, while nano-carriers P0 had very little effect on inducing apoptosis. Also, within 48 hours and at a concentration of 100 µg/ml, about 8.14%, and 8.17% of nano-carriers P1 caused early apoptosis of MCF-7, and MDA-MB231 cells, respectively. Within 72 hours and at a concentration of 50 µg/ml, about 5.87%, and 9.92% of nano-carriers P1 caused early apoptosis of MCF-7, and MDA-MB231 cells, respectively. Also, within 72 hours and at a concentration of 100 µg/ml, about 15.91%, and 21.20% of nano-carriers P1 caused early apoptosis of MCF-7, and MDA-MB231 cells, respectively.

Investigation of Apoptosis by Hoechst-33342 Staining

The Hoechst-33342 staining was used to survey apoptotic variations of nuclei following treatment with various doses of P1. At 50 µg/ml and an upper concentration of P1, apoptotic bodies were observed in the nuclei of cells (Fig. 7). These bodies were not observed in treated cells at 25 µg/ml concentrations and fewer of P1 nano-carriers.

Drug delivery systems (DDS) are designed as methods by which agents and drugs are delivered to desired goals such as cancer cells performed by a variety of carriers [27]. Nanotechnology-based drug delivery systems can improve the distribution and accumulation of drugs in the target tissue and correct the treatment due to the unique properties of nanoscale materials [28]. Polymeric micelles are an absorbing platform for increasing the solubility and bioavailability of insoluble/low-soluble drugs in water. The many benefits of polymer micelles, such as long-term blood circulation, increased solubility, drug protection, controlled release, increased bioavailability, etc., have led to the expansion of its use in the medical industry [29]. Moreover, many drug-loaded nano-carriers destroy cancer cells through the induction of apoptotic cell death [30, 31]. Breast cancer is the most common type of cancer among women. Nowadays, breast cancer is treated by surgery and chemotherapy, but usually, patients prefer chemotherapy [32]. Many drugs and toxic compounds can induce the apoptosis phenomenon in alive cells. The apoptosis phenomenon is a type of programmed cell death. Apoptosis is involved in cancerous cell death in tumors that were treated with different anticancer agents such as chemotherapy, immunotherapy, and radiation therapy [33]. Docetaxel is one of the anticancer agents that is widely used in the chemotherapy for treatment of breast cancer [34]. Docetaxel is an effective toxoid for the micro polymerization of microtubules that is widely used to treat a variety of cancers, including breast, stomach, ovarian, and other cancers [35, 36]. However, the use of this drug is associated with problems due to its side effects and also its rapid excretion from the blood caused by its low solubility. All of these factors reduce the penetration of the DTX into the cell [37]. It seems that DTX encapsulation by polymer nanomaterials can solve this problem to some extent and improve its performance and reduce its side effects. The studies showed that compared to normal cells, cancer cells absorb more substances due to the over-expression of effx channels and pumps because of their need for more uncontrollable growth [38, 39]. On the other hand, due to the inefficiency of these channels in selective action due to structural defects and defective exocytosis, these cells will be more vulnerable to drugs and chemotherapy [40, 41]. Cem Varan and Erem Bilensoy developed a core-shell DTX-loaded PCL-PEG-PCL with a positive charge by using a chitosan coating. Results analyzing showed DTX cytotoxic effects on the glioma cell line clearly [42]. Another study showed the inhibitory effects of cisplatin (CDDP) and methotrexate (MTX) loaded in PCL-PEG-PCL within a slow-releasing on lung cancer cell line [43]. Also, Kheiri et al. designed loaded artemisinin (a natural anti-malarial) PCL-PEG-PCL micelles and studied its effect on MCF-7 & and 4T1 cell lines. Their results showed specific toxicity to both cancer cell lines [9]. Guo et al. prepared curcumin-loaded PCL-PEG-PCL by the interfacial precipitation method to enhance the targeting of the drug delivery to the liver [44]. Singh et al. worked on enhancing drug delivery by designing vancomycin-loaded PEG-PCL-PEG. Cytotoxicity studies showed the biocompatibility of vancomycin-loaded PEG-PCL-PEG and antibacterial efficacy [45]. In the present study, to increase solubility and bioavailability, DTX was encapsulated in a biocompatible, biodegradable, and safe polymer mycelium from PCL-PEG-PCL. Numerous parameters, such as organic solvent type, water phase volume, drug concentration, rate, and stirring time, can affect encapsulation efficiency. The size of synthesized nano-carriers is one of the most important and influential factors in the transfer and delivery of pharmaceutical agents to target sites [46]. The defense system easily absorbs and removes large particles. Therefore, these dimensions must be synthesized in the appropriate range. The synthesized nano-carriers with dimensions less than 250 nm can be easily transported in the circulatory system [47]. The nanocarriers designed in this study have the appropriate size for use in biological systems so that the size of P1 is 177.5 nm and the size of P0 is 158.4 nm on average. The particle surface charge (potential of Zeta) is another important factor in the transfer and delivery of drug agents loaded into nano-carriers [48]. Also, the surface charge is an important indicator of the stability of nano-carriers. The negative charge of the nano-carriers synthesized in this study (DTX/NCs) is -10.21 mv. Because of the high absolute amount of negative charge in the PCL-PEG-PCL copolymer structure, some of it is modified by the positively charged PEG block. Also, The length of the hydrophobic ester chain in the PCL-PEG-PCL structure is one of the influential factors in the hydrolysis of the copolymer, so through increasing the length of this chain, the hydrolysis of the copolymer slows down and this leads to the slow release of drug from the nano-carrier. The efficiency of the encapsulation of synthesized nano-carriers was reported to be about 82.5%, which is high efficiency. The results show that DTX/PCL-PEG-PCL nano-carriers can release 34% of the encapsulated drug at pH 7.4 and 39% of it at pH 5.5 in a controlled manner within 14 days. The rapid release of the drug in the early stages could be due to the loading of a certain amount of drug agents on the surface of the synthesized nano-carriers. As the pH decreases (similar to the pH in the microenvironment of cancer cells), the remaining positively charged amine groups act similarly to protein, creating a repulsive force. Therefore, during the decomposition of the nano-carrier, the shell swells and dissolves in the environment, leading to the rapid release of some of the encapsulated drugs. In general, PCL-PEG-PCL nano-carriers can be considered a valuable carriers for DTX transmission. According to the results obtained, it was shown that all groups prevent cell proliferation, which depends on drug dose and time. The cell viability assay by the MTT method showed that P1 nano-carriers had less toxicity in the first 48 hours than 72 hours. The apoptosis assay by flowcytometry technique indicated apoptotic cell death has also occurred, and this was confirmed by the observation of apoptotic bodies by Hoechst-33342 staining. Based on the results obtained by flowcytometry, it can be concluded that the occurrence of apoptosis is definite, because the results showed that the induction of apoptosis is dependent on concentration and time, with increasing concentration from 50 µg/ml to 100 µg/ml and time from 48 hours to 72 hours, the rate of both early and late apoptosis by P1 nano-carriers increased. In contrast, P0 nano-carriers lacking DTX had very little effect on the induction of apoptosis. In all the mentioned results, cytotoxicity of P1 increased in a concentration and time-dependent manner, so that over time from 48 hours to 72 hours, cytotoxicity increased. These results indicate that slow release of the drug from P1 has occurred, and the released DTX induced apoptotic cell death. As a result, DTX is effectively encapsulated by PCL-PEG-PCL nano-carriers, which can increase the solubility and bioavailability of the drug by high penetration into the cell. As the MTT results confirm, P1 nano-carriers resulted in lower cell viability than the free drug. These findings suggest that DTX-induced cell death is associated with apoptotic features and most importantly, our results confirm that PCL-PEG-PCL can act as a drug carrier and prevent rapid drug excretion by gradual releasing. Also, in all in vitro studies of the designed nano-carrier effects, fibroblast cells showed the greatest resistance to the effect of the drug, which could be due to the normal structure of the efflux channels and pumps compared to cancer cells.

This study showed that nano-carriers synthesized by the nano-precipitation method are suitable for DTX delivery. Nano-carriers loaded with DTX have a spherical structure and have proper zeta dimensions and potential. The drug's encapsulation efficiency and slow release make it suitable for use in biological systems. The results showed that P1 significantly inhibited cell growth and its proliferation compared to the free drug, depending on the dose and the time used due to the increased solubility of the drug in encapsulated conditions. So that, the nano-carrier P1 caused early apoptosis of MCF-7, and MDA-MB231 cells, while nano-carrier P0 had very little effect on inducing apoptosis. Based on the results, it can be concluded that controlled drug release from the designed nano-carriers has been achieved. Also, these results may be used to complete the identification of molecular mechanisms of DTX-induced apoptosis and pave the way for the use of this nano-carrier in clinical use for drug delivery.

Author contributions

All authors contributed to the study’s conception and design. Khojastehfar and Mahjoub conceived of the presented idea and designed the study. Khojastehfar, Hakemi, and Nataj performed the experiments. Mahjoub, Zabihi, Mostafazadeh, and Janbabaei contributed to the analysis of the data. Khojastehfar wrote the manuscript. Mahjoub, Zabihi, Mostafazadeh, and Janbabaei reviewed and edited the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated during the current study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

The authors gratefully acknowledge cooperation from Babol University of Medical Sciences.

Funding

This study was funded by the Babol University of Medical Sciences, Iran (IR.Babol MU.724132929).

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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Editorial decision: Returned Without Review
03 Jul, 2023
Editor assigned by journal
30 Jun, 2023
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30 Jun, 2023

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Encapsulated Docetaxel in Synthesized PCL-PEG-PCL Nano-Carriers as a Strategy for Controlled Drug Delivery in Breast Cancer

Status:

Version 1

Abstract

Background

Methods and Results

Conclusion

Figures

Introduction

Materials and Methods

Basic Materials

Synthesis of DTX-loaded Polymeric Micelles

Characterization of Nano-carriers

Drug Loading Content (%DL) and Encapsulation Efficiency (%EE)

Cell Culture

Detection of Apoptosis

Annexin V-FITC/Propidium Iodide Kit

Fluorescent Staining by Hoechst-33342

Results

Encapsulation Efficiency (EE) and Drug Loading (DL)

Cell Viability Assay

Apoptosis Assessment

Flowcytometric Assay by Annexin-V/PI Labeling

Investigation of Apoptosis by Hoechst-33342 Staining

Discussion

Conclusion

Declarations

Acknowledgments

References

Status:

Version 1