Introduction

In the past decade, advances in diagnostics and therapeutics have offered patients with cancer possibilities for improved treatment and quality of life. Immunotherapies and phototherapies are widely discussed and have shown promising results (Banchereau and Palucka 2018; Dai Phung et al. 2019; Tran et al. 2016, 2018). However, current treatment strategies do not sufficiently meet patient needs, especially those who cannot afford treatments. Therefore, chemotherapy is likely to remain a standard treatment for primary and metastatic cancers for years to come; however, new approaches are required to minimize systemic toxicity and drug resistance.

Combination chemotherapies containing two or more different active agents have demonstrated significant advantages in cancer treatment, as these agents employ different mechanisms of action against cancer cells (Pushpalatha et al. 2017; Zhang et al. 2016). However, the outcomes of these treatments are still not sufficient as anticancer drugs prevailing in blood circulation result in systemic adverse events. Therefore, controlled drug-release systems responding to the specific properties of the tumor microenvironment, such as acidic pH and hypoxia, might offer some advantages, including drug distribution into target sites (Dai Phung et al. 2020b; Kanamala et al. 2016; Ruttala et al. 2018).

In a previous study, the combination of paclitaxel (PTX) (a diterpenoid derivative) and dihydroartemisinin (DHA) (an artemisinin-derived compound) decreased the treatment dose of PTX while enhancing its anticancer efficacy (Dai Phung et al. 2020a). In this study, to develop an affordable and effective nano-carrier, PTX and DHA were loaded into ethyl cellulose (EC)-based nanoparticles (NPs), and the NP surface was covered by a lecithin (LC) layer. Owing to its biocompatibility and relatively low cost, EC, a hydrophobic polymer, is widely used for controlled release dosage (Pang et al. 2019) and has attracted increasing attention as a base for nano-formulations (Abbaspoor et al. 2018). EC is stable over a wide pH range and facilitates a similar release pattern of encapsulated drugs under different pH conditions. LC is stable at neutral pH but hydrolyzes at acidic pH. We hypothesized that LC could serve as a supporting coating material to avoid the burst release of the drug in a normal physiological environment, while also degrading at low pH to accelerate drug dissolution.

The NPs were formulated using the solvent evaporation method followed by storage under a variety of conditions. NP characteristics were determined using dynamic light scattering (DLS), electron microscopy (EM), and Fourier-transform infrared spectroscopy. The release of the drug was examined under different pH conditions to confirm our hypothesis. Flow cytometry and fluorescence microscopy were used to examine cellular uptake, and cytotoxicity studies were conducted to investigate the effect of drugs and formulations on cancer cells. Ultimately, an in vivo study was performed to evaluate the ability of formulations to inhibit tumor growth in an animal model.

Experimental Section

Materials

DHA was purchased from Nanjing Jingzhu Bio-Technology Co., Ltd. (China). PTX was obtained from Fujian South Pharmaceutical Co. (China). LC (from soybean) was purchased from Kanto Chemical (China). Ethyl cellulose (Ethocel™ E20, viscosity: 18.0–22.0 mPa.s, Ethoxyl content: 48.0–49.5% wt), Tween 80, PEG 400, Poloxamer 188, and polyvinyl alcohol were provided by Xilong Scientific (Shanghai, China). Ethyl cellulose was characterized as having a degree of substitution of 2.5–2.6 and molecular weight of 51.9 ± 10 kDa (Aguilar-Zárate et al. 2019; Davidovich-Pinhas et al. 2014).

Preparation of PTX/DHA NPs

PTX/DHA-loaded EC/LC NPs were prepared using the conventional o/w emulsion–solvent evaporation method. Drugs and carriers (EC and LC) were dissolved in 5 mL of dichloromethane (DCM), and the solution (oil phase) was dropped slowly into 30 mL of water containing stabilizers at different concentrations (water phase) at a rate of 2.5 mL/min. The emulsion was homogenized by sonication at 100 W using the high-intensity probe ultrasonic processor Vibra-Cell (Sonics and Materials, Newtown, CT, USA) and magnetic stirring at 1200 rpm (IKA RCT Basic IKAMAG, Germany) for 5 min in an ice-cold water bath (4–5 °C). The formed emulsion was then stirred continuously for 2 h to evaporate the DCM. The obtained nanosuspension was washed three times by ultrafiltration (molecular weight cutoff [MWCO]: 10 kDa; Millipore, Billerica, MA, USA) to remove the free drug and produce the final suspension of EC/LC NPs. PTX/DHA-loaded EC NPs were prepared using the same procedure as described for PTX/DHA-loaded EC/LC NPs but without LC in the oil phase. The lyophilized samples were produced via lyophilization using sucrose (10%) as the cryo-protector.

The formulation stability was determined under three different conditions: room temperature for 2 weeks, 4 °C for 1 month, and freeze–thaw cycles. For the freeze/thaw cycle test, the nanosuspension was placed in a − 70 °C freezer for 12 h and allowed to thaw at room temperature; the freeze/thaw cycle was repeated three times. The NP size before and after the test was recorded.

NP size and morphology characterization

The size of the NPs was determined by DLS using a Zetasizer Nano ZS90 (Malvern, Worcestershire, UK). The morphology of the NPs was examined by transmission electron microscopy (TEM; JEOL JEM1010, USA) under an accelerating voltage of 100 kV. The NP suspension was stained at a ratio of 1:1 (v/v) with uranyl acetate, placed on a carbon-coated copper grid, and dried in air before being observed by TEM.

Fourier-transform infrared spectroscopy

Transmittance spectra were collected under controlled environmental conditions using a Jasco FT/IR-6100 spectrophotometer using the KBr pellet technique. The lyophilized NPs were mixed with KBr and tableted before measurement. The spectra were recorded in the 400–4000 cm−1 wavelength range with 16 scans and a 4 cm−1 resolution.

Encapsulation efficiency and drug loading

The NP suspension (2 mL) was placed in a centrifuge tube containing an ultrafiltration membrane (MWCO 10 kDa). Centrifugation was conducted at 5000 rpm for 30 min at 10 °C. The solution at the bottom of the ultrafiltration tube was collected, and the free drug was quantified by high-performance liquid chromatography (HPLC). The drug entrapment efficiency (EE) and loading capacity were calculated using the following equations:

EE (%) = (Total drug—Free drug) / Total drug × 100.

Loading capacity (%) = (Total drug—Free drug) / Total nano × 100.

In vitro drug release

The in vitro release profiles of PTX and DHA were determined using the dialysis method. Briefly, 2 mL of the NP suspension was sealed in a dialysis bag (MWCO: 14 kDa; Membrane Cell, Chicago, IL, USA). The dialysis bag was then placed in 30 mL of the release medium (phosphate-buffered saline [PBS] containing 0.5% Tween 80, pH 5.0, and pH 7.4) at 37 °C with shaking at 100 rpm. At different time points, 1 mL of the release medium was collected and replaced with 1 mL of fresh release medium.

The released PTX and DHA amounts were determined by HPLC (Agilent 1260 Infinity LC system) using a Zorbax Eclipse XDB C18 column (5 μm, 250 × 4.6 mm). A mixture of acetonitrile and phosphate buffer (0.03 M, pH 3.0) (57:43, v/v) was used as the mobile phase at a flow rate of 0.7 mL/min. The injection volume was set to 50 μL. The drugs were detected at 210 nm using a UV detector.

Cellular uptake studies

Human lung carcinoma (A549) and Lewis lung carcinoma (LLC) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM L-glutamine supplemented with 10% fetal bovine serum (FBS; GIBCO) in an incubator at 37 °C and 5% CO2.

For the flow cytometry study, A549 and LLC cells were incubated in 6-well culture plates for 24 h at 37 °C and then incubated with 1 ppm of the formulation at 37 °C. After 30, 60, and 90 min of incubation, cells were collected, washed twice with PBS, and fixed with 4% formaldehyde for 10 min at room temperature. The cells were then washed with PBS, re-dispersed in 1 mL of PBS, and assessed using flow cytometry.

For fluorescence detection, cells were incubated in 6-wells plates for 24 h at 37 °C with study samples at 1 ppm. After 30, 60, and 90 min of incubation, cells were washed and fixed with 4% formaldehyde. After removing the formaldehyde and washing the cells, 2 mL of Hoechst 33,342 (5 µg/mL) was added to each culture well. After 30 min, cells were washed and observed under a fluorescence microscope (Ziess_Axio).

In vitro cytotoxicity study

Using a previously reported method (Skehan et al. 1990), in vitro cytotoxicity was assessed. The formulations were diluted to appropriate concentrations. The cells (A549 and LLC) were detached using trypsin and seeded into a 96-well plate, formulations were added, and cells were incubated for 72 h. Wells without reagents but with TBUT (180 μL) were used as the day 0 control. After 1 h, cells in the control wells were fixed with trichloroacetic acid (20%). After 72 h, the cells were fixed with TCA for 1 h, stained with sulforhodamine B (SRB) for 30 min at 37 °C, washed three times with acetic acid, and allowed to dry at room temperature. Unbuffered Tris base solution (10 mM) was added and gently shaken for 10 min to dissolve the SRB. The optical density was measured at a wavelength of 540 nm using an ELISA Plate Reader (Biotek).

In vivo study

BALB/c mice (8–9 weeks old) were used for the in vivo study. LLC cells cultured in DMEM supplemented with 10% FBS were inoculated into the mice flank at 2 × 106 cells per animal. After 5 days of tumor inoculation, 45 mice were randomly divided into five groups: EC-nano, free drugs, EC/LC NPs (dose: 5 mg/kg), control (without treatment, drinking water ad libitum), and EC/LC NPs (dose: 2.5 mg/kg). Formulations were intravenously administered on days 0, 3, 7, and 10, and the tumor size was monitored weekly. The volume of the tumor was calculated using the formula V = a × b2 / 2, where V = volume, a = length, and b = width. At the end of the study, mice were euthanized to isolate tumors, and blood was collected to determine the effect of the sample on hematological parameters and liver and kidney enzymes. The study protocol was approved by the ethics committee of medicine and pharmacy research at the Military Medical University (Hanoi, Vietnam) (approval number: 019B/20, dated August 06, 2020).

Results

Preparation and characterization of PTX/DHA NPs

PTX/DHA-loaded EC/LC or EC NPs were prepared by the emulsion-solvent evaporation technique. The process parameters, including the sonication time, sonication power, and aqueous phase volume, were optimized (Fig. S1). Polymers play a crucial role in the formation of NPs on which the drug can be loaded. A polymer-to-drug ratio that is too low may keep the drug from being fully encapsulated by the carrier, whereas if the ratio is too high, it will be difficult for the drug at low loading to be uniformly distributed in the NPs. The increase in the polymer/drug ratio increased the NP size but slightly decreased the polydispersity index (PDI) (Fig. 1a). This could be because a higher polymer concentration results in a higher viscosity of the inner phase, leading to a minor reduction in the dispersed force (Budhian et al. 2007). A polymer:drug ratio of 3:3 was selected for further development.

Fig. 1
figure 1

The optimization of EC nanoparticle preparation. Particle size (bar) and polydispersity index (symbol) following changes in a EC/drug ratio, b LC/EC ratio, c surfactant type, and d Tween 80 concentration. The results are presented as means ± standard deviations, n = 3. EC, ethyl cellulose; LC, lecithin

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LC is a hydrophobic mixture of natural phospholipids; it is used widely in the food and pharmaceutical industries and is considered a safe, biocompatible excipient. LC is also used as a surfactant to increase the stability and encapsulation of drugs into carriers (Jabri et al. 2018). When LC was added to the inner phase, the NP size decreased, but the PDI increased (Fig. 1b). LC not only increases drug encapsulation into the NP but also makes the system more stable. LC is a component of the cell membrane, is biocompatible, and helps improve NP bioavailability (Martin-Banderas et al. 2013).

Surfactants act as stabilizers of emulsions and prevent NP aggregation. In our experiment, Tween 80, Poloxamer 188, PVA, and PEG 400 were investigated at a 1.5% concentration. Tween 80 appeared to be the best and formed NPs of 134.3 ± 1.1 nm in size, with a PDI of 0.197 ± 0.009 (Fig. 1c). Using different Tween 80 concentrations showed that an increase in Tween 80 reduced NP size (Fig. 1d). Maintaining the surfactant at the medium level results in micelles being formed from the released drug and the excess surfactant (Sharma et al. 2016).

The final formulation was made using the following parameters: organic phase (5 mL of DCM solution containing 5 mg of PTX, 10 mg of DHA, 15 mg of EC, and 15 mg of LC), aqueous phase (30 mL of 1.5% Tween 80 solution), mixing rate of 2.5 mL/min, sonication time of 5 min, sonication power of 100%, and organic solvent evaporation at room temperature for 2 h.

Characteristics of optimized formulations

The morphology of NPs was examined using TEM and scanning electron microscopy (EM). The NPs were spherical, and their size was consistent with that determined using DLS (Fig. 2a, b).

Fig. 2
figure 2

Characteristics of the optimized formulation. Morphology analysis of EC/LC nanoparticles: a TEM image, scale bar = 200 nm; b SEM image, scale bar = 500 nm.c The stability of the nano-system under different conditions. d Fourier-transform infrared spectroscopy results of materials and nanoparticles. e Entrapment efficiency and loading capacity of nanoparticles. f The release of PTX in the medium at pH 5.0 and 7.4. g The release of DHA in the medium at pH 5.0 and 7.4. The results are presented as means ± standard deviations, n = 3. EC, ethyl cellulose; LC, lecithin; TEM, transmission electron microscopy; SEM, scanning electron microscopy; PTX, paclitaxel; DHA, dihydroartemisinin

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The NPs were stored under various conditions, including room temperature, 4 °C, and freeze/thaw cycles. The NP size and PDI remained similar under all conditions with a size of approximately 130 nm and PDI below 0.2, indicating the stability of the system for long-term storage (Fig. 2c).

The IR spectrum showed the functional groups of the formulation components (Fig. 2d). The characteristic absorptions in the IR spectrum of DHA were observed at 847, 876, and 1093 cm−1 (C-O–O-C bond) and 3374 cm−1 (O–H bond). The characteristic PTX IR spectrum showed absorption bands at 1735 (C = O bond of ester), 1647 (C = O bonds of amide), and 1244 (C-N bonds) cm−1. The polymer characteristic absorption appears at 1281 cm−1 (C–O–C bond of ether), whereas that of LC at 1746 (C = O bond of ester) and 1066 cm−1 (phosphate group). All these characteristic absorptions were observed in the IR spectra of the physical mixture as well as that of the formulated NPs, indicating no interaction between the drugs and excipients.

The addition of LC did not affect the EE and loading capacity of PTX, but did affect DHA. The EE of DHA increased from 50.1% to 61.1%, whereas the loading capacity increased from 10.5% to 14.2% (Fig. 2e). Notably, PTX is practically insoluble (solubility < 0.7 μg/mL) and DHA is very slightly soluble (solubility 134.8 μg/mL). Therefore, most PTX was encapsulated inside the EC core, whereas DHA was detected only on the surface or at the edge of the EC NPs. The presence of LC generates a suitable hydrophobic/hydrophilic balance for improved DHA accumulation. This feature has also been reported elsewhere (Jabri et al. 2018).

Intriguingly, the LC layer on the surface of NP generated interesting NP characteristics, such as delayed PTX and DHA release at pH 7.4, but at acidic pH 5.0, the release pattern of both drugs was quite complicated and similar to that of EC NPs (Fig. 2f, g). Briefly, after 24 h, about 40% of PTX was released from EC NPs at both pH conditions and EC/LC NPs at pH 5.0, whereas only about 20% of PTX was released at pH 7.4. The pattern of DHA release did not change, possibly owing to the localization of DHA mostly at the edge of the systems, which is affected by the outer environmental conditions (Fig. 2f). Even after the initial similar release, DHA release continued to be delayed at pH 7.4; this might be explained by the deeper encapsulation of DHA in the EC core. It is known that LC becomes unstable under acidic conditions and may trigger the release of drugs from NPs (Baptista et al. 2003; Haidar et al. 2017).

Cell studies

Uptake of NP by cancer cells was evaluated using flow cytometry and confocal imaging (Fig. 3, Fig. S2). Figure 3 shows the presence of NP in the cytoplasm, indicating the uptake of carriers. Higher fluorescence levels were observed in EC/LC NP-treated cells than in EC NP-treated cells, which might be attributed to the similarity between LC and the cell membrane, resulting in a more favorable uptake of EC/LC NPs. After 30 min, the fluorescence signal in cells was weaker than that after 60 and 90 min, with the fluorescence signal levels being similar at the two later time points. These results suggest a gradual increase in NP uptake during the first 60 min.

Fig. 3
figure 3

Cellular accumulation of nanosystems. The uptake of EC NPs and EC/LC NPs into A549 and LLC cancer cells determined by a flow cytometry and b fluorescence microscopy. EC, ethyl cellulose; LC, lecithin; NPs, nanoparticles

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Consistent with the uptake of NPs, EC NPs and EC/LC NPs inhibited cell growth at a similar rate of about 80% in both cancer cell lines after 72 h of treatment. In vitro cytotoxicity results indicated that a higher drug concentration had a greater impact on cell viability (Fig. 4). The results also suggested that PTX played the main role in inhibiting cancer cell growth.

Fig. 4
figure 4

In vitro cytotoxicity of free drugs, EC NPs, and EC/LC NPs in A549 and LLC cancer cells. The results are presented as mean ± standard deviation, n = 6. EC, ethyl cellulose; LC, lecithin; NPs, nanoparticles

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In vivo study

The anti-tumor study was conducted using five groups: EC NP, free drug, EC/LC NP (dose: 5 mg/kg for three formulations), EC/LC NP (dose: 2.5 mg/kg), and control group (Fig. 5). Body weight and tumor size were monitored throughout the study (Fig. 5, Table S1). The mice body weights on days 0 and 7 of the formulation-treated groups were not significantly different from those of the control (p > 0.05). On day 14, body weights in the EC NP-, EC/LC NP-, and free drug-treated groups were lower than those in the control group. At the end of the study (day 21), body weights in all four treated groups were significantly lower than those in the control group (p < 0.05), possibly owing to rapid tumor growth (Fig. 5c).

Fig. 5
figure 5

In vivo anti-tumor study. a Tumor volume of tumor-bearing mice in five treatment groups: control, free drugs (PTX + DHA), EC NPs, and EC/LC NPs each at the dose of 5 mg/kg, and EC/LC NPs at the dose of 2.5 mg/kg. b Inhibition by formulations of the tumor-growth rate. c The body weight of mice throughout treatment. The results are presented as means ± standard errors, n = 9. *p < 0.05, **p < 0.01, one-way analysis of variance with Tukey’s multiple comparisons tests. EC, ethyl cellulose; LC, lecithin; NPs, nanoparticles; PTX, paclitaxel; DHA, dihydroartemisinin

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The EC/LC NP-treated group showed the best tumor suppression of 18.8%, 34.9%, and 27.4% with 5 mg/kg on days 7, 14, and 21 of the experiment, respectively. A lower dose of 2.5 mg/kg decreased tumor inhibition to only 19.8% on day 21 (Fig. 5b, Fig. S3). A dose of 5 mg/kg in the EC NP-treated group decreased tumor volume at 14 and 21 days compared with the control, but the difference was not statistically significant (p > 0.05). On days 14 and 21, tumor-size inhibition in the EC NP-treated group was 14.1% and 12.3%, respectively, compared with that in the control group; free drugs inhibited tumor growth by only 4.1% on day 14, followed by a tumor growth increase. Tumors were isolated at the end of the study and weighed. Free drugs reduced tumor weight by only 5%, whereas EC/LC NPs of the same dose reduced it by 31.3%.

The hepato-parameters changed significantly after the free drug administration: aspartate aminotransferase (AST): 606.03 vs 1587.85 U/L and alanine aminotransferase (ALT): 30.07 vs 50.45 U/L. The encapsulation of drugs into EC NPs and EC/LC NPs mitigated the magnitude of this change to 975.33 and 979.6 U/L (AST) and 42.17 and 36.3 U/L (ALT), respectively (Table 1). Perhaps the hydrophilic head of LC affected this change. Kidney function remained constant as judged by the creatinine level. Other biochemical parameters of the treated groups were unchanged (Table S2).

Table 1 The hepato- and nephro-parameters after the course of treatments
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Discussion

EC is commonly employed for the development of pharmaceutical dosage forms owing to its high compatibility and stability (Adeleke 2019), although the issue with EC non-biodegradability remains. The safety of EC with regard to kidney functions, indicated in this study, might be a positive feature to consider for further pharmaceutical development of this material. However, EC is ready to be used as a drug delivery carrier with or without chemical modification, as evidenced by its recent application in nanotechnology (Leitner et al. 2020; Wang et al. 2020; Xie and Li 2017).

Chemotherapy remains indispensable for treating anticancer. However, limitations that need to be overcome are drug resistance and the lack of target-specific drug delivery, which leads to systemic toxicity. A variety of strategies have been introduced, such as new materials, combination therapies, and new treatment regimens. Further development costs time and money; two means that patients with cancer may not have. In this study, the combination of already approved PTX and DHA has been exploited for possible treatment enhancement. Moreover, the use of LC and EC, which are common recipients, could make the therapy affordable.

In this study, EC NPs of small size and narrow distribution were successfully fabricated using an optimized process. PTX was encapsulated more efficiently than DHA because of its higher hydrophobicity; the solubility of PTX is < 0.7 μg/mL, whereas that of DHA is 134.8 μg/mL (Dai Phung et al. 2020a). This difference also suggests that PTX is located inside the inner hydrophobic core of EC NPs, whereas DHA might be distributed on the hydrophilic and hydrophobic interfaces near the NP surface. The addition of LC increased the EE of DHA (Fig. 2e) as it offered more amphiphilic space to incorporate and prevent PTX burst release during the initial time after administration. At a lower pH, LC likely dissociated from the NP surface, which induced an increase in the release of both PTX and DHA. Moreover, owing to the compatibility of LC with the cell membrane, its presence might augment the cellular uptake of EC/LC NPs compared to that of EC NPs (Fig. 3a). However, over time, most of the two types of NPs entered cells and exhibited similar activity (Fig. 5). PTX appeared to be the main player of the drugs in killing cancer cells, implying that if it is released freely, it would be harmful to healthy cells. The controlled release of PTX under physiological conditions is, thus, critical to reduce side effects and maximize drug action on the main target, the tumor. In vivo, the potent killing of cancer cells by EC/LC NPs takes place mainly where the acidic environment of the tumor favors the unique properties of drug delivery, as indicated by the significant reduction in tumor volume with minimal undesirable impact on organs and the biochemical status of the body.

pH‐responsive drug delivery systems have been introduced and developed since a long period of time (Zhu and Chen 2015), especially as cancer treatment, considering the acidic nature of tumors (Hossen et al. 2019; Liu et al. 2014). Regarding EC, some studies have attempted to formulate this type of carrier, such as EC-graft-PDMAEMA, EC-graft-PAA, EC-graft-polystyrene, EC-graft-poly(2-hydroxyethyl methacrylate), and EC-graft-poly(ethylene glycol) methyl ether methacrylate (EC-g-P(PEGMA)) (Yuan et al. 2012). However, in most of these carriers, EC was bonded chemically with a pH-responsive polymer. This approach requires more effort to maintain safety and promote clinical translation. In this study, a simple procedure was employed, making the preparation easier and potentially more feasible for scaling up. In addition, a combination therapy is an indispensable approach in oncology because it offers more efficacy and less toxicity (Mokhtari et al. 2017). In our study, PTX and DHA were combined, as done in a previous study, but using a different design. In the previous study, PTX and DHA were loaded into a PTX-PEG-DHA co-polymer, forming a micelle that released similar amounts of both the drugs in media with two different pH. These drugs demonstrated a synergistic effect in inhibiting colon cancer cells. When encapsulated into the nano-system, they inhibited tumor growth more efficiently than free drugs (Dai Phung et al. 2020a). In this study, PTX and DHA with different aqueous solubilities were encapsulated into a hydrophobic EC and an amphiphilic LC, which resulted in a quicker release of DHA, a less toxic drug, in both pH conditions, while PTX was released in greater amount in the acidic environment compared with that in the other medium. This modification was also applied on a new type of cancer—lung cancer which offers PTX and DHA loaded EC/LC nanoparticles being a potential approach for cancer treatment.

Conclusions

Here, we prepared and tested EC NPs with favorable properties, including a size of about 130 nm, PDI of less than 0.2, and long-term stability. The presence of LC in the NPs appears to offer better drug EE; it can also be hydrolyzed in an acidic environment, resulting in enhanced drug release, whereas the drug remains inside the NP under normal pH conditions. After incubating NPs with cancer cells, they were detected in the cytoplasm after 30 min, with NP accumulation increasing with incubation time. The uptake of NPs appeared to inhibit cell growth in vitro. Most importantly, the intravenous administration of drug-loaded EC/LC NPs inhibited tumor growth compared with the carrier alone, other formulations, or free drugs. This result suggests a new method of using the old and cost-effective EC and LC for the development of affordable combined chemotherapies to treat cancer.