The Synergistic Anticancer Effect of Dual Drug- (Cisplatin/Epigallocatechin Gallate) Loaded Gelatin Nanoparticles for Lung Cancer Treatment

Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei City 110, Taiwan Department of Radiation Oncology, Taipei Medical University Hospital, Taipei City 110, Taiwan International Ph. D. Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei City 110, Taiwan Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, Taipei City 110, Taiwan Department of Industrial Pharmacy, College of Pharmacy, University of the Philippines Manila, Manila, 1000 Metro Manila, Philippines Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, No. 35, Keyan Road, Zhunan Town, Miaoli County 35053, Taiwan Research Center of Biomedical Device, College of Biomedical Engineering, Taipei Medical University, Taipei City 110, Taiwan International Ph. D. Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei City 110, Taiwan


Introduction
Lung cancer is one of the most common cancers. It has high mortality, worldwide, with estimated numbers of over two million new cases being diagnosed and more than 1.7 million deaths annually [1]. Currently, platinum-based chemotherapy regimens are used for broad-based treatment of lung cancer patients. The four platinum-containing compounds, cisplatin, carboplatin, oxaliplatin, and nedaplatin, have become common anticancer drugs [2]. Compared to other anticancer drugs, cisplatin can be used for many types of cancer, such as cancers of the lung, head, neck, and cervix, and can prolong the survival time of patients [2]. Cisplatin (cis-diammine-dichloro-platinum or cCDDP) has a heavy metal complex, with a central platinum atom, surrounded by two chlorine atoms and two molecules of ammonia. The cytotoxic effect of cisplatin involves its reaction with DNA to form interstrand and intrastrand cross-linking, which inhibits DNA replication and RNA transcription, leading to DNA breakage and ultimately triggering apoptosis [3,4]. However, cisplatin resistance is the major cause of therapeutic failure, so five-year survival rates of lung cancer are still too low to satisfy [5,6]. Common side effects of cisplatin include tiredness, malaise, vomiting, nephrotoxicity, and hearing loss, which can also lead to patient weakness and treatment failure [7]. Therefore, ideally, a therapeutic agent is needed that can overcome chemoresistance and avoid the side effects of cisplatin, for maximum damage to cancer cells and a lasting therapeutic effect.
Green tea is one of the most popular beverages in the world. It contains various polyphenols, mainly catechins, which include epicatechin (EC) and epigallocatechin (EGC) as hydroxy derivatives, epicatechin gallate (ECG), and (−)-epigallocatechin-3-gallate (EGCG) [8,9]. EGCG is one of the most abundant polyphenols in green tea. It contains three hydroxyl substituents on the B ring and a gallate moiety esterified on the third carbon (C3) on the C ring. These structures contribute to its activity as an antioxidant and iron chelation [10]. EGCG has several pharmacological and biological properties, including antioxidant, antibacterial, cardioprotection, antiatherosclerosis, anti-inflammation, neuroprotection, and anticarcinogenesis [11,12]. Accumulating evidence has demonstrated that EGCG inhibits tumor growth, invasion, angiogenesis, cancer stemness, metastasis and affects multiple signaling pathways by binding to various molecular targets, such as mitogenactivated protein kinase (MAPK), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), and epithelial-mesenchymal transition (EMT) [13]. EGCG was also reported to enhance the effects of chemotherapy and target therapy in lung cancer cells [14]. Therefore, we hypothesize that therapeutic agents, in combination with EGCG, may provide an effective way to overcome drug resistance, exemplified by the resistance of tumor cells to cisplatin.
Nanomedicine is a field that combines nanotechnology, pharmaceutical, and biomedical sciences to develop drugs loaded in nanocarriers, with higher therapeutic effects and minor side effects, for the treatment of diseases, especially cancer [15]. Nanocarriers have unique properties, such as nanoscale size and a high ratio of surface area to volume, and have the potential to modulate the pharmacokinetics and pharmacodynamics of drugs, thereby enhancing their therapeutic effectiveness. Loading the drug into the nanocarrier can enhance its stability in the body, prolong the circulation time of the compound in the blood, and control the rate of drug release. Due to specific physiological conditions in the cancerous tissue, there can be enhanced permeability and an enhanced retention effect (EPR) [15,16]. In addition to having characteristics such as high permeability and a long lasting effect for cancer treatment, nanoparticle-based drug delivery systems also have many unique properties that can improve the antitumor response. For example, the encapsulation of a hydrophobic drug in hydrophilic nanocarriers can overcome the problem of solubility and chemical stability of anticancer drugs [17]. Active targeting or cell membrane targeting chimeric peptides of nanoparticles which can be achieved by surface modification of specific antibodies or peptides on nanoparticles can recognize and target cancer cells enhancing tumor eradication capacity [18][19][20]. Furthermore, for modulation of drug release, metal-based nanocarriers that can be pH-triggered, near-infrared (NIR) light-responsive, or photosensitive have been designed for drug release in high local concentrations at a suitable site, in tumor cells [21,22]. Multifunctional nanocarriers that combine different functional antitumor drugs and are used for codelivery of multiple medications by nanoparticles to exhibit synergistic antitumor efficiency were also investigated [17,19,21]. Drug-loaded NPs can change the biodistribution of the drug, causing accumulation in the tumor area. The effectiveness of cisplatin-loaded gelatin nanoparticles for lung cancer treatment has been demonstrated in previous studies, both in vitro and in vivo [23,24]. To reduce the resistance to cisplatin, we developed a dual drug delivery system based on cisplatin/EGCG-loaded gelatin nanoparticles (GE-Pt NPs), to exert synergistic antitumor activity. As mentioned above, multifunctional nanocarriers are an approach which involves the loading of two or more drugs into a single well-designed nanocarrier, which uses different mechanisms or targets multiple pathways to improve the therapeutic outcome [25]. Unlike in the case of a single regimen, multiple drugs can produce broad and sustained cellular toxicity that can provide the opportunity for overcoming intrinsic or acquired drug resistance [25][26][27]. Cisplatin and EGCG can work together to kill cancer cells by different mechanisms enhancing their toxicity to cells for treating tumorous cells/ tissue. In this study, the optimal parameters for the preparation of GE-Pt NPs, to achieve high drug loading rates and slow drug release profiles, were investigated. The anticancer effect and the cellular uptake of the GE-Pt NPs were investigated in A549 cancer cells and compared to cisplatin solution, to evaluate GE-Pt NP's toxicity.

Materials and Methods
The same equation was also used for calculating the EE (%) of EGCG. The released profiles of EGCG and cisplatin from the GE and GE-Pt NPs were suspended in phosphate-buffered saline (PBS) at 37°C and were evaluated by a dialysis method. The colloidal suspension of GE NPs and GE-Pt NPs, of known EGCG and cisplatin concentration, was placed inside a dialysis bag and dialyzed against PBS at 37°C. The EGCG and cisplatin released outside the dialysis bag were sampled at indicated time intervals (0, 5, 10, 15, 30, …, 1200, and 1400 minutes). The EGCG and cisplatin concentrations outside the dialysis bag were measured by the same methods as used in the EE examination.

Cell Viability Test.
To study the anticancer effect of the GE and GE-Pt NPs, for lung cancer treatment, human lung adenocarcinoma cells (A549) were used. The A549 cells were grown in Nutrient Mixture F-12 Ham medium with 10% fetal bovine serum and 1% PSN supplement. Cells were grown in an incubator at 37°C in a 5% CO 2 environment. The cell viability was determined by the CCK-8 assay. A549 cells were seeded in a density of 5 × 10 3 cells/well in 96-well plates and incubated overnight. Then, various concentrations of EGCG, cisplatin, GE, and GE-Pt NPs colloidal suspensions were prepared by diluting stock formulations with culture media for EGCG (5, 20, and 80 μg/mL) and cisplatin (2, 8, and 32 μg/mL). At the indicated date (days 1, 3, and 7), the cell viability was detected by using the CCK-8 kit with a WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) reduction assay. The working WST-8 agent was added into each well at 110 μL per well and then incubated for 3 h at 37°C. A microplate reader (Multiskan GO Microplate Spectrophotometer, Thermo Scientific, Waltham, MA, USA) was used to read the absorbance optical density (OD) at 450 nm. The percentage of viable cells was calculated in comparison to control cells, cultured with medium only. For the group treated for 7 days, the culture medium was replaced on day 3 by the addition of fresh medium alone. All the experiments were performed in six repeats (n = 6) and carried out in three batches. The statistical analyses were performed using Student's t-test or one-way analysis of variance (ANOVA) using Excel (Microsoft). A probability (p) value ≤ 0.05 is statistically significant.

Intracellular Drug Accumulation.
For the quantification of intracellular cisplatin accumulation, A549 cells were grown in 24-well culture plates at a concentration of 1:5 × 10 5 cells per well. After incubation overnight, the cells were 3 Journal of Nanomaterials cocultured with cisplatin solution or GE-Pt NPs at the same cisplatin concentration (15 μg/mL) and then incubated at 37°C for 6 and 24 hours. In the control group, the cells were cultured in the normal medium. At the indicated time, cells were washed 3 times with PBS and then detached by trypsinization. The cell pellets were collected and digested by 50% HCl solution at 80°C for 60 min, to release platinum (Pt) ions. The samples were diluted to the proper concentration for analysis. The intracellular Pt concentration in each tested condition was quantified (n = 3) using an inductively coupled plasma mass spectrometer, ICP-MS Thermo X-SERIES 2 ICP-MS (Thermo Scientific, Waltham, MA).

Characterization of Dual Drug-Loaded Nanoparticles (GE-Pt NPs)
3.1.1. Size and Zeta Potential Results. The schematic drawing of the formation of gelatin/EGCG self-assembling nanoparticles (GE NPs) is shown in Figure 1(a). Due to the electrical attraction, these two components are forced together into aggregating particles; then, cisplatin is conjugated with the carboxyl group of gelatin, via ion exchange with the -NH 2 on cisplatin [24]. The colloidal solution of GE and GE-Pt is revealed in Figure 1(b), showing a good condition of semitransparent suspension without precipitation ( Figure 1).
The monodispersion of GE and GE-Pt NPs can also be confirmed from the DLS results by its narrow peak distribution, as shown in Figure 2(a). The morphologies of GE and GE-Pt NPs were revealed by TEM, shown in Figure 2(b), round and spherical structures of the particles were observed, with no aggregation, and the particle size of these two NPs was around 50~100 nm. Some smaller particles were found, but this may due to dehydration during the preparation of TEM samples. Figure 3(a) shows the FTIR pattern of the raw materials (gelatin, EGCG, and cisplatin) and their nanoparticles (GE, GE-Pt). The spectra of the gelatin reflecting the C=O bending band at 1640 cm -1 and the amide band (N-H) at 1550 cm -1 were observed [31]. In the gelatin, a weak carboxyl group stretching vibration was noted at 1160 cm -1 [31,32]. Characteristic peaks of EGCG were found at wavelengths of 1700 (-COO), 1620 (C=C aromatic ring), and 1234 (-OH aromatic ring) [28,33,34]. The peak of cisplatin (NH) was found at 1299 cm -1 . The amine peaks of cisplatin were seen at 1625 cm -1 (NHN) for asymmetric bending, 1316 cm -1 (NHN) for symmetric bending, and 1299 cm -1 (NHN) for symmetric bending [35]. Although GE and GE-Pt NPs had EGCG and cisplatin incorporated, the EGCG and cisplatin amount in total gelatin-based NPs was too low to be detected and distinguished by FTIR. Their FTIR patterns were similar to that of gelatin. Thermal analysis was used to identify the metal (Pt) component of the cisplatin.

FTIR and TGA Results.
The result of the TGA assay is provided in Figure 3(b). The weight loss between 50 and 200°C in the GE is due to the adsorbed water lost during heating. Weight lost from 200 to 450°C is due to the decomposition of proline in gelatin. Glycinin was lost from gelatin between 450 and 620°C. All of the organic components were lost around 800°C, and the total weight loss reached 100% with no residues above 700°C. A similar weight loss pattern was found in GE-Pt from 50 to 200°C. There is a dramatic weight loss seen in GE-Pt above 200°C, from the Cl-NH 2 decomposition of cisplatin, since cisplatin decomposes from 225 to 249°C [36]. The fraction remaining above 700°C (~26%) was the platinum from the cisplatin, loaded into GE-Pt, since it has been previously reported that the residual weight in CDDP, above 1000°C, is Pt [37]. From this result, we confirmed the cisplatin loading in GE-Pt NPs, by TGA, calculated as 0.57 mg/mL cisplatin.

Dual Drug Releasement from Variant Nanoformulations.
The in vitro release of EGCG and cisplatin from the two NPs (GE or GE-Pt) was determined by placing them in a dialysis bag, followed by immersion in phosphate buffer (PBS, pH 7.4) at 37°C for 1400 min. Figure 4(a) shows the accumulated EGCG release rate from EGCG solution, GE, and GE-Pt NPs as 78:2 ± 0:1%, 45:0 ± 0:2%, and 33:6 ± 0:2%, respectively, at 120 min ( * p < 0:05 compared with EGCG solution). The release rate slowed after 240 min and later reached a sustainable and stable rate of EGCG release for more than 1400 min. The EGCG had been completely released by the EGCG solution group at 960 min. Meanwhile, the EGCG release rate in these two nanoformulations was <60% with a slow EGCG release pattern. At 1440 min, the release of cisplatin from GE-Pt NPs was 30:1 ± 0:3% compared with that from cisplatin solution ( * p < 0:05). For the same amount of time, the release of cisplatin solution was higher (37:8 ± 0:1%). The rapid initial cisplatin release, from both formulations, was followed by a slower cisplatin release, which continued for up to 24 hours.

Dual Drug-Loaded NPs for Treating Lung Cancer Cells
3.2.1. Anticancer Effect of Gelatin/EGCG Self-Assembled Nanoparticles (GE NPs). The anticancer activity of EGCG has been widely reported, both in vitro and in vivo. In this test, A549 cells were treated with EGCG solution and gelatin/EGCG nanoparticles (GE NPs) to examine the cellular effect in different EGCG conditions. Low (5 μg/mL), medium (20 μg/mL), and high (80 μg/mL) concentrations of EGCG and GE NPs were tested to determine cell viability, to evaluate their potential anticancer effect. An EGCG concentration of 80 μg/mL showed high toxicity to A549 cells, both in solution and in nanoform (GE NPs). It caused low cell viability 4 Journal of Nanomaterials (between 13 and 41%, see Figures 5(a)-5(c)) depending on the period of treatment (1-7 days). If 20 μg/mL was used, the EGCG solution was more effective for inhibiting cancer proliferation than GE NPs ( * p < 0:05), in all culture periods, due to quick access in the medium. However, in the lowest EGCG-treated group (5 μg/mL), GE NPs were more effective than free EGCG in reducing cell viability on day 3 (98:6% ± 2:3%, * p < 0:05) and day 7 (93:7 ± 10:9%).   Journal of Nanomaterials However, at lower EGCG concentrations (<20 μg/mL), EGCG is not effective for inhibiting A549 cell viability, either in solution or in nanoform.
The comparison of two nanomedicines (GE and GE-Pt) is shown in Figure 7. With dual drug loading in one of the nanoparticles, GE-Pt shows a significant increase in anticancer effect by reducing cell viability for 7 days of treatment (24:0 ± 2:9%, * p < 0:05, Figure 7). For EGCG, even in nanoform (GE), the cell viability was decreased to 62:0 ± 11:0% on day one, but no further effect was seen after a longer culture period (days 3 and 7) during which cell viability rose to around 94%.

Intracellular Platinum Content Increased by
Treating with Nanoformulation. In order to examine cisplatin accumulation in the cell, the concentration was measured by ICP-MS (Figure 8). The platinum concentration in the cells was 6.4 ppb for the cells treated with cisplatin solution and 14.2 ppb for the cells cultured with GE-Pt after 1 hour of treatment ( * p < 0:05). After 3 hours, the platinum concentration was slightly increased (~11.0 ppb) in the group treated with the cisplatin solution. However, the platinum concentration in cells treated with GE-Pt NPs for 3 hours reached  Journal of Nanomaterials 40.1 ppb (2.8-fold compared with the GE-Pt at 1 h). The platinum concentration of cells in GE-Pt NPs was 3.6-fold higher than that in the cisplatin solution, after 3 h of treatment, which is a significant difference ( * p < 0:05). The difference is evidence that the nanoparticles (GE-Pt NPs) are transporting drugs (cisplatin) more efficiently into the cells, compared with free cisplatin.

Discussion
Cisplatin resistance and the severe side effects of treatment are major causes of treatment failure in lung cancer patients.
Here, we provide a novel strategy for antitumor treatment by developing dual drug-containing nanoparticles for treating lung cancer, to overcome the chemoresistance of cisplatin.  Nanomedicine is an emerging field that combines nanotechnology, pharmaceutical, and biomedical sciences and has the capacity to improve therapeutic efficacy and reduce side effects [15,16]. In this study, gelatin was used as the carrier component because of its nontoxicity, low cost, ready availability, biodegradability, and biocompatibility [38][39][40]. Various models of chemoresistance to cisplatin have been reported, concerning decreased DNA-adduct formation, enhanced DNA repair, decreased susceptibility to induction of apoptosis, inhibition of efflux transporters, generation of reactive oxygen species (ROS), increased detoxification of the drug, and increased drug inactivation by sulfurcontaining molecules [26]. Therefore, coadministration of two therapeutic agents, involved in different mechanisms, may offer a synergistic therapeutic effect, leading to the eradication of resistant cancer cells.  Journal of Nanomaterials The EGCG exhibits its antitumor effect in lung cells, including the inhibition of cell proliferation, migration, and angiogenesis [13,41]. However, EGCG has some pharmacochemical limitations, such as poor drug stability and membrane permeability [42], resulting in low bioavailability and effectiveness for treating cancer cells. Oxidation of the polyphenol structure leads to EGCG degradation [43,44]. Chemoprevention is a promising approach for cancer management, especially through the use of naturally occurring phytochemicals capable of impeding the process of one or more steps in carcinogenesis [45]. Siddiqui et al. encapsulated the green tea polyphenol epigallocatechin-3-gallate (EGCG) in polylactic acid-(PLA-) polyethylene glycol (PEG) nanoparticles. They observed that encapsulated EGCG retains its biological effectiveness with over 10-fold dose advantage for reducing the viability of human prostate cancer (PCa) cells. This could serve as a basis for the use of nanoparticle-mediated delivery, to enhance bioavailability and limit any unwanted toxicity of chemopreventive agents, EGCG [45]. In our study, the EGCG in nanoform (GE NPs) was more effective in reducing the viability of A549 cells than the EGCG solution on days 3 and 7 of treatment ( Figure 5) at low EGCG concentration.
Cisplatin is one of the first-line chemoagents for various kinds of cancer treatment, including lung cancer [24,46]. Cisplatin is a conventional treatment for most advanced non-small-cell lung cancer (NSCLC) patients, but it has no significant therapeutic effect due to chemoresistance. Its clinical use is also limited due to severe adverse effects, including nephrotoxicity. Different types of nanoparticles with cisplatin encapsulation have been developed to increase the therapeutic effect of cisplatin, with fewer side effects. Gelatin nanoparticles (GP), loaded with cisplatin and decorated with biotinylated epidermal growth factor (bEGF), have previously been developed (GP-Pt-bEGF). These have a lower half-maximal inhibitory concentration (IC 50 = 1:2 μg/mL) for the inhibition of A549 cell growth, and in vivo testing shows that GP-Pt-bEGF has higher antitumor activity and lower toxicity than cisplatin solution, in a subcutaneous model [24]. The investigation of cisplatin-loaded poly   Journal of Nanomaterials (lactic-co-glycolic acid) (PLGA) nanoparticles demonstrated an average particle size and zeta potential of 284.8 nm and -15.8 mV, respectively. The release profile of cisplatin from PLGA NPs showed a biphasic characteristic: an initial burst release on day 1 followed by a controlled release phase [47]. A similar cisplatin release pattern was seen in our GE-Pt NPs (Figure 4(b)).
The size of the NPs also plays a key role in efficient drug delivery into cells. The adsorption of NPs onto the cell membrane leads to an increase in drug concentration near the cell surface, offering a gradient that would favor drug influx into the cells [48]. Cisplatin-PLGA NPs exhibited in vitro anticancer activity against A549 cells, comparable to that of cisplatin solution [47]. Alavi   11 Journal of Nanomaterials (butylcyanoacrylate) (PBCA) NPs significantly increased its cytotoxicity. The IC 50 of this cisplatin-PBCE NPs was around 10~15 μM in the lung cancer cell line (LCC1) [49], and the cisplatin-PBCE NPs significantly enhanced the therapeutic effects of the drug in vivo, evidenced by the survival time of lung cancer-bearing mice compared to the cisplatin solution receiver group [49]. The cytotoxicity of the cisplatin NPs may partly be caused by their efficient uptake by target cells. This study has shown that a high platinum concentration was acquired in A549 cells cocultured with the GE-Pt NPs (Figure 8), suggesting that cisplatin loaded into NPs is more effective in delivery into cancer cells than free cisplatin for the reduction of cancer cell viability, with fewer side effects. The GE-Pt is much more effective than cisplatin or cis-platin+EGCG solution (Figure 6), which could be due to the addition of EGCG.
Cisplatin exerts its anticancer effect through multiple steps. Therefore, resistance to cisplatin has been attributed to multiple mechanisms, such as decreased cisplatin accumulation and increased nucleotide excision repair (NER) [26,50]. Thus, EGCG might overcome cisplatin resistance via the regulation of downstream molecules, in each of these distinct resistance mechanisms. For example, a mechanism that increases cisplatin uptake and accumulation in cancer cells is the involvement of copper transporter 1 (CTR1), a membrane protein participating in copper homeostasis, in cisplatin uptake [26]. EGCG could induce the expression of CTR1, leading to the accumulation of cellular cisplatin and cisplatin-DNA adducts and enhanced sensitivity of ovarian cancer cells to cisplatin. These findings provide a new strategy for green tea polyphenol, EGCG, as an adjuvant for the treatment of ovarian cancer [51]. Excision repair crosscomplementing rodent repair deficiency, complementation group 1 (ERCC-1) is a single-strand DNA endonuclease, which participates in the NER system that repairs cisplatininduced DNA lesions [26]. High expression of ERCC-1 in clinical samples correlates with cisplatin responsiveness and patient survival [52,53]. Heyza et al. found that EGCG is a potent inhibitor of ERCC-1 activity, which inhibits DNA repair ability and enhances the therapeutic efficacy of cisplatin [54]. These findings support EGCG as an ideal candidate to modulate various molecules in cisplatin resistance mechanisms. The result in Figure 7 reveals that the GE-Pt NPs highly reducing the A549 cell viability may be explained by these mechanisms.
It is reported that cisplatin, combined with EGCG, shows a synergistic inhibitory effect in an in vitro lung cancer model, and the effective doses of cisplatin and EGCG were 12 μM and 100 μM, respectively [14]. In vivo xenografts also gave a similar result; with the injection of dual drugs several times during the treating period, tumor size was dramatically reduced [5,14,55]. In our study, cisplatin and ECGC were coloaded into one nanoparticle, to form GE-Pt NPs. Even at low drug concentration (cisplatin/EGCG at 2 μg/mL + 5 μg/mL), GE-Pt NPs possessed high cytotoxicity compared to the cisplatin solution and cisplatin+EGCG solution (Figures 6 and 7). Also compared with a previous study, gelatin nanoparticles with cisplatin (cDDP) loading (GPs-CDDP) have no effect for reducing cell viability (~100%) of A549 at 5 μg/mL cisplatin after one-day treatment [24]. In this study, when A549 cells treated with low concentration of GE-Pt (EGCG 5 μg/mL+cisplatin 2 μg/mL) for the same time period, their cell viability downed to 40%. The cisplatin concentration in GE-Pt NPs (2 μg/mL) here was lower than GPs-CDDP (5 μg/mL), but with higher toxicity to A549 cells. These results indicate that dual drug-loaded NPs (GE-Pt NPs) provide a stable, sustainable release of cisplatin/EGCG ( Figure 4) and an improved uptake for a better accumulation of cisplatin in cells (Figure 8). And the synergistic effect of cisplatin/EGCE was shown, in effective tumor cell eradication, even for a very low dose. The combination therapies of cisplatin with EGCG together can be considered to overcome drug resistance and reduce the toxicity of cisplatin for lung cancer treatment.

Conclusions
The gelatin nanoparticles with cisplatin and EGCG loading (GE-Pt) were designed and prepared to overcome the chemoresistance of cisplatin by synergistic action for the effective killing of lung cancer cells. In this study, we successfully synthesized spherical GE-Pt NPs in nanosize (~75 nm), possessing a positive charge (+20 mV). The encapsulation rate of cisplatin and EGCG in GE-Pt was about 64% and 89%, respectively. Even in a low concentration of GE-Pt NPs (EGCG at 5 μg/mL and cisplatin at 2 μg/mL), these GE-Pt NPs still exhibited significant inhibition of cancer cell viability. These effects were caused, in part, by the ease of uptake into cells, facilitated by endocytosis. The synergistic effect of the combination with cisplatin, EGCG, and nanomedicine (GE-Pt NPs) was shown to reduce A549 cell viability in vitro to 24% after 7 days. This provides an effective anticancer formulation for further evaluation in an animal model.

Data Availability
The data used to support the findings of this study are included in the article.

Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.