Rational Fabrication of Folate-Conjugated Zein/Soy Lecithin/Carboxymethyl Chitosan Core-Shell Nanoparticles for Delivery of Docetaxel.

The objective of this work is to design and fabricate a natural zein-based nanocomposite with core-shell structure for the delivery of anticancer drugs. As for the design, folate-conjugated zein (Fa-zein) was synthesized as the inner hydrophobic core; soy lecithin (SL) and carboxymethyl chitosan (CMC) were selected as coating components to form an outer shell. As for fabrication, a novel and appropriate atomizing/antisolvent precipitation process was established. The results indicated that Fa-zein/SL/CMC core-shell nanoparticles (FZLC NPs) were successfully produced at a suitable mass ratio of Fa-zein/SL/CMC (100:30:10) and the freeze-dried FZLC powder showed a perfect redispersibility and stability in water. After that, docetaxel (DTX) as a model drug was encapsulated into FZLC NPs at different mass ratios of DTX to FZLC (MR). When MR = 1:15, DTX/FZLC NPs were obtained with high encapsulation efficiency (79.22 ± 0.37%), small particle size (206.9 ± 48.73 nm), and high zeta potential (-41.8 ± 3.97 mV). DTX was dispersed in the inner core of the FZLC matrix in an amorphous state. The results proved that DTX/FZLC NPs could increase the DTX dissolution, sustain the DTX release, and enhance the DTX cytotoxicity significantly. The present study provides insight into the formation of zein-based complex nanocarriers for the delivery of anticancer drugs.

Introduction

Over the past decades, various nanomaterial-based drug delivery systems (DDS) have been developed to improve the pharmacokinetic and pharmacodynamic profile of therapeutics, especially in the field of cancer treatment.[1−3] Among them, protein-based nanocarriers have recently gained increasing attention due to their unique advantages, e.g., ease of availability, biodegradability, extraordinary drug binding capacity, and the presence of numerous functional groups available for chemical modifications.[4−6]

Zein is a hydrophobic plant protein extracted from corn gluten meal whose average hydrophobicity is 50 times larger than those of albumin and fibrinogen.[7,8] Although it has high hydrophobicity, zein behaves as an amphiphile that can easily self-assemble into different shapes and structures including microspheres, films, fibers, nanoparticles, and composites with other natural polymers.[9,10] Moreover, the zein molecule has a very special bricklike structure that provides sufficient space for drug entrapment.[11,12] Due to the high hydrophobicity, zein-based DDS can sustain drug release without treatment with chemical cross-linkers.[13,14] Obviously, zein possesses many favorable features for drug delivery and is becoming popular among various research groups.

However, pure zein-based DDS tend to aggregate in aqueous solutions with a neutral pH or at physiological pH, because the isoelectric point of zein is 6.2–6.8,[15,16] and pure zein-DDS are usually insufficient in the drug loading, membrane permeability, site-specific delivery, and drug release profiles.[12,13,17] Also, the protein nature of zein may cause immunogenicity in vivo.[18] To reduce these limitations, it was reported to be an effective strategy to coat zein nanoparticlse with other macromolecules, e.g. phospholipids,[19,20] polysaccharides,[21−23] proteins,[24,25] etc. In addition, the -NH2 and COOH groups of the zein molecule were usually conjugated with targeting ligands (e.g., folic acid[26] and lactoferrin[27]) to fulfill the site-specific drug delivery.

Docetaxel (DTX), a semisynthetic derivative of paclitaxel, is one of the most efficient anticancer drugs.[28] However, its clinical application is greatly restricted by its poor aqueous solubility, low permeability, and undesirable side effects.[29] To overcome these drawbacks, much attention has been focused on novel nanomaterial-based DTX formulations, such as intelligent polymeric micelles,[30] solid lipid nanoparticles,[31] chitosan-based nanoparticles,[32] hybrid nanocarriers,[33,34] and inorganic nanoparticles.[35,36] Many of them exhibited significantly enhanced solubility, targeting, and antitumor activity in preclinical studies. Nonetheless, it remains challenging to design and fabricate an effective DTX nanoformulation that can be used in clinical and commercial applications. Up to now, only a few DTX nanoformulations have entered clinical trials, and none have been approved in the market.[29,37]

Recently, chondroitin sulfate/zein[38] and phosphatidylcholine/zein[39] hybrid nanoparticles have been reported for DTX delivery. These studies demonstrated that zein incorporation increased the DTX loading capacity, sustained the DTX release, and improved the antitumor efficacy. In our previous work, folate-conjugated zein (Fa-zein) was synthesized and verified as an attractive carrier for sustained and targeted delivery of anticancer drugs.[26] On these bases, we intend to design and fabricate a rational zein-based nanocarrier with multilayer core–shell structure for DTX delivery in this study. Scheme depicts our design protocol. That is, zein is selected as an ideal carrier to form an inner hydrophobic core. DTX is expected to be loaded in the inner core by hydrophobic interaction and/or hydrogen bonding. Specifically, the -NH2 group of zein is conjugated with folic acid to achieve a high tumor accumulation efficiency, and to improve the stability and redispersibility, soy lecithin (SL) and carboxymethyl chitosan (CMC) are selected as the coating components to form an outer shell. SL is expected to act as a linker or interlayer; that is, its hydrophobic part can embed in the inner core while its hydrophilic part is coated with CMC.

Scheme 1

Proposed Structure of a Designed Zein-Based Nanocarrier for DTX Delivery

Different drug loading methods have been reported to produce zein-based DDS, including solvent evaporation,[40] phase separation,[41] flash nanoprecipitation,[42] electrohydrodynamic atomization,[43] spray drying,[44] the supercritical antisolvent technique,[45] the built-in ultrasonic dialysis process (BUDP),[46,47] etc. Usually, the coprecipitation of particles or solid dispersions of drug and carrier are produced in these present methods. It remains challenging to fabricate nanoparticles with multilayer or core–shell structures, especially when the composite carriers have different polarity and solubility. In this study, we are developing a simple, rational, and scalable method to fabricate the designed DTX loaded Fa-zein/SL/CMC ternary nanoparticles (DTX/FZLC NPs) with multilayer core–shell structures. Investigations on the particle fabrication via this novel process were conducted in detail. The complexation mechanism of the FZLC ternary nanocomposite was discussed by means of various characterization methods. The influences of the SL and CMC contents on the stability and redispersibility of the obtained nanoparticles were evaluated. After that, DTX/FZLC NPs were prepared at different ratios of DTX to FZLC. The encapsulation efficiency, DTX releasing profiles, and in vitro cytotoxicity of DTX/FZLC NPs were systematically studied.

Experimental Section

Materials

Folic acid (Fa, mass fraction purity > 0.97) was purchased from Shanghai Boao Biotechnique Co. Ltd., China. Zein (Z3625) was purchased from Sigma-Aldrich Shanghai Trading Co. Ltd., China. Docetaxel (DTX, mass fraction purity > 0.98), soy lecithin (SL, mass fraction purity > 0.98), and water-soluble carboxymethyl chitosan (CMC, carboxylation degree ≥ 0.80) were purchased from Shanghai Macklin Biochemical Co. Ltd., China. All other chemicals were of analytical grade and used as received.

Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin, penicillin, and streptomycin were all purchased from Life Technologies (Grand Island, NY, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan).

Synthesis of Fa-Zein

Fa-zein was synthesized and purified according to our previous work.[26] In brief, the γ-carboxylic group of Fa was activated and conjugated with the amino groups of zein through amide coupling chemistry. The initial molar ratio of Fa to zein was selected as 40:1, where the average molecular weight of zein was assumed to be 20 kDa. The sample after reaction was purified by washing with 0.2 M PBS (pH = 7.8) and then acidized by diluted HCl (pH = 4.0). Finally, pure Fa-zein was obtained by freeze-drying (BK-FD10S freeze drier, Hainan Cheng Ming Industrial Co., Ltd., China).

The structure of the final Fa-zein was confirmed by the 1H NMR spectrum (Bruker AV 600, Bruker, Switzerland), which was in accordance with that reported in our previous work.[26] The conjugation degree was 4.47 (Fa/zein, molar ratio), which was quantified by a UV-spectrophotometer (UV-7200, Shimadu Instrument Co., Ltd., China) according to our previous work.[26]

Fabrication of DTX/FZLC NPs

In this study, a novel process was established to prepare DTX/FZLC NPs with multilayer core–shell structures. This process combines spray drying with antisolvent precipitation technologies, which is named the atomizing/antisolvent precipitation (AAP) process. A simple preparation scheme for the fabrication of DTX/FZLC NPs by the AAP process is illustrated in Scheme .

Scheme 2

Schematic Diagram for Fabrication of DTX/FZLC NPs by the AAP Process

The operating procedure is described as follows. The solution of Fa-zein, SL, and/or DTX was atomized first by compressed air through a nanosprayer with a 0.7 μm membrane cap. To achieve a fine atomization effect, the solution flow rate was set as 1.5 mL/min, and the atomizing pressure was controlled at 100 kPa in this study. As the jets of solution atomized, fine droplets were formed and introduced into the antisolvent phase (CMC aqueous solution). Mutual mass transfer between the fine droplets and the antisolvent phase then occurred spontaneously. During this processing, the polarity of these fine droplets changed from hydrophobic to hydrophilic, causing Fa-zein, SL, and/or DTX molecules to aggregate and self-assemble into nanoparticles. CMC molecules would adsorb on the surface of these nanoparticles by electrostatic and/or hydrogen bond interactions. Finally, the dispersion was freeze-dried (BK-FD10S freeze drier, Hainan Cheng Ming Industrial Co., Ltd., China) for 24 h to yield powdered-form nanoparticles.

To investigate the influences of SL and CMC molecules on the properties of the obtained nanoparticles, FZL (composite of Fa-zein and SL) and FZLC (composite of Fa-zein, SL, and CMC) samples were prepared separately: (1) For the preparation of FZL samples, the solution phase was 20 mL of an ethanol–water (70:30, v/v) solution of Fa-zein (100 mg) and SL (0 mg, 10 mg, 20 mg, 30 mg, 40 mg, and 50 mg), and the antisolvent phase was 100 mL of distilled water. (2) For the preparation of FZLC samples, the solution phase was 20 mL of an ethanol–water (70:30, v/v) solution of Fa-zein (100 mg) and SL (30 mg), and the antisolvent phase was 100 mL of an aqueous solution of CMC (10 mg, 20 mg, 30 mg, 40 mg, and 50 mg).

For the preparation of DTX/FZLC NPs, the nanocarriers were composed of 100 mg of FA-zein, 30 mg of SL, and 10 mg of CMC. DTX (5.6 mg, 7 mg, 9.3 mg, 14 mg, and 28 mg) was added to the solution phase to reach different mass ratios of drug to carrier (MR), i.e., 1:5, 1:10, 1:15, 1:20, and 1:25.

Characterization Methods

Turbidity Measurement

The turbidity measurements were performed with a UV/vis spectrophotometer (UV-7200, Shimadu Instrument Co., Ltd., China), where the absorbance (Abs) of the samples was recorded at 600 nm as the turbidity value.[48,49] All measurements were conducted at 25 °C and repeated three times.

Particle Structure Analyses

The particle size and zeta potential of samples were measured using a Zeta-sizer Nano-ZS90 (Malvern Instruments Ltd., Worcestershire, UK) with a dynamic light scattering instrument and a microelectrophoresis instrument, respectively. Before each measurement, the samples were suspended in distilled water and stirred ultrasonically for 5 min to disperse effectively and avoid multiple particle effects. All measurements were carried out at 25 °C, and each sample was analyzed in triplicate.

The morphology of the freeze-dried products was observed by scanning electron microscopy (SEM, Verios G4 UC, Thermo Scientific, USA). Samples were prepared by spreading the products on an aluminum stub using double-sided adhesive carbon tape and then sputter-coating with gold under high vacuum conditions, and to evaluate the redispersibility, the products were ultrasonically dispersed in distilled water first. The core–shell structure of particles was observed by transmission electron microscopy (TEM, JEM-2100, JEOL Ltd., Japan). Before observation, the aqueous dispersion of each sample was dropped on a 200-mesh carbon-coated copper grid and dried naturally.

The chemical structure and intermolecular interaction of freeze-dried products were characterized by a Fourier transform infrared (FT-IR) spectrophotometer (Frontier, PerkinElmer, USA). Data were collected on the transmittance mode over a frequency region of 4000–400 cm–1 with a resolution of 4 cm–1. Samples were prepared by dispersing the products in dry KBr and pressing the mixture into disc form. The solid state of DTX in freeze-dried products was analyzed by an X-ray diffractometer (XRD, D8 ADVANCE, Bruker AXS, Germany) with Cu Kα radiation generated at 40 mA and 40 kV. The samples were scanned in the 2θ angle range between 5° and 60°. The thermal behavior of samples was measured by a differential scanning calorimeter (DSC, Q100, TA Instruments, USA). Approximately 5 mg samples were pressed and loaded on standard aluminum pans and heated from 25 to 200 °C at a rate of 10 °C/min.

Determination of the Encapsulation Efficiency

For an evaluation of the DTX loading, 10 mg of freeze-dried sample was mixed with 10 mL of PBS (pH 7.4), vortexed for 15 s, and then immediately filtered using a filter with a pore size of 0.22 μm to obtain the supernatant. The free DTX in DTX/FZLC NPs was obtained by calculating the DTX content in the supernatant. Another 10 mg of freeze-dried sample was thoroughly dissolved in 80% acetonitrile/water (v/v) to measure the total DTX in the DTX/FZLC NPs.

The DTX content was determined using a high-performance liquid chromatography system (HPLC, Waters e2695, USA) equipped with a C18 column (4.6 mm × 250 mm, 5 μm) as described before.[50,51] The mobile phase was a mixture of methanol, acetonitrile, and water (23:36:41, v/v/v). The peak detection was performed at a 229 nm wavelength using a quantity of 20 μL injection volume at a flow rate of 1 mL/min. The concentration of DTX present in the samples was determined by comparing the peak area with the standard curve. The encapsulation efficiency (EE) was used to evaluate the DTX content that was entrapped into the DTX/FZLC NPs and calculated as follow:Each experiment was carried out in triplicate.

In Vitro Drug Release

DTX release from DTX/FZLC NPs was carried out in 0.05 M PBS (pH = 7.4) using a dialysis method as described before.[52] Briefly, an aliquot of DTX/FZLC NPs was dispersed into a dialysis bag with 4 mL of PBS (pH = 7.4) and suspended in 200 mL of release medium and gently shaken at 100 rpm in a water bath (37.0 ± 0.5 °C). At predetermined intervals, 2 mL of dissolution sample was withdrawn and compensated with an equal volume of the fresh medium maintained at the same temperature. The concentration of DTX in the sampled solution was determined by HPLC as described in section . The cumulative drug percentage released from the sample (Cr) was calculated as the ratio of the amount of drug released at time t to the initial amount used.

In Vitro Cell Viability Assay

The cell viability was analyzed through the CCK-8 assays using MCF-7 and SKOV-3 cancer cells as described before.[53,54] Cells were cultured in DMEM, supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a humidified incubator with 5% CO2.

The cell viability experiment was performed in 96-well plates at an initial density of 5000 cells/well with 100 μL of medium. The cells were incubated for 24 h before experiments. Then, the cells were washed with fresh medium and treated with raw DTX, FZLC NPs, and DTX/FZLC NPs at different concentrations. After further incubation for 24 or 48 h, 10 μL of CCK-8 was added to each well, and the plates were incubated for another 2 h. Then, the absorbance of each well was measured by a microplate reader (Multiskan MK3, Thermo Fisher Scientific Inc., USA) at 450 nm with background subtraction at 630 nm. The cell viability was expressed as a percentage compared to the untreated control cells.

Statistical Analysis

Data were expressed as means and standard deviations for at least three independent experiments. Statistical comparison was carried out using SPSS software (SPSS Inc., Chicago, IL, USA). P values < 0.05 were considered statistically significant.

Results and Discussion

Fabrication and Characterizations of FZLC NPs

For the commonly used phase separation methods, zein-based nano-/microparticles were formed by mixing the solution with bulk antisolvent directly.[12] Differently, the solution was atomized into the antisolvent phase for our AAP process, meaning that particles were formed from the droplets, much like the spray drying[44] and supercritical antisolvent processes.[45] Thus, there is probably an interface between the solution droplets and the antisolvent phase before the particle formation. Initially, we carried out a series of experiments to assess the influences of the components in solution and/or the antisolvent phase on the particle formation. As shown in Figure , three different phenomena were observed.

Figure 1

Phenomena and schematic diagram for particle formation via the AAP process with different components: (a) Fa-zein, (b) Fa-zein/SL, and (c) Fa-zein/SL/CMC.

A film was formed for pure Fa-zein via the AAP process. This phenomenon is mainly caused by the strong hydrophobicity of Fa-zein molecules, resulting in the formation of droplets with a hydrophobic surface, as reported by Dong et al.[55] These hydrophobic droplets were rejected into the aqueous phase and then agglomerated together to form films on the interface.

Aggregated particles were obtained for Fa-zein/SL via the AAP process. As a natural small molecular surfactant, SL may tune the wettability of pure Fa-zein droplets and help them to break the interfacial resistance. The study of Dai et al.[19] found that the addition of SL changed the secondary structure of zein in ethanol–water solution, and the presence of SL significantly decreased the zeta potential of zein/SL composite colloidal nanoparticles. The decreased charge went against the colloidal stability. Accordingly, Fa-zein/SL droplets were easily introduced into the aqueous phase and formed small particles, but these particles tended to aggregate and precipitate on the bottom.

Dispersed particles were produced for Fa-zein/SL/CMC via the AAP process. It was reported that coating with CMC was able to add a negative charge to the particle surface and, hence, modify the physicochemical properties of these particles.[56,57] During the particle formation via the AAP process, CMC molecules around the Fa-zein/SL droplets would be adsorbed and coated on the particle surface spontaneously, which enhanced the steric exclusion and electrostatic repulsion among formed particles, resulting in good dispersity and stability.[58] Thus, CMC in the aqueous phase avoided the aggregation of formed particles effectively, and the suspension of dispersed particles was formed.

Results of the Turbidity Measurement

The influences of the SL and CMC contents on particle formation were further investigated via the turbidity measurement. Freshly made suspensions of FZL with different SL dosages and of FZLC with different CMC dosages before freeze-drying were measured, and the results are shown in parts a and b, respectively, of Figure . The SL and CMC contents show a great effect on the sample turbidity. As shown in Figure a, the initial turbidity of the FZL suspensions increases along with the increase in SL dosage before 30 mg and then tends to balance. As a control, the solution of 30 mg of SL without Fa-zein was atomized into the aqueous phase. The absorbance of the obtained SL suspensions is only 0.062. Thus, the higher turbidity means the more counts of FZL composite colloids in the suspensions. During storage, the turbidity of the FZL suspensions decreases and becomes very low after 12 h. The change of turbidity can also be intuitively observed from Figure c. These results indicate that adding SL can help the entrance of Fa-zein droplets into the aqueous phase to form suspensions of FZL composite colloids, but these colloids are unstable and tend to aggregate and precipitate. On the other hand, it can be found from Figure b that the initial turbidity of the FZLC suspensions decreases along with the increased CMC dosage. The decreased turbidity is probably because of the fact that the CMC molecules in the aqueous phase are bound to the surface of the Fa-zein/SL droplets, resulting in forming a vesicle-like structure and inhibiting the rapid formation of colloidal nanoparticles. After 1 h of storage, there is an obvious upturn in the turbidity of the FZLC suspensions with CMC dosage over 20 mg. These phenomena suggest that adding CMC may hinder the particle aggregation and enhance the particle stability, but too high a CMC content is unfavorable for the flash fabrication of colloidal nanoparticles.

Figure 2

Turbidity measurement of freshly made suspensions of (a) FZL with different SL dosages at 0 and 12 h of storage and (b) FZLC with different CMC dosages at 0 and 1 h of storage. (c) Visual observation of freshly made FZL suspensions with different Fa-zein/SL mass ratios: (1) 100:10 at 0 h of storage; (2) 100:30 at 0 h of storage; (3) 12 h of storage; Turbidity variation (d) and visual observation (e) of freeze-dried FZL and FZLC powder after being redispersed in deionized water with storage time.

From the above results, a suitable mass ratio of Fa-zein/SL and Fa-zein/SL/CMC was selected as 100:30 and 100:30:10 for the following preparation of FZL and FZLC samples, respectively. The water redispersibility of freeze-dried nanoparticles is an obstacle for their further applications. To address this issue, a certain amount of freeze-dried FZL or FZLC powder was dispersed in distilled water and stirred ultrasonically for 5 min. Their redispersibility and stability were then evaluated according to the turbidity measurements. Figure d shows the variation in their relative absorbance (RA) with storage time. RA declines rapidly for the FZL dispersion but declines slightly for the FZLC dispersion during 12 h of storage. The change of turbidity is also observed from Figure e, where the FZL dispersion becomes clear after 2 of h storage but the FZLC dispersion is still cloudy after 12 of h storage. These results suggest that freeze-dried FZLC powder can achieve an acceptable redispersibility and stability, which largely is thanks to the good solubility and strong steric repulsion of CMC.[56]

Results of the SEM and TEM Characterizations

The particle morphology was observed though the SEM and TEM characterizations. As shown in Figure , the freeze-dried powder of Fa-zein is an irregular film (Figure a), FZL is aggregated particles (Figure b) that are hard to be further dispersed in water (Figure e), and FZLC is interconnected nanoparticles (Figure c). Clumped and connected zein-based multicomposite nanoparticles were observed in many previous studies, especially when coating with water-soluble components.[25,56,59,60] From Figure d and f, it can be clearly seen that these connected FZLC NPs can be well dispersed into individual nanoparticles in water, and the individual FZLC NPs are nanospheres with core–shell structure. According to the AAP procedures and properties of composite materials, it can be reasonably assumed that the core is mainly composed of hydrophobic Fa-zein molecules and the shell is composed of the water-soluble CMC molecules, while the amphiphilic SL molecules act as linkers and form the interlayer.

Figure 3

SEM images of (a) a freeze-dried powder of Fa-zein, (b) FZL, (c) and FZLC. (d) SEM image of a FZLC dispersion. TEM images of (e) FZL and (f) FZLC dispersions.

Results of the FT-IR Characterization

FT-IR spectroscopy was used to investigate the structural characteristics of FZLC NPs further. As shown in Figure , Fa-zein exhibits absorption peaks at 3308 cm–1 (O–H stretching), 1654 cm–1 (amide I band), and 1539 cm–1 (amide II band);[26] LC exhibits absorption peaks at 1735 cm–1 (C=O stretching) and 1089 cm–1 (P=O stretching);[19] CMC exhibits absorption peaks at 3443 cm–1 (-NH2 and -OH stretching), 1632 cm–1 (-NH bending), 1415 cm–1 (-COO stretching), and 1323 cm–1 (-C–O stretching).[61] Most of the characteristic bands of Fa-zein and LC are observed in the spectrum of FZL powder (Figure d), but there are some slight shifts of these characteristic peaks. The changes might be due to the hydrogen bond interaction between the peptide bond (CO–NH) of Fa-zein and the P=O bond of phospholipids during the formation of complexes.[19,62] As shown in Figure e, FZLC powder exhibits a broad -OH stretching vibration peak between 3419 and 3313 cm–1, which might shift from the 3308 cm–1 of FA-zein and the 3442 cm–1 of CMC, and other characteristic bands of Fa-zein, LC, and CMC can be observed with sight changes. These phenomena confirm that the FZLC NPs were successfully composed by Fa-zein, LC, and CMC molecules according to some intermolecular interactions.

Figure 4

FT-IR spectra of (a) raw Fa-zein, (b) raw LC, (c) raw CMC, and freeze-dried powder of (d) FZL and (e) FZLC.

Fabrication and Characterizations of DTX/FZLC NPs

DTX/FZLC NPs were prepared at different mass ratios of DTX to FZLC (MR), and the results are summarized in Table . It can be seen that MR has a great effect on the EE and particle size and a slight effect on the zeta potential. EE decreases with the increased DTX content. This is because a high DTX content means the carriers around the drug decreased, resulting in more drug molecules being exposed to the surface of the DTX/FZLC NPs. When MR ≤ 1:15, the EE is larger than 79%, meaning that most of the DTX molecules have been entrapped into DTX/FZLC NPs. In the study of Lee et al.,[38] DTX was loaded into chondroitin sulfate/zein NPs and the EE was 64.2 ± 1.9%. A relatively high EE in our study is due in large part to the novel particle fabrication process. The particle size of DTX/FZLC NPs presents a unimodal distribution with narrow range, besides that obtained at MR = 1:5. When MR ≤ 1:15, it shows a negligible effect on the particle size, and the particle size of the obtained DTX/FZLC NPs is around 200 nm. The zeta potential of DTX/FZLC NPs is around −40 mV. The high surface charge can enhance the repulsion and decrease the aggregation between DTX/FZLC NPs, which is beneficial to their clinical application.

Table 1

Results of DTX/FZLC Samples Prepared at Different MR Values

Sample	DTX:FZLC (MR, w/w)	Total DTX (g/100 g)	EE (%)	Size (nm)	Zeta potential (mV)
DTX/FZLC-1	1:5	18.83 ± 3.41	70.11 ± 0.50	257.8 ± 95.84	–42.6 ± 4.09
				83.58 ± 15.49
DTX/FZLC-2	1:10	6.59 ± 1.63	73.27 ± 0.52	254.4 ± 21.78	–42.9 ± 3.99
DTX/FZLC-3	1:15	5.99 ± 0.12	79.22 ± 0.37	206.9 ± 48.73	–41.8 ± 3.97
DTX/FZLC-4	1:20	3.83 ± 0.20	82.38 ± 0.29	208.7 ± 54.29	–40.3 ± 4.21
DTX/FZLC-5	1:25	3.53 ± 0.28	86.75 ± 1.61	203.4 ± 71.06	–39.2 ± 5.48

The morphologies, size, and zeta potential distribution of typical samples are shown in Figure . The raw DTX is rodlike (Figure a). When MR is 1:5, the obtained DTX/FZLC NPs are irregular (Figure b), and uncoated DTX nanocrystals can be seen from the TEM image (Figure d); also, the size distribution presents double peaks (Figure g). A similar phenomenon was also observed in our previous work;[63] the drug molecules easily formed nanocrystals at high drug loading. Notably, when MR decreased to 1:15, the morphology of the obtained DTX/FZLC NPs (Figure c, e, and f) is similar to that of unloaded FZLC NPs (Figure c, d, and f), the size distribution only presents a single peak at 206.9 ± 48.73 nm (Figure h), and the zeta potential is −41.8 ± 3.97 mV (Figure i). These phenomena suggest that DTX can be successfully encapsulated into FZLC NPs at low MR, and MR = 1:15 is selected to prepare DTX/FZLC NPs for the following characterizations.

Figure 5

SEM images of (a) raw DTX, (b) DTX/FZLC-1, and (c) DTX/FZLC-3. TEM images of (d) DTX/FZLC-1, (e) DTX/FZLC-3, and (f) amplified DTX/FZLC-3. Particle size distributions of (g) DTX/FZLC-1 and (h) DTX/FZLC-3. (i) Zeta potential distribution of DTX/FZLC-3.

The obtained DTX/FZLC NPs were further characterized by XRD, DSC, and FT-IR. As shown in Figure a, the characteristic diffraction peaks of DTX all disappear in the XRD pattern of DTX/FZLC NPs, which implies that DTX is most likely in an amorphous state in the FZLC matrix, rather than in a crystalline form. From Figure b, it can be seen that the characteristic endothermic peak of DTX around 169.2 °C also disappears in the DSC curve of DTX/FZLC NPs, which further confirms the XRD result that DTX is molecularly dispersed in the FZLC matrix. The absence of the endotherm peak of drugs also provides evidence of encapsulation.[64]Figure c displays the FT-IR spectra of typical samples. As a control, the physical mixture sample (DTX + FZLC) was prepared by mixing DTX with FZLC (1:15, w/w) directly. The spectral pattern of DTX/FZLC NPs is differed from that of DTX + FZLC, where the characteristic absorption bands of DTX almost disappear. These phenomena further suggest that DTX probably exists in the inner core of DTX/FZLC NPs, and its characteristic absorption bands are covered by the FZLC matrix.

Figure 6

XRD spectra (a) and DSC curves (b) of raw DTX, FZLC, and DTX/FZLC NPs. (c) FT-IR spectra of raw DTX, DTX + FZLC, and DTX/FZLC NPs.

In Vitro Drug Release Behavior

The in vitro release profiles of raw DTX, DTX + FZLC, and DTX/FZLC NPs are shown in Figure . First, it can be seen that DTX/FZLC NPs enhance the DTX dissolution significantly. The percentage of DTX dissolved from DTX/FZLC NPs is about 66% after 24 h, compared to about 20% from raw DTX or DTX + FZLC. The increased DTX dissolution is mainly attributed to the amorphous state of DTX in DTX/FZLC NPs. For the amorphous solid, there is no crystal lattice energy to disrupt during dissolution, resulting in a faster dissolution rate and extent relative to the crystalline state.[65,66]

Figure 7

In vitro release profiles of raw DTX, DTX + FZLC, and DTX/FZLC NPs. The illustration correlates the drug release data of DTX/FZLC NPs with the Higuchi model.

Second, it can be found that DTX/FZLC NPs sustain the DTX release rate successfully, where DTX can be of sustained release more than 24 h. The DTX release from DTX/FZLC NPs may involve two possible mechanisms, i.e., the dissolution diffusion of the drug from the matrices and matrix erosion resulting from degradation of the FZLC. As shown in the illustration of Figure , the first 60% of DTX release data can be successfully correlated using the Higuchi model as follows:where the power = 0.5 means that the drug diffusion corresponds to a Fickian diffusion mechanism.[67] This fact indicates that the release of DTX from DTX/FZLC NPs is more consistent with a diffusion mechanism than a matrix erosion mechanism. This phenomenon can be attributed to the amorphous state of DTX, the small particle size, and slow matrix erosion of DTX/FZLC NPs.

In Vitro Antitumor Activity

The in vitro cytotoxic effects of raw DTX, unloaded FZLC, and DTX/FZLC NPs for MCF-7 and SKOV-3 cells are shown in Figure . Particularly, the concentration of unloaded FZLC was equal to that of the corresponding DTX/FZLC NPs group. It can be seen that unloaded FZLC shows no toxic effect on the activity of both MCF-7 and SKOV-3 cells at all concentrations, up to 48 h of incubation.

Figure 8

In vitro cell cytotoxicity of FZLC, raw DTX, and DTX/FZLC samples with different concentrations in MCF-7 cells incubated for (a) 24 h or (b) 48 h and in SKOV-3 cells incubated for (c) 24 h or (d) 48 h.

On the other hand, it can be seen that the cytotoxicity induced by DTX/FZLC NPs is notably higher than that of raw DTX in both MCF-7 and SKOV-3 cells, especially at high DTX dosages. The different cytotoxicities might be due to their varied DTX dissolution and release rates. As mentioned above, the loading in the FZLC matrix can enhance the DTX dissolution greatly, which will achieve a higher DTX equilibrium concentration at high dosages, resulting in higher cell cytotoxicity compared to raw DTX. Due to the limited DTX dissolution, the cell cytotoxic effects of raw DTX increase slightly with increased DTX dosage, but it can be seen that the cell cytotoxic effects of DTX/FZLC NPs increase obviously with increased DTX concentration and incubation time, which proves the controlled and sustained efficacy of the NPs’ formulation.

Conclusion

In this study, Fa-zein/SL/CMC ternary biocomposites with core–shell nanostructures were successfully fabricated for DTX delivery through a novel and rational AAP process. The AAP process has the advantages of atomizing and antisolvent self-assembly. During the AAP process, SL and CMC play different roles in the particle fabrication. That is, the surfactant SL can eliminate the interfacial resistance and introduce Fa-zein droplets into the antisolvent phase, while water-soluble and negative CMC can enhance the steric exclusion and electrostatic repulsion among formed particles, resulting in good dispersity and stability. According to the turbidity measurement, a suitable mass ratio of Fa-zein/SL/CMC was selected as 100:30:10. The freeze-dried FZLC powder can achieve an acceptable redispersibility and stability in water. The individual FZLC NPs are nanospheres with core–shell structures, which are composed of Fa-zein, LC, and CMC molecules according to intermolecular interactions. DTX was successfully encapsulated into FZLC NPs. When MR = 1:15, the EE, size, and zeta potential of the obtained DTX/FZLC NPs are 79.22 ± 0.37%, 206.9 ± 48.73 nm, and −41.8 ± 3.97 mV, respectively. The results of XRD, DSC, and FT-IR studies indicate that DTX is molecularly dispersed in the inner core of DTX/FZLC NPs. The in vitro release assay indicates that DTX/FZLC NPs can enhance the DTX dissolution significantly and sustain the DTX release for more than 24 h, where the DTX release mainly corresponds to a Fickian diffusion mechanism. The cytotoxicity assay shows the safety of the FZLC carrier. Loading in the FZLC matrix can enhance the DTX cytotoxicity significantly in both MCF-7 and SKOV-3 cells. These results suggest that the novel AAP process is suitable for the fabrication of biocomposites with multilayer core–shell structures, and the designed FZLC NPs hold promising potential as natural vehicles for anticancer drugs.