Biocompatible green tea extract-stabilised zinc nanoparticles encapsulated by poly(butyl-2-cyanoacrylate) with control release profile and antioxidative capacity

Abstract

Green tea extract-stabilised Zn nanoparticles (GT-Zn NPs) encapsulated by poly(butyl-2-cyanoacrylate) (PBCA@GT-Zn NPs) were developed for control release of Zn. The crucial parameters, concentrations of GT-Zn NPs and the monomer n-butyl-2-cyanoacrylate (BCA), which affected the size, polydispersity indexes (PDI) and encapsulation efficiency (EE %), were evaluated to achieve the smaller size, higher EE (%) and stably and uniformly dispersed NPs. The optimised PBCA@GT-Zn NPs with <100 nm diameter (46.38 ± 3.46 by SEM, 86.91 ± 3.66 by DLS), PDI (0.159 ± 0.042), zeta potential (−20.56 ± 2.53 mV) and EE (89.31 ± 1.09 %) were obtained. In vitro release of Zn ions was conducted at pH 7.4, showing that Zn from the self-made tablets and lyophilised PBCA@GT-Zn NPs is released significantly slower than that of GT-Zn NPs. Additionally, the PBCA@GT-Zn NP tablets are pH-responsive, non-toxic and antioxidative, suggesting that they could be used as safe food material or drug carriers, contributing to relieve Zn deficiency.

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Introduction

Zinc (Zn) is an essential trace element nutritionally important to mammals and can regulate body metabolism as the component of metalloenzyme to generate important biological effects, such as increasing growth rate, improving appetite, and treating dermatitis and allotriophagia. (Narváezcaicedo et al., 2018; Zhang & Zhao 2015). An estimated 31% of the global population is at risk of Zn deficiency in 2014 (Patel & Turner 2018). The disease associated with Zn deficiency is high, leading to an estimate of roughly half million deaths per year in infants and children under 5 years of age (Krebs et al., 2014). Presently, a major obstacle of the common oral administration of Zn is poor absorption with bioavailability ranging 14–60%. Numerous approaches, such as fortification of food and development of inorganic, organic and chelated forms of Zn, have failed to overcome the problem of poor oral absorption (Patel & Turner 2018). Therefore, an efficient delivery strategy is much needed to overcome these defects and relieve Zn deficiency. Here, we tried to use biodegradable poly(butyl-2-cyanoacrylate) (PBCA) to prepare Zn NPs due to the fact that previous studies have shown the excellent appearance in oral drug delivery of PBCA NPs, making it an ideal material for a control release pattern (Bagad & Khan 2015).

However, Zn is hardly to be incorporated into PBCA NPs because Zn ions can participate in chemical reactions with the polymers or other substances when it gets into touch with PBCA. Therefore, co-delivery systems have been generated, making use of embedding multiple NPs or drug species into polymers or encapsulating multiple therapeutic agents into NPs (Duan et al., 2012). This co-delivery polymeric nanocarrier system can retard the release of incorporated molecules by two procedures, release from the NPs and subsequent release from the degradation of polymeric NPs.

Tea is one of the most popular beverages. Growing evidence has suggested that consumption of tea can result in enhancing immune function, lowering blood pressure, reducing the risk of cancer and stroke, and preventing dental cavities and gingivitis (Nune et al., 2009; Liang et al., 2017). In China, green tea is overproduced every year and development of approaches to fully use this valuable resource is highly needed. Increasing interests have been attracted to use green tea extract (GT) to create metallic NPs due to the simplicity of large-scale preparation and low toxicity (Nune et al., 2009; Moulton et al., 2010; Shahwan et al., 2011). Green tea is a type of non-fermented tea, sharing the most similar chemical constituents to those of fresh tea leaves including polyphenols, purine alkaloids, phenylpropanoids and new flavoalkaloids. (Ke et al., 2019; Wu et al., 2019; Gaur et al., 2020). The main ingredient of GT is tea polyphenol (TP). But TP-Zn NPs have low oral bioavailability (Zhang & Zhao 2015). Therefore, we used GT other than TP to prepare the co-delivery nanocarrier system for Zn NPs in this paper.

On the basis of above analyses, we developed PBCA NPs delivery system containing GT-Zn complex. GT-complexed Zn NPs (GT-Zn NPs) were synthesised and then encapsulated by PBCA, leading to form PBCA@GT-Zn NPs. The NPs with high entrapment efficiency (EE) and small size were achieved. The synthesised NPs were characterised in terms of size, morphology, composition and structure. Compared with that of GT-Zn NPs, release profile of Zn from the formulation of PBCA@GT-Zn NPs was slowly sustained. Furthermore, the antioxidation and cytotoxicity of Zn NPs were investigated, indicating that PBCA@GT-Zn NPs have high safety with preferable antioxidative capacity. To the best of our knowledge, this is the first systematic study on the sustained and prolonged Zn release via a green synthesis and interfacial spontaneous polymerisation, and also provides a PBCA encapsulated and GT-complexed delivery system for control release of Zn to relieve Zn deficiency, a new approach to make full use of the overproduced green tea through preparing the Zn-supplemented nanostyle of green tea infusion.

Materials and methods

Materials

Green tea Lu’an GuaPian (Camellia sinensis var. sinensis) leaves (Cheng et al., 2018) were from State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University (Hefei, China). Zn acetate dihydrate (Zn(Ac)₂.2H₂O) and the monomer n-butyl-2-cyanoacrylate (BCA) were purchased from Saen Chemical Technology Co., Ltd. (Shanghai, China) and Beijing Kangpaite Enterprise Co., Ltd., (Beijing, China), respectively. Dextran^® 70, Pluronic F68 (Poloxamer-68, F68), ethyl acetate with analytical grade and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were all obtained from Sigma Co. Ltd. (Shanghai, China). Acetone and other reagents (analytical grade) were purchased from Tianmen Co., Ltd (Anhui, China). EBM-2 endothelial cell was obtained from the cell bank of Chinese Academy of Sciences (Shanghai, China). Ultrapure water with a resistivity of 18.2 Ω was used, supplied by laboratory water purification system from High-tech Instruments Co., Ltd (Shanghai, China). All the reagents are of analytical grade, which were used without further purification.

Sample preparation

Five gram of green tea leaves was crushed and blended in 100 mL of ultrapure water by stirring them at 1000 r.p.m. for 1 h. The blended tea powder solution was then vacuum-filtered using vacuum pump from High-tech Instruments Co., Ltd (Shanghai, China) to remove solid particles. The liquid was further filtered through 0.22 µm membrane. 0.10 M Zn acetate solution was added dropwise to the tea extract at a ratio of 1:1 with continuous stirring (1000 r.p.m.). After 2-h treatment, the green solution was obtained, centrifuged and washed three times with ultrapure water. The collected samples were freeze-dried for further developing GT-Zn NPs.

Subsequently, GT-Zn NPs encapsulated in PBCA (PBCA@GT-Zn NPs) were synthesised according to the interfacial spontaneous polymerisation method (Yao et al., 2017, Fig. 1). Briefly, the organic phase of 0.06% (w/v) GT-Zn NPs, 0.25% (v/v) BCA monomer, and 4.0 % (v/v) ethyl acetate were mixed into 10 mL acetone. The mixture was injected dropwise into 10 g ultrapure aqueous solution, including 0.6% (w/v) F68 as emulsifier and 1.0 % (w/v) dextran^® 70 as steric stabilizer. The polymerisation process was carried out under magnetic agitation (~1000 r.p.m.) at room temperature for 2.5 h to overcome BCA rapid polymerisation. The resulted colloidal suspension is about 10 mL after removing acetone by rotovapor at around 35.0 °C under vacuum. Then, the suspension liquid was stirred at a rate of ~1000 r.p.m. for 30 min. After filtering through 3- to 5-μm filter membrane, the PBCA@GT-Zn NP colloidal suspension was obtained and stored in the fridge at 4 °C. To optimise the prepared PBCA@GT-Zn NPs, various concentrations of GT-Zn NPs and BCA were used.

Characterisation by dynamic laser scattering, SEM, TEM, EDX, UV–vis (UV), FT-IR spectroscopy, X-ray diffraction and Zn EE%

Characterisation was based on the methods as reported by us recently (Yao et al., 2019, Appendix S1: Methods 1.1-1.6).

In vitro release

The Zn ions release response in vitro from GT-Zn NPs or PBCA@GT-Zn NPs was monitored using pressed disc (Wang et al., 2016) and dialysis bag method (Bagad & Khan 2015) with modification (Appendix S1: Method 1.7).

DPPH assay

The DPPH radical scavenging assay was performed via the reported method with modifications (Siripatrawan & Harte 2010, Appendix S1: Method 1.8).

Cell viability assay

The in vitro cytotoxicity of the prepared NPs was assessed in human EBM-2 endothelial cells by a standard MTT viability assay as described previously (Zhang & Zhao 2015, Appendix S1: Method 1.9).

Statistical analysis

All assays were determined at least in triplicate, and the values are presented as mean ± SD, unless otherwise specified. Statistical differences were determined by one-way analysis of variance (ANOVA) with the Turkey test applied post hoc for paired comparisons (SPSS 17.0 (SPSS inc., Chicago, IL, USA) and Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA)). Data with P < 0.05 are considered significant.

Results and discussion

Characterisation of the prepared nanoparticles

In order to optimise crucial factors to prepare the NPs (the concentrations of GT-Zn NPs and the monomer BCA), particle size, PDI, zeta potential and EE (%) were used as indexes to prepare PBCA@GT-Zn NPs (Table 1).

Particle size characterised by SEM, TEM, DLS and zeta potential

In the absence of BCA, the average particle size of GT-Zn NPs was 39.91 ± 6.28 nm detected by SEM and 119.87 ± 9.91 nm by DLS. As the BCA content increased from 0.15 to 0.40 % (v/v), the average colloidal size of samples E (prepared by 0.15%), C (0.25%) and F (0.40%) increased from 44.29 ± 3.15 to 50.18 ± 4.02 nm by SEM and from 79.11 ± 2.67 to 103.51 ± 2.19 nm by DLS (Table 1), suggesting GT-Zn NPs were loaded in PBCA by interfacial spontaneous polymerisation. The core-shell structure of PBCA@GT-Zn NPs was confirmed by the TEM images (marked by red circles, Fig. 2d). In our study, pH is not adjusted (around 5.9 in our water phase) for the sake of avoiding the disassociation of GT-Zn NPs in acidic conditions (Yi et al., 2014). Due to the fact that PBCA was coated on the surface of GT-Zn NPs, the mean diameter in SEM and DLS of PBCA@GT-Zn NPs is larger than that of the empty PBCA NPs (Samples without GT-Zn NPs) by SEM (42.67 ± 2.61 nm) and DLS (64.27 ± 1.64 nm) (Table 1, P < 0.05).

With increasing content of GT-Zn NPs from 0.03 to 0.09 (%, w/v), the average size of the prepared PBCA@GT-Zn NPs (samples B, C and D) is around 46–47 nm by SEM and 82–86 nm by DLS, indicating that the variable contents of GT-Zn NPs have not affected the diameter significantly (Table 1, P > 0.05). Without BCA, the PDI of GT-Zn NPs is 0.382 ± 0.205, revealing that GT-Zn NPs tend to aggregate (Fig. 2a, c). However, all the PBCA@GT-Zn NPs had a small PDI at around 0.2, suggesting a homogenous dispersion for PBCA@GT-Zn NPs. Zeta potential of the independent GT-Zn NPs was − 5.09 ± 2.66 mV (Fig. 2e). As BCA concentration increased from 0.15 to 0.40 % (v/v), the zeta potential of PBCA@GT-Zn NPs decreased from − 15.74 ± 4.51 to − 24.21 ± 2.49 mV (samples E, C and F) resulted from a few free carboxylic groups and and/or adsorption of other anions on the particle surface (Wu et al., 2009).

The stability of GT-Zn NPs and PBCA@GT-Zn NPs in PBS (pH = 7.4) buffer presented by size distribution was investigated. GT-Zn NPs remained stable for at least 5 days when stored in PBS buffer (Fig. 2f). When the reaction time was increased to 6 days, the size of GT-Zn NPs dramatically increased to about 209.37 ± 8.62 nm, and aggregation of critical nuclei occurred. However, the average colloidal size of PBCA@GT-Zn NPs remained unchanged (less than 100 nm) at least for 1 week. Particles with a size smaller than 100 nm could increase their transportation via endocytosis through the gastrointestinal cells due to preferential internalisation (Banerjee et al., 2016). Therefore, considering an oral administration of PBCA@GT-Zn NPs, diameter below 100 nm is advantageous. As the amounts of GT-Zn NPs increase from 0.00 to 0.09% (w/v), zeta potential of PBCA@GT-Zn NPs increased from − 25.49 ± 1.35 to − 18.52 ± 1.72 mV (samples A, B, C and D). The unencapsulated Zn ions and GT-Zn NPs on the surface of PBCA@GT-Zn NPs may have reduced the zeta potential significantly.

EE%

An increase in the amount of BCA monomer from 0.15 to 0.40 % (v/v) in organic solution led to an increase in the EE (%) of Zn from 81.32 ± 2.36 to 90.81 ± 0.95 (%), while an increase in GT-Zn NPs from 0.03 % to 0.09 % (w/v) resulted in a decrease in the EE (%) of Zn from 90.89 ± 2.03 to 73.06 ± 2.48 (%) (Table 1).

Morphological observation and others

According to the above analyses, physicochemical properties such as particle size, PDI, zeta potential and EE (%) of the optimum formulation are those of the sample C (Table 1). The optimised average size of PBCA@GT-Zn NPs was about 46.38 ± 3.46 nm with the rather spherical morphology in SEM (Fig. 2b), in accordance with the TEM measurement (~46 nm, Fig. 2d), Moreover, the SEM and TEM micrographs show that the PBCA@GT-Zn NPs are well separated from each other, conforming to the lower PDI than 0.2. The hydrodynamic diameter and size distribution of GT-Zn NPs measured by DLS is 119.87 ± 9.91 nm (Table 1 and Fig. S1), suggesting that green tea phytochemicals (catechins) are capped on Zn NPs (Nune et al., 2009). The average particle dimension of PBCA@GT-Zn NPs detected by DLS is ~90 nm, larger than those from SEM and TEM. A probably accepted excuse is that DLS measurements include the dendron surface PBCA or/ and F68 structure and the solvation layer around the NPs in the solution (Moussodia et al., 2010).

UV analysis

In Fig. 3a, the UV of GT, GT-Zn NPs, PBCA@GT-Zn NPs and PBCA NPs was recorded. GT-Zn NPs exhibited a significant reduction of the UV absorption peak at 274 nm, similar to the characteristic absorption of GT, suggesting the interaction of Zn²⁺ with the GT resulted from developing of GT-Zn NPs. Moreover, the colour transition to green after 2 h of incubation in Fig. 1 further validated the reduction of metallic Zn to GT-Zn NPs (Zhang & Zhao 2015). After the BCA self-polymerisation, a lower absorbance of GT-Zn NPs at 280 nm was recorded in the UV of PBCA@GT-Zn NPs, since PBCA NPs have no absorption spectrum at 280 nm, indicating the successful loading of the GT-Zn NPs into PBCA NPs, accompanied by a colour change for the solution, gradually from green to milky colloidal suspension (Fig. 1). In addition, the red shift from 274 to 280 nm elaborated the larger dimension of PBCA@GT-Zn NPs than that of GT-Zn NPs and the inclusion of both GT-Zn NPs and PBCA in the fully synthesised NPs.

FT-IR spectra analysis

To elaborate the formation structure of GT-Zn NPs and PBCA@GT-Zn NPs, FT-IR spectra of all samples are presented in Fig. 3b-f. Compared the spectrum of GT (Fig. 3b) with those of the prepared Zn NPs (Fig. 3d–f), the peak at 3436 shifted to 3418 cm⁻¹ in the IR of GT-Zn NPs, indicating the interaction of Zn with the OH groups from the molecules in GT (Wiseman et al., 1997; Nasrollahzadeh et al., 2016). The water-soluble polyphenols in GT were responsible for the bioreduction and capping of the Zn NPs (Kumar et al., 2013), which confirmed the formation of green tea-stabilised Zn NPs. The characteristic peak of C≡N (marked by the arrow) identified in the spectra of PBCA@GT-Zn NPs (Fig. 3e, 2251 cm⁻¹) and blank PBCA NPs (Fig. 3f, 2252 cm⁻¹) supported that C≡N did not participate in any chemical reaction during the polymerisation (Behan et al., 2001). Interestingly, the main characteristic bands at about 2900, 2252, 1754, 1637 and 1264 cm⁻¹ corresponding to C–H alkyl, C≡N, C = O, C = C and C–O stretching vibration, respectively, were also observed in the PBCA@GT-Zn NPs curve, suggesting that PBCA capped the GT-Zn NPs. Additionally, the characteristic peak of the − OH group in GT-Zn NPs (Fig. 3d) and PBCA@GT-Zn NPs (Fig. 3e) at ~3400 cm⁻¹ was relatively lower than that of GT (Fig. 3b) alone, indicating that GT was conjugated to the surface of Zn NPs through this functional group. Moreover, the − OH peak of PBCA@GT-Zn NPs was lower than that of GT-Zn NPs, further confirming the encapsulation of Zn NPs in PBCA.

The structure and crystalline phase of the prepared GT-Zn NPs and PBCA@GT-Zn NPs were further analysed by XRD (Fig. 3g). Details can be found in Fig. S4 and related results and discussions.

The PBCA@GT-Zn NPs with encapsulation of GT-Zn NPs by PBCA were more complete and stable than GT-Zn NPs, which was further confirmed by the analyses of the ¹H NMR spectra of GT (Fig. 2a), GT-Zn NPs (Fig. S2b), PBCA@GT-Zn NPs (Fig. S2c) and PBCA (Fig. S2d) (Ji et al., 2017).

In vitro Zn control release

To detect the release of Zn ions, a Zn-free PBS solution was selected as release medium and the ultracentrifugation at 18k r.p.m. was applied to separate GT-Zn NPs. Fig. 4 presents the cumulative percentage of in vitro release at which Zn is released from the prepared NPs in PBS under acidic (pH 4.5) and neutral conditions (pH 7.4). GT-Zn NPs and PBCA@GT-Zn NPs had similar release profile, characterised by an initial burst release within 10 h followed by a sustained release. The initial fast release from GT-Zn NPs was probably attributed to the presence of free Zn and Zn weakly bound by GT on the surface of GT-Zn NPs (Zhang & Zhao 2015). The release of Zn from the NPs under different pH values reached a plateau after 72 h.

For PBCA@GT-Zn NPs, PBCA carriers had shown more delayed release (Bagad & Khan 2015). Compared with GT-Zn NPs, the initial burst of PBCA@GT-Zn NPs was reduced, mainly ascribed to encapsulation of GT-Zn NPs in PBCA. In view of the convenient carrying to avoid repeated medication, release of Zn from the prepared NP tablets was conducted (Fig. 4a). As reported, most tumours/cancer tissues have lower extracellular pH values (pH 6.0–7.0) than those of normal tissues and the bloodstream (pH 7.4), decreasing even to pH 4.0 inside the cell lysosome compartments (Schmaljohann, 2006). Therefore, pH 4.5 release medium was also applied to assess Zn release. The cumulative release of Zn from the tablets increased to 70–90% at pH 4.5, which was significantly more than that at pH 7.4 (about 50–60%). The main reason is that negative charges on the surface of NPs decreased under the acidic condition, resulting in weaker binding between Zn and GT (Yi et al., 2014), and also, more Zn was disassociated from the NPs with the decrease of the pH value. As shown in Fig. S5, the tablets of GT-Zn NPs in PBS (pH 4.5) are approaching complete breakage after 72 h, while PBCA@GT-Zn NPs keep about 50% disc tables morphology. The same tendency was also observed at pH 7.4. Considering that dextran^® 70 is a neutral polysaccharide (Wu et al., 2009), the dextran^® 70 in the PBCA@GT-Zn NPs also probably plays the porogen effect since polysaccharide is water-soluble and biodegradable (Lima et al., 2012).

Combined with above-mentioned, the mechanism of GT-Zn NPs and the excipients to adjust the release behaviour of Zn is schematically presented in Fig. 4c. Release of Zn from GT-Zn NPs is the dissociation of GT and Zn with the degradation of NPs. The degradation of GT-Zn NPs may be ascribed to the weakening of binding between GT and Zn and the oxidation of GT. For being encapsulated into the polymeric NPs, Zn release attributes to both the gradual leaching of dextran^® 70 generated channels in the PBCA interior and boost degradation of PBCA NPs, and then release of Zn and/or GT-Zn NPs with disintegration. In Fig. S5, the appearance of yellow colour in PBS (pH 7.4) solutions may result from the oxidation of the released TPs form GT. TP (the main ingredients of GT) is stable in acidic conditions, but not at pH ˃ 7.0 (Zeng et al., 2017). However, the intensity of yellow colour in PBCA@GT-Zn NPs medium was lower than that of GT-Zn NPs, indicating that PBCA@GT-Zn NPs overcame the oxidation of free GT and delayed its release rate. Based on the analyses of the PBCA@GT-Zn NP tablets, it is not hard to draw a conclusion that both the formulations entrapment of GT-stabilised Zn into polymeric NPs exhibited a biphasic release pattern with an initial burst effect followed by a controlled release. Lyophilised PBCA@GT-Zn NPs showed slower release of Zn than GT-Zn NPs. Therefore, PBCA@GT-Zn NPs in tablets with pH-responsive release behaviours and sustained and controlled release profiles are suitable for use as delivery carrier.

In vitro antioxidative activity

The free radical scavenging activity of the prepared NPs against DPPH is presented in Table S2. Among the determination concentrations, the inhibition of DPPH radical by GT-Zn NPs and PBCA@GT-Zn NPs is increased with the concentration ranging from 0.1 to 1.0 mg mL⁻¹, indicating that the antioxidant activity probably increased with the enhancement of the release of Zn and GT from NPs. The PBCA@GT-Zn NPs had stronger antioxidation capacity than that of GT-Zn NPs (P < 0.05) while the blank PBCA NPs had no antioxidation. The higher DPPH radical scavenging activity of PBCA@GT-Zn NPs was probably from the fact that the encapsulation of GT stabilised Zn in PBCA, protecting GT from oxidation and thus leading to enhanced antioxidation (Zhang & Zhao 2015).

In vitro cytotoxicity

To further evaluate the safety of PBCA@GT-Zn NPs, in vitro cytotoxicity of the prepared NPs was examined against EBM-2 endothelial cells (Table S2). At the concentration from 0.1 to 1.0 mg mL⁻¹, the cell viability of PBCA NPs is higher than 95% after 48 h. PBCA@GT-Zn NPs and GT-Zn NPs had similar trends of cell viability. The lower the concentration, the higher the cell viability of the NPs. And the in vitro cytotoxicity of all samples was lower than 15% and had no significant difference. The above facts suggested that the PBCA@GT-Zn NPs are non-toxic and could be used for the control release of Zn and GT to treat Zn deficiency.

Conclusion

A novel core-shell encapsulation of GT-stabilised Zn in PBCA NPs was developed via a green synthesis and interfacial spontaneous polymerisation. The optimised formulation (sample C, Table 1) indicates PBCA@GT-Zn NPs have a size <100 nm with uniformly dispersed, PDI < 0.2, negative zeta potential (−20.56 ± 2.53), high stability and EE (%) around 90%. Characterisation of the Zn NPs confirmed the formation of GT-Zn NPs using GT and Zn ions, and verified that GT-stabilised Zn was coated by PBCA NPs. In vitro Zn release evaluation indicated that the prepared PBCA@GT-Zn NPs can provide sustained and controlled Zn release. Moreover, the tablets of PBCA@GT-Zn NPs were pH-responsive for the release of Zn. Further in vitro assays suggested that PBCA@GT-Zn NPs had effective antioxidation activity and biocompatibility (non-toxicity). These results demonstrated that PBCA@GT-Zn NPs could be used as a potential safe Zn delivery carrier to relieve Zn deficiency.

Acknowledgments

This study was financially supported by Natural Science Foundation of China (Grant No. 31972462), Anhui Provincial Key Research and Development Plan (201904a06020011), and A Key Grant from Department of Sciences and Technology of Anhui (Grant No. 17030701017) to XZ.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Ethics approval was not required for this research.

Data availability statement

Research data are not shared.

References

Author notes