Mitochondria play an important role in genesis and development of tumor, and are also drug targets. Herein, we developed a multifunctional celastrol (cela) nanoparticles with a positive core and a negative outer layer. Firstly, the mitochondrial targeted material: triphenyl phosphonium-tocopherol polyethylene glycol succinate (TPP-TPGS, TT) was synthesized, and prepared TT/PLGA@cela nanoparticles (NPs). Then, the positive charge on the surface was neutralized using tumor targeted and pH sensitive chondroitin sulfate-folic acid (CS-FA) material to obtain CS-FA/TT/PLGA@cela NPs. Characterization revealed CS-FA/TT/PLGA@cela NPs to be globular particles with smooth surfaces and an average diameter of 100 nm. This construct could improve the uptake in 4T1 cells. After CS-FA/TT/PLGA@cela NPs entered cancer cells, CS-FA was degraded, then the positively charged TT/PLGA@cela NPs were exposed and completed lysosomal escape, finally localizing to mitochondria. Subsequently, in the alkaline environment of mitochondria, cela is released to kill cancer cells. Meanwhile, the results of the mitochondrial respiration test and mitochondrial membrane potential assay demonstrated that CS-FA/TT/PLGA@cela NPs exerted mitochondrial injury and damage. Moreover, the NPs remarkably enhanced proapoptotic protein expression in 4T1 cells. Importantly, this nanoplatform was able to achieve excellent anti-cancer effects in vivo. Together, the results indicated that the mitochondria-targeting CS-FA/TT/PLGA@cela NPs potentially represent a signifcant advancement in breast cancer treatment.
Research Article
Development of multifunctional celastrol nanoparticles to enhance antitumor effects by interfering with the function of mitochondria in breast cancer
https://doi.org/10.21203/rs.3.rs-1983232/v1
This work is licensed under a CC BY 4.0 License
Version 1
posted
You are reading this latest preprint version
mitochondria targeting
enhance antitumor effects
celastrol
breast cancer
Breast cancer is the most commonly diagnosed cancer among women all over the world, representing approximately 30% of all new cancer diagnoses in women and it is also the second leading cause of death after lung cancer [1, 2]. Chemotherapy is still one of the main treatments for breast cancer [3]. Celastrol (cela), also known as tripterine, is a quinone methyl triterpene, which has the potential function of treating breast cancer [4]. According to previous studies, cela inhibited breast cancer by oxidative stress, mitochondrial dysfunction and up-regulating microRNA-15a [5, 6]. However, its clinical translation is hindered owing to two disadvantages that are low water solubility [7], which limits its bioavailability, and high systemic toxicity stemming from its parochial therapeutic index [8, 9]. Therefore, it is very important to develop a delivery system that can reduce the toxicity and increase the efficiency of cela.
Cela could act on mitochondrial respiratory chain (MRC) complex I and induce cell apoptosis via ROS-dependent mitochondrial pathways [9–11]. Mitochondria also have significant roles in regulating apoptosis, calcium handling, and phospholipid synthesis [12]. These various functions make mitochondria vital for all cell types, but clinical evidence indicates that energy-intensive cells-cancer cell is notably susceptible to mitochondrial dysfunction [12]. We speculate that promoting the tumor cells mitochondria targeting of cela may further increase its anticancer efficacy while alleviating the adverse effects.
Mitochondrial targeting therapy is an extremely wise choice for cancer cells [13]. Mitochondrial cell membrane has relatively strong negative charge, and some literatures have reported that TPP has a good affinity with mitochondria due to its large positive charge. In Francesco et.al research, dodecyl was applied into modify TPP, has been used as a treatment of breast cancer [14]. Besides, In Lee report, they prepared TPP conjugates of glycol chitosan, via the Michael reaction and amidation method which showed antitumor activity [15]. According to the structural characteristics of mitochondria, Singh et.al designed complex (TPP-TPGS) of delocalized lipophilic cationic tocopheryl polyethylene glycol succinate (TPGS) and triphenylphosphine (TPP) [11]. These studies have achieved certain curative effects, but positively charged nanoparticles have high nonspecific internalization rate and short blood circulation half-life [16–18].
Hence, we designed the NPs with a positive core and a negative outer layer. Biodegradable and FDA approved polylactic acid glycol acid (PLGA) was used as the carrier of cela. Mitochondria-targeting TPP-TPGS modified drug carriers in the drug delivery systems (TT/PLGA@cela NPs). In order to target tumor more accurately, folic acid (FA) was modified by connecting with chondroitin sulfate (CS) through amide bond (CS-FA). To avoid blood clearance, the TT/PLGA@cela NPs were coated with CS-FA. Through this method, we prepared CS-FA/TT/PLGA@cela NPs that first targeted the tumor. Then, chondroitin sulfate was degraded in the tumor micro acid environment. The TT/PLGA@cela NPs targeted the mitochondria and released cela in the mitochondria, causing mitochondrial apoptosis and killing tumor cells. Firstly, the CS-FA/TT/PLGA@cela NPs were characterized by a variety of methods. Furthermore, the release characteristics, cellular uptake, and in vivo anti-tumor activity of the NPs were investigated. In addition, the mechanisms, including lysosomal escape behavior, mitochondrial localization and mitochondrial damage were elucidated.
Materials
Chondroitin sulphate sodium salt from bovine cartilage was obtained from Qingdao Wanyuanshan Biotechnology Co., Ltd. (Mw = 30-50 KDa, Qingdao, China). PLGA was obtained from Jinan Daigang Biomaterial Co., Ltd. (Mw = 30 kDa, Jinan, China). TPP-COOH, TPGS and cela were purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. 384-well tissue culture plates and confocal dishes were supplied from NEST Biotechnology Co., Ltd. (Shanghai, China). Cell Titer-Glo Luminescent was procured from Promega Biotech Co., Ltd. (Beijing, China). Fetal bovine serum (FBS), RPMI 1640 cell culture medium (incomplete) and trypsine (0.25%) were procured from Nanjing Senbeijia Biological Technology Co., Ltd. DAPI, MitoTracker Red, LysosomeTracker Red, and JC-1 were brought from Shanghai Biyuntian Biotechnology Co., Ltd. Dialysis membranes was obtained from the Shanghai Yuanye Biotechnology Co., Ltd. All other chemicals and reagents were of analytical purity grade.
Synthesis of TPP-TPGS
The synthesis of TPP-TPGS was illustrated in Scheme 1. First, TPP-COOH (100 mg, 0.22 mmol, 1 equiv) were added to 3 mL of SOCl2 and stirred magnetically at 80 oC for 5 h. SOCl2 which in the reaction solution was removed by vacuum decompression. Next, TPGS (660 mg, 0.44 mmol, 2 equiv) was dissolved in dichloromethane (5 mL) with TEA (92 uL, 0.66 mmol, 3 equiv), and then the mixture was slowly dropped into the product of the previous step and stirred at 25 oC for 5 h. Subsequently, the reaction solution was dialysed in deionised water for 3 days with dialysis membrane of MWCO 1000 Da and freeze-dried for 48 h (62% yield). Finally, compound was confirmed by 1H NMR (TPP-TPGS).
Synthesis of CS-FA
The synthesis route was showed in Scheme 1. In brief, FA (1000 mg, 2.27 mmol, 1 equiv), TEA (347 mg, 3.39 mmol, 1.5 equiv), Boc-1,4-Diaminobutane Hydrochloride (470 mg, 2.5 mmol, 1.1 equiv) and EDCI (254 mg, 2.27 mmol, 1 equiv) were dissolved in 30 mL of dimethyl sulfoxide and stirred at 25 °C for 12 h. Then, methyl tert butyl ether (MTBE) and acetone (7:3, v/v, 200 mL) were added to the reaction solution to obtain precipitation, and wash precipitation with ethyl acetate (EA) to give FA-NH-Boc.
FA-NH2 was synthesized according to the following procedure. To a solution of FA-NH-Boc (2100 mg, 3.4 mmol) dissolved in dichloromethane (10 mL) and trifluoroacetic acid (5 mL) was added. The mixture was stirred at 25 oC for 3 h. MTBE and acetone (1:1, v/v, 200 mL) were added to the reaction solution to obtain precipitation, and wash precipitation with EA. The crude product was purified by High pressure preparation and purification system to obtain FA-NH2 (50% yield). Compound was confirmed by 1H NMR (FA-NH2).
CS (200 mg, 0.42 mmol, 1 equiv), EDCI (161 mg, 0.84 mmol, 2 equiv) and NHS (193 mg, 1.68 mmol, 4 equiv) were dissolved in formamide (50 mL) and stirred at 25 °C for 3 h. Then, FA-NH2 (43 mg, 0.084 mmol, 0.2 equiv) and 233 μL TEA (170 mg, 1.68 mmol, 4 equiv) were added to the reaction solution and stirred at 25 °C for 12 h. The precipitate was removed by centrifugation, and the supernatant extract was dialysed in deionized water for 3 days with dialysis membrane of MWCO 15000 Da. The final solution was freeze-dried for 48 h, and a yellow CS-FA powder was obtained (50% yield). Compound was confirmed by 1H NMR (CS-FA).
Preparations and characterizations of CS-FA/TT/PLGA@cela NPs
Nanoparticles were prepared by single emulsion solvent evaporation method, as shown Scheme 2a. In short, 70 mg of polymer (TPP-TPGS) and 20 mg of TPGS were solubilized in 20 mL of water (W phase). Separately, 5 mg of cela and 40 mg of PLGA were dissolved in 5 mL of acetone to form the organic oil phase (O phase). Under moderate magnetic stirring, the prepared O phase was dropped into the aqueous phase. After 2 h of stirring, acetone was removed by vacuum decompression. The mixed solution was centrifuged at 14200 rpm for 30 min to obtain NPs suspension in water (TT/PLGA@cela NPs).
The negatively charged CS-FA was used to modify the prepared positively charged drug loaded NPs through electric interaction. Brief, CS-FA solution with a concentration of 16 mg/mL was added to the nanoparticle solution suspended in water (TT/PLGA@cela NPs) and stirred for 12 hours to finally obtain CS-FA/TT/PLGA@cela NPs. Labeled coumarin 6 (C6) nanoparticles were fabricated in the same manner.
Subsequently, size distribution of NPs and cela release performance from NPs in different conditions were measured by a Malvern Zetasizer Nano ZS90, TEM and UV. The drug encapsulation efficiency (EE, %) and drug loading (DL, %) of cela-loaded NPs were calculated by the following equations [19].
Measurement of pH-triggered drug release in vitro
To test pH-triggered cela release of the NPs, the NPs solution was dialyzed in phosphate buffer saline (PBS) with various pH (pH 5.0, pH 7.4, pH 8.0) at 37 ℃ under magnetic stirring. The TT/PLGA@cela NPs or CS-FA/TT/PLGA@cela NPs solution (1.0 mL) was dialyzed against a 10.0 mL buffer (MWCO 15000 Da) [20]. 1 mL of the medium were collected and added 1 mL new buffer solution at prescribed time intervals. The cela concentrations of the samples were detected by a UV.
Cell Culture
Mouse derived breast cancer cell line (4T1 cells) were purchased from ATCC. Cells were cultured in RPMI 1640 medium with 10% FBS, and 1% sodium pyruvate in a 5% (v/v) CO2 / air mixture at 37 °C.
Cell viability
In vitro cytotoxicity was assessed using the Cell Titer-Glo Luminescent Cell Viability Assay. The cytotoxicity of cela, PLGA@cela NPs, TT/PLGA@cela NPs, CS-FA/TT/PLGA@cela NPs and CS-FA/TT/PLGA NPs were determined using the Cell Titer-Glo Luminescent Cell Viability method. 4T1 cells were seeded in 384-well plates at 400 cells per well; after 12 h, the 2 μL culture medium at the equivalent drug concentration ranging from 0 to 0.5 μg/mL in each well was added and incubated for 72 h. 20 μL provided by the kit single reagent was added to the well plates and allowed to react for 10 min before chem-iluminescence detection. The viability of cells (%) was determined according to the fluorescence value.
Cellular uptake and endocytosis pathways
4T1 cells were seeded in confocal microscopy dishes (2 × 105 cells per well) for 12 h and then incubated with 100 µg/mL FA for 30 min. CS-FA/TT/PLGA@C6 NPs (10 µg/mL) were added and incubated at 37 °C for 4 h. Control cells were treated without FA, followed by incubation with CS-FA/TT/PLGA@C6 NPs (10 μg/ mL) or TT/PLGA@C6 NPs (10 μg/mL) at 37 °C for 4 h. The solution was removed, and the cells were washed 3 times with PBS. The cells were fixed with 1% paraformaldehyde for 30 min. Then, the nuclei of the cells were dyed with DAPI for 20 min. After incubation, the 4T1 cells were imaged by laser confocal microscopy (CLSM).
The possible endocytosis mechanism of CS-FA/TT/PLGA@C6 NPs was studied by incubating 4T1 cells with specific endocytosis inhibitors. 4T1 cells were incubated with 100 mM sodium azide, 10 μg/mL chlorpromazine, 5 μg/mL colchicine, 0.3 mg/mL amiloride or 5 mg/mL methyl-β-cyclodextrin (M-β-CD) in serum-free medium at 37 °C for 30 min, and then incubated with C6 nanoparticles suspension at 37 °C for 4 h. Then, intracellular fluorescence content was detected by enzyme labeling instrument.
Lysosomal escape behavior
4T1 cells were cultured in confocal microscopy dishes. After 12 h, 4T1 cells were incubated with PLGA@C6 NPs, TT/PLGA@C6 NPs and CS-FA/TT/PLGA@C6 NPs for 4 h. The solution was removed, and the cells were washed 3 times with PBS; the cells were incubated with a lysosome red fluorescence probe, LysoTracker Red, for 30 min and were fixed with 1% paraformaldehyde for 30min. Then, the nuclei of the cells were dyed with DAPI for 20 min. After incubation, the 4T1 cells were imaged by CLSM.
Mitochondrial localization and oxygen consumption rates assay
4T1 cells were cultured in confocal microscopy dishes. After 12 h, 4T1 cells were incubated with PLGA@C6 NPs, TT/PLGA@C6 NPs and CS-FA/TT/PLGA@C6 NPs for 6 h. The solution was removed, and the cells were washed 3 times with PBS; the cells were treated with a mitochondrial red fluorescence probe, MitoTracker Red, for 30 min and were fixed with 1% paraformaldehyde for 30 min. Then, the nuclei of the cells were dyed with DAPI for 20 min. After incubation, the 4T1 cells were imaged by CLSM.
Oxygen consumption rates (OCR) was analyzed by using the Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technology). 4T1 cells were seeded in 96-well seahorse microplate at a density of 12,500 cells per well 16 h then treated with free cela and cela-loaded NPs for 4h or 8 h. Before the assay, cells were washed 3 times with assay medium. Then the plate was incubated for 1 h in a non-CO2 incubator before measuring the OCR. Three baseline OCR measurement were recorded before injecting oligomycin A (1 μM) to inhibit ATP synthase, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 1.0 μM) to uncouple the mitochondria and yield maximal OCR, and rotenone (0.5 μM) and antimycin A (0.5 μM) to prevent mitochondrial oxygen consumption through inhibition of Complex I and Complex III, respectively. According to measurements, indices of mitochondrial function were determined.
Mitochondrial membrane potential assay
The mitochondrial membrane potential (MMP) was measured by the JC-1 Detection Kit according to the instructions [21]. 4T1 cells were cultured in confocal microscopy dishes then treated for the 12 h with free cela and cela-loaded NPs. Control was treated with FBS-free medium alone. Cells were incubated with JC-1 for 30 min at 37 ℃. After that, the 4T1 cells were imaged by CLSM.
Western blotting
4T1 cells were inoculated into in a 6-well plate at 2 × 105 cells/well at 5 % CO2 at 37 °C for 24 h and treated with cela and cela-loaded NPs at a final concentration of 0.3 μg/mL for another 24 h. Controls samples were treated by adding blank medium. Western blotting was used to analyzed the protein levels in breast cancer cells. Antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The membrane was washed 3 times with TBST, and the enhanced chemiluminescence detection kit was used to observe the signal.
Antitumor studies and toxicity analysis
To investigate the anti-tumor effect of the NPs, 4T1 breast cancer cells were inculated in BALB/c mice to induce tumors and to conduct an in vivo pharmacodynamic experiment. In short, cultured mouse 4T1 cells (1 × 107 cells/mL) were collected and 0.1 mL 4T1 cells were seeded into the armpits by hypodermic injection. When the tumor volume reaches 50-60 (mm3), the mice were stochastically divided into 4 groups (n=5), and they were given saline (control), cela (2 mg/kg), TT/PLGA@cela NPs (2 mg/kg) and CS-FA/TT/PLGA@cela NPs (2 mg/kg) day about by intravenous injection.
Tumor volume and weight of mice were determined every 2 days until 14 days. The relative volume of the tumor was calculated as tumor length × width2/2 (mm3). Then, the mice were sacrificed and their tumors were removed for photographe and measurement. Tumors and major organs were fixed overnight in 4% paraformaldehyde and sections were stained with TUNEL and HE respectively.
Furthermore, to test whether cela and cela-loaded NPs had toxic effects on the blood system, the healthy mice were stochastically divided into 4 groups (n=3), and they were given saline (control), cela (2 mg/kg), TT/PLGA@cela NPs (2 mg/kg) and CS-FA/TT/PLGA@cela NPs (2 mg/kg) by intravenous injection. Routine blood examination was conducted 1,7, and 14 days after administration in mice of each group [22].
Statistical analysis
Data were statistically analyzed using one-way analysis of variance (ANOVA). A p-value < 0.05 was considered statistically significant.
Synthesis and characterization of CS-FA and TPP-TPGS
To enhance the tumor targeting of the NPs, CS-FA was obtained by FA modification of the carboxyl of CS (Scheme 1). The characteristic peak of aromatic protons in FA were at 8.00-9.00 ppm while peak of proton hydrogen of the CS was at 4.00-5.00 ppm. Ultimately, the aromatic ring of FA (8.00–9.00 ppm) and the characteristic peak of CS (4.00-5.00 ppm) existed in CS-FA, as shown in Fig. 1a, indicating successful synthesis of CS-FA.
TPP-TPGS was synthesized to further target subcellular organelle mitochondria. Fig. 1b exhibits the 1H NMR spectra of TPP-COOH, TPGS and TPP-TPGS. The characteristic peak of aromatic protons in TPP-COOH were at 7.70-7.80 ppm while peak of protons from polyethylene glycol in TPGS were at 3.54 ppm. The peak of TPGS in 3.30 ppm was caused by water in the solvent, which wasn’t the characteristic peak of TPGS [23]. The 1H-NMR spectrum of TPP-TPGS showed characteristic peaks of TPP-COOH at 7.70-7.80 ppm and peaks of TPGS at 3.54 ppm, indicating that TPP-TPGS had been successfully synthesized.
Preparation and characterization of the NPs
The preparation, delivery, lysosomal escape, mitochondrial action and anti-cancer effects of CS-FA/TT/PLGA@cela NPs were depicted in Scheme 2. CS-FA/TT/PLGA@cela NPs were prepared by single emulsion solvent evaporation method and characterized by TEM and Dynamic Light Scattering (DLS). All NPs were spherical, and had a uniform particle size of about 70-100 nm (Fig. 2a, c, Additional file 1: Fig. S1). The average diameter of CS-FA/TT/PLGA@cela NPs was approximately 100 nm, which was larger than that of TT/PLGA@cela NPs. This may be caused by the outer of CS-FA. Meanwhile, diameter of PLGA@cela NPs was larger than that of TT/PLGA@cela NPs, it may be that the existence of TPP-TPGS makes the structure more compact. In addition, the zeta potentials of PLGA@cela NPs and TT/PLGA@cela NPs were -20.2 mV and 22.3 mV (Additional file 1: Fig. S2), respectively. It indicated the positive charge of TPP-TPGS could increase the zeta potential. At the same time, the zeta potential of the CS-FA/TT/PLGA@cela NPs was extremely decreased compared with that of the TT/PLGA@cela NPs, demonstrating negatively charged CS-FA could reduce the zeta potential.
To determine the capacity of the nano-loaders, the drug loading and encapsulation efficiency were detected by UV. The drug loading and encapsulation efficiency of CS-FA/TT/PLGA@cela NPs were 36.1±2.1 % and 75.4±2.8 %, respectively. These results were not significantly different from the corresponding values of TT/PLGA@cela NPs (Additional file 1: Table S1). In addition, the NPs did not significantly change particle size (Additional file 1: Fig. S3) or zeta potential (Additional file 1: Fig. S4) at 4 °C for 15 days. The stability results provide a basis for the preservation methods of subsequent animal experiments.
Measurement of pH-triggered drug release in vitro
To investigate the correlation between the release rate in response to alkaline pH and the physicochemical properties of the drug, the pH-triggered cela release rate was measured by an in vitro drug release method. As shown in Fig. 2d, e, in a buffered medium simulating mitochondrial pH around 8.0, the TT/PLGA@cela NPs and CS-FA/TT/PLGA@cela NPs exhibited rapid release behavior, with 95% of cela released within 24 h. However, the TT/PLGA@cela NPs and CS-FA/TT/PLGA@cela NPs exhibited sustained release at pH 5.0 and 7.4 with the drug release rate 20% and 56% respectively. It confirmed that TT/PLGA@cela NPs and CS-FA/TT/PLGA@cela NPs could response to weakly alkaline pH environment of tumor cell mitochondria, reduce drug leakage in the cytoplasm and lysosome, and promote the rapid release in mitochondrial. Furthermore, TT/PLGA@cela NPs and CS-FA/TT/PLGA@cela NPs had similar release, demonstrating that CS-FA did not affect the weakly alkaline response of nanoparticle mitochondria. Consistent with the previous study of Tan et al. [19], at pH 8.0, the high solubility of cela may lead to the weak interaction between cela and the hydrophobic core of nanoparticles, which ultimately leads to the rapid release of drugs.
Cellular uptake and endocytosis pathways
CLSM can be used to study the cellular uptake of NPs. As shown in Fig. 3a, the fluorescence intensity of CS-FA/TT/PLGA@C6 NPs was significantly stronger than that of TT/PLGA@C6 NPs. It indicated that the outer CS-FA played an essential role in promoting the absorption of NPs, conferring the ability of tumor targeting. In addition, when CS-FA/TT/PLGA@C6 NPs was added to 4T1 cells that contained saturated FA, the cellular uptake efficiency of the NPs decreased due to the competitive binding of free FA on the cancer cells. This result was mainly due to the excessive free FA that could competitively block the uptake of CS-FA/TT/PLGA@C6 NPs, indicating that excessive free FA inhibited cell binding and uptake through specific competitive binding with receptor on 4T1 cell. This result demonstrated that CS-FA/TT/PLGA@C6 NPs was more conducive to the cellular uptake due to the targeting of FA.
NPs can be ingested by phagocytosis or pinocytosis. Endocytosis can occur through giant pinocytosis, endocytosis mediated by grid protein or cell membrane cave-like invagination, or independent endocytosis [24,25]. Here, various inhibitors were selected to study different absorption mechanisms [26]. Sodium azide is an active transport inhibitor with comprehensive energy consumption [27]. Methy-β-Cyclodextrin is used as a specific endocytosis inhibitor mediated by cell membrane caveolae or lipid raft [28,29]. Chlorpromazine is used to inhibit reticulin dependent endocytosis [30]. Amiloride hydrochloride is a Na+/H+ exchange inhibitor of microcytosis, while colchicine inhibits the formation of macropinocytosis [31]. As shown in Fig. 3b, co-incubation with sodium azide inhibited the uptake of CS-FA/TT/PLGA@C6 NPs in 4T1 cells (P<0.01), suggesting that cell uptake was active transport. At the same time, chlorpromazine also affected the uptake of CS-FA/TT/PLGA@C6 NPs in 4T1 cells (P<0.01). However, the fluorescence intensity of CS-FA/TT/PLGA@C6 NPs in the other inhibitors treated group was similar to that in the control group, indicating that the endocytosis mechanism of CS-FA/TT/PLGA@C6 NPs was not closely related to other ways. Therefore, clathrin-mediated endocytosis was the main endocytic pathway for CS-FA/TT/PLGA@C6 NPs, which may be related to FA mediated internalization of nanoparticles.
Assay of cytotoxicity in vitro
Inhibition of cancer by NPs was investigated in vitro by proliferation assays. The potential therapeutic efficacy of NPs was investigated in 4T1 cells by Cell Titer-Glo Luminescent Cell Viability Assay. Cela and cela-loaded NPs had concentration-dependent cytotoxicity in 4T1 cells. With the increase of cela concentration, the inhibitory effect on cancer cells increased. As shown in Fig. 3c, PLGA@cela NPs, TT/PLGA@cela NPs and CS-FA/TT/PLGA@cela NPs exhibited enhanced anticancer activity with the IC50 value much lower than free cela (Additional file 1: Table S2). At the maximum concentration of 0.5 μg/mL cela, the cell survival-rates of cela, PLGA@cela NPs, TT/PLGA@cela NPs, CS-FA/TT/PLGA@cela NPs and CS-FA/TT/PLGA NPs were 60.7%, 40.5%, 5.3%, 1.2% and 100.0%, respectively. The results showed that the cytotoxicity order of NPs: CS-FA/TT/PLGA@cela NPs > TT/PLGA@cela NPs > PLGA@cela NPs > cela > CS-FA/TT/PLGA NPs. CS-FA/TT/PLGA@cela NPs had the strongest cytotoxicity. Moreover, the cytotoxicity of CS-FA/TT/PLGA NPs in cancer cells was low, indicating that these carriers are safe and biocompatible with tissues and cells.
Lysosomal escape behavior
After the NPs are absorbed by cancer cells, they must quickly escape lysosomes to produce antitumor effects. Therefore, CLSM was used to study the interaction between the lysosomes and the NPs. As shown in the Fig. 4a, C6 fluoresced green, lysosomes were stained red, and nuclei were stained blue. When various nanoparticles were incubated with cells for 4 h, PLGA@C6 NPs were captured by lysosomes, which could be proved by the co-localization of red and green fluorescence into yellow fluorescence. In contrast, in the nanoparticle map of TT/PLGA@C6 NPs and CS-FA/TT/PLGA@C6 NPs, the yellow fluorescence weak, and the green fluorescence was dominated, indicating the escape of nanoparticles from lysosomes. It was reported that TPP escaped lysosomes quickly because its positive charge could reduce the stability of lysosomal membrane [25,32]. Our results are consistent. Moreover, compared with PLGA@C6 NPs, CS-FA/TT/PLGA@C6 NPs showed lysosomal escape behavior. The result might be that CS-FA was degraded under the acidic microenvironment of tumor, TT/PLGA@C6 NPs exposed and successfully achieved lysosomal escape [11,33-35].
Co-localization of mitochondria and oxygen consumption rates assay
After escaping from lysosomes, laser confocal was used to detect the presence of nanoparticles in mitochondria. Three different nanoparticles were incubated with 4T1 cells for 6 h, and then mitochondria were stained. C6 emitted green fluorescence, mitochondria were stained red, and nuclei were stained blue. The amalgamated yellow signal, which came from the green fluorescent C6 labeled nanoparticles colocalized with red fluorescent-tagged mitochondria, was imaged by CLSM, as shown in Fig. 4b. In the case of the treatment with PLGA@C6 NPs, the clear separation of green and red signals showed that nanoparticles were not localized to mitochondria. In contrast, in the case of the treatment with TT/PLGA@C6 NPs, CS-FA/TT/PLGA@C6 NPs, the mitochondria in the merged image were yellow, manifesting a quite good overlap between the green signal of C6 and the red signal of the mitochondria, which indicated the two NPs were located at mitochondria. It suggested that the CS-FA layer was removed from the nanoparticles to expose TT/PLGA@C6 NPs, which furnished for accurate mitochondrial targeting. Moreover, the merged yellow fluorescence of CS-FA/TT/PLGA@C6 NPs was stronger than that of TT/PLGA@C6 NPs, which might be caused by the former cells absorbing more. These delivery process resulted in specific accumulation of the nanoparticles in the mitochondrial fraction.
To further investigate the effects of CS-FA/TT/PLGA@cela NPs on mitochondrial respiration in 4T1 cells, we used the XF cell Mito stress test kit. Oligomycin (ATP coupling agent), FCCP (uncoupling agent), mycin (antimicrobial), and rotenone (mitochondrial inhibitor) were added at predetermined time points, and OCR of mitochondria at various stages was detected using an XF96 analyzer. In the concentration dependent test (Fig. 5a), OCR of 4T1 cells was affected at all concentrations. Compared with the negative control group, treatment with cela or cela-loaded NPs showed that the basal respiratory value decreased with the increase of cela concentration. After adding oligomycin, the ATP coupling efficiency of CS-FA/TT/PLGA@cela NPs treated cells was significantly lower than that of the control group and the free cela group. This result showed that nanoparticles could disrupt ATP synthesis by blocking the proton channel in F0 region and affecting the uncoupling electron transfer of ATP synthase [36]. The addition of FCCP led to the disappearance of the restriction of electron transport on the proton gradient, and the OCR increased sharply to reach the maximum oxygen consumption. However, the OCR after CS-FA/TT/PLGA@cela NPs treatment was also lower than that of other groups. Moreover, the inhibition of mitochondrial respiration TT/PLGA@cela NPs was stronger than that of PLGA@cela NPs. After treatment with 0.3 μg/mL free cela, PLGA@cela NPs, TT/PLGA@cela NPs, CS-FA/TT/PLGA@cela NPs, the coupling efficiency of cells decreased by 3% ,10%, 15% and 40% respectively, the maximum oxygen consumption of cells decreased by 54%, 58%, 62% and 85% respectively, and the respiratory reserve capacity of cells decreased by 65%, 71%, 73% and 97% respectively, compared with the negative control group. It could be found that the inhibition of CS-FA/TT/PLGA@cela NPs on mitochondrial respiration was the strongest at the concentration of 0.3 μg/mL. Meanwhile, at the concentration of 0.5 μg/mL, the same was true.
The respiration of cells treated with the NPs for 4 and 8 h was also determined. Compared with the negative control group with treatment for 4 h (Fig. 5b), free cela, PLGA@cela NPs, TT/PLGA@cela NPs and CS-FA/TT/PLGA@cela NPs decreased the coupling efficiency of cells by 1%, 18%, 40% and 48% respectively, the maximum oxygen consumption by 21%, 71%, 85% and 87% respectively, and the respiratory reserve capacity by 23%, 83%, 97% and 98% respectively. Meanwhile, with treatment for 8 h, it was also found that CS-FA/TT/PLGA@cela NPs had good inhibition on the mitochondrial respiration of cancer cells.
These results revealed that free cela could interfere with the correct function of mitochondria. The results are consistent with those reported by Tan et.al [19,35], which indicate that cela acts directly on mitochondria changing the mitochondrial membrane permeability. The results of the mitochondrial respiration test demonstrated that the inhibition of CS-FA/TT/PLGA@cela NPs on the mitochondrial respiration of cancer cells was more significant than that of free cela and PLGA@cela NPs. This result suggested that CS-FA/TT/PLGA@cela NPs exerted mitochondrial injury and damage in the respiratory function of cells, resulting in cell death.
Mitochondrial membrane potential assay
The marked event in the early stage of mitochondrial injury is the decrease of MMP [37]. JC-1 is a fluorescent which accumulates in the mitochondrial membrane in two forms (monomers or dimers), depending on mitochondrial membrane potential. To probe cela and cela-loaded NPs mediated changes in the mitochondrial membrane potential, the mitochondrial membrane potential of 4T1 cells was assessed using JC-1 detection probes [38,39]. As shown in Fig. 6, the control group cells showed strong red fluorescence with a high MMP, whereas the cells treated with CS-FA/TT/PLGA@cela NPs exhibited green fluorescence owing to the monomer form of JC-1. At the same time, PLGA@cela NPs displayed weak damage to mitochondria due to the lack of mitochondrial targeting. Besides, compared with TT/PLGA@cela NPs, damage to mitochondria of CS-FA/TT/PLGA@cela NPs was greater. This is probably because CS-FA/TT/PLGA@cela NPs were better absorbed in 4T1 cells, and a large amount caused greater damage to mitochondria. Thus, CS-FA/TT/PLGA@cela NPs treatment enhanced the depolarization of the MMP.
Apoptosis related protein expression
Western blotting was used to measure the activities of caspase3 [40]. When cells are apoptotic, caspase3 is sheared and converted into activated cl-caspase3. Therefore, as shown in Fig. 7, CS-FA/TT/PLGA@cela NPs significantly enhanced the activities of cl-caspase3 in 4T1 cells. The expression of cl-caspase3 protein in TT/PLGA@cela NPs group was lower than that in CS-FA/TT/PLGA@cela NPs group which may be due to CS-FA promoting more internalization of NPs. Moreover, the activated cl-caspase3 protein expression of the NPs treated group was higher than that of the free cela group. Bax functions as a regulator of apoptosis in cell [40]. In this study, the CS-FA/TT/PLGA@cela remarkably enhanced proapoptotic protein (Bax) expression in 4T1 cells (Fig. 7). At the level of detecting apoptotic protein, it also proved that CS-FA/TT/PLGA@cela NPs could promote tumor cell apoptosis and further inhibited tumor potential which may be due to the dual role of CS-FA and TPP-TPGS.
Antitumor effects on the breast cancer
To evaluate the antitumor effect of cela-loaded NPs, different formulations with the same dose of cela were analyzed in the breast cancer‑bearing mouse models. As shown in Fig. 8a,b, tumor growth in the treatment groups was slower than that in the control group within 14 days of treatment. Compared with that of the cela group, the tumor-inhibition rate of the TT/PLGA@cela NPs group was significantly increased, mainly due to the nanoparticles enhancing the solubility and mitochondrial localization of cela. CS-FA/TT/PLGA@cela NPs displayed the strongest antitumor activity and notably inhibited tumor growth (Fig. 8d). The highest tumor inhibition rate of 76.28% was achieved on day 14 in mice treated with CS-FA/TT/PLGA@cela NPs, which was higher than that achieved using cela or TT/PLGA@cela NPs (Fig. 8c). It corresponds to the results of in vitro experiments with the strongest cell absorption and cytotoxicity. Changes in the body weights of the mice were monitored during the treatment period. As shown in Fig. 8e, the body weights of mice in the NPs group did not change significantly at 14 days, indicating that the NPs were safe. Meanwhile, the weight of mice in the cela group decreased significantly, which should be caused by the toxicity of cela. Besides, HE staining was performed on the heart, liver, spleen, lung and kidney of mice, and the results are shown in the Fig. 9a. HE staining of the main organs of administration of NPs groups showed that none of the groups exhibited obvious damage compared with the control group, indicating the safety and nontoxicity of NPs. However, there was significant inflammatory cell infiltration in the liver and kidney in the cela group. Meanwhile, the purpose of blood routine analysis was to check whether the material cause the blood system’s abnormality. Routine blood examination was conducted 1, 7 and 14 days after administration in mice of each group. From Additional file 1: Fig. S5, for control group or nanoparticles administration group, data did not show significant difference. Nevertheless, as shown in the Additional file 1: Fig. S5, platelets decreased significantly one day after cela administration. Apparently, cela-loaded NPs reduced the toxicity by the cela and influence on other organs and extended the circulation time in the blood thus increasing cela accumulation in the tumor tissue. What is more, TUNEL assay apoptosis in tumor sections confirmed that the number of positive cells was higher in cela-loaded NPs group compared with cela group (Fig. 9b). Meanwhile, the Fig. 9b indicated that CS-FA/TT/PLGA@cela NPs induced more apoptosis than TT/PLGA@cela NPs in tumors.
CS-FA/TT/ PLGA@cela NPs could target tumor cells. After entering tumor cells, CS-FA degraded and exposed positively charged TT/PLGA@cela NPs. Then successful lysosome escape, TT/PLGA@cela NPs entered mitochondria, released cela in mitochondria. So as to induce mitochondrial apoptosis and enhance the anti-tumor effect.
We synthesized the mitochondrial targeted material TPP-TPGS, tumor targeted and pH sensitive material CS-FA. Then developed a multifunctional cela nanoparticles with a positive core and a negative outer layer. CS-FA/TT/PLGA@cela NPs preferentially targeted to tumor cells, and showed good mitochondrial localization ability. TEM findings revealed CS-FA/TT/PLGA@cela NPs to be spherical with an average diameter of 100 nm. Firstly, CS-FA/TT/PLGA@cela NPs could increase the uptake rate in tumor cells. Next, the nanoparticles could escape from lysosomes and co-localization of mitochondria. Lastly, CS-FA/TT/PLGA@cela NPs was able to achieve excellent anti-cancer effects in vivo. Meanwhile, the CS-FA/TT/PLGA NPs had good biocompatibility and were nontoxic. In addition, mechanism studies showed that CS-FA/TT/PLGA@cela NPs could cause mitochondrial injury and damage, induce expression of apoptosis protein. In conclusion, the results of this study provided an effective method for developing a drug delivery system that targeted tumor cells, further localized mitochondria and enhanced antitumor effects.
Cela: Celastrol; TPGS: tocopheryl polyethylene glycol succinate; TPP: triphenylphosphine; PLGA: polylactic acid glycol acid; FA: folic acid; CS: chondroitin sulfate; CS-FA: chondroitin sulfate-folic acid; TPP-TPGS, TT: triphenyl phosphonium-tocopherol polyethylene glycol succinate; NPs: nanoparticles; EA: ethyl acetate; MTBE: methyl tert butyl ether; C6: coumarin 6; EE: encapsulation efficiency; DL: drug loading; PBS: phosphate buffer saline; M-β-CD: methyl-β-cyclodextrin; CLSM: laser confocal microscopy; OCR: Oxygen consumption rates; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; MRC: mitochondrial respiratory chain; MMP: mitochondrial membrane potential.
Acknowledgements
Not applicable.
Author contributions
YQ and ZW: investigation, conceptualization, methodology, data analysis, formal analysis, writing—original draft. XW: data analysis, formal analysis. TZ: conceptualization, methodology. YH: investigation, methodology, data analysis. DW: investigation, data analysis, formal analysis. HS: investigation, supervision,. LZ and YZ: supervision, resources, methodology, data analysis, formal analysis, project administration, writing—review and editing. All authors read and approved the final manuscript.
Funding
This research was supported by the National Natural Science Foundation of China (21877061).
Availability of data and materials
Most of the datasets supporting the conclusions of this study are included within the manuscript and the additional files. The datasets used or analyzed during the study are available on reasonable request.
Ethics approval and consent to participate
Animal experiments involved above were performed in accordance with the guidance approved by the Ethics Committee of Nanjing Normal University, which were approved by the Institutional Animal Care and Use Committee (IACUC-20210250).
Consent for publication
All authors agree to be published.
Competing interests
The authors declare no competing financial interest.
- Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer Statistics, 2021. CA Cancer J Clin. 2021;71:7-33.
- Viale PH. The American Cancer Society's Facts & Figures: 2020 Edition. J Adv Pract Oncol. 2020;11:135-136.
- Taghian NR, Miller CL, Jammallo LS, O'Toole J, Skolny MN. Lymphedema following breast cancer treatment and impact on quality of life: a review. Crit Rev Oncol Hematol. 2014;92:227-34.
- Wagh PR, Desai P, Prabhu S, Wang J. Nanotechnology-Based Celastrol Formulations and Their Therapeutic Applications. Front Pharmacol. 2021;12:673209.
- Zuo A, Zhao P, Zheng Y, Hua H, Wang X. Tripterine inhibits proliferation, migration and invasion of breast cancer MDA-MB-231 cells by up-regulating microRNA-15a. Biol Chem. 2019; 0469.
- Shrivastava S, Jeengar M K, Reddy V S, Reddy G B, Naidu V G. Anticancer effect of celastrol on human triple negative breast cancer: possible involvement of oxidative stress, mitochondrial dysfunction, apoptosis and PI3K/Akt pathways. Exp Mol Pathol. 2015;98:313-27.
- Yang H, Pan Z, Jin W, Zhao L, Xie P, Chi S, Lei Z, Zhu H, Zhao Y. Preparation, characterization and cytotoxic evaluation of inclusion complexes between celastrol with polyamine-modified β-cyclodextrins. J Incl Phenom Macro. 2019;95:147-157.
- Zhang X, Zhang T, Zhou X, Liu H, Sun H, Ma Z, Wu B. Enhancement of oral bioavailability of tripterine through lipid nanospheres: preparation, characterization, and absorption evaluation. J Pharm Sci. 2014;103:1711-9.
- Shi J, Li J, Xu Z, Chen L, Luo R, Zhang C, Gao F, Zhang J, Fu C. Celastrol: A Review of Useful Strategies Overcoming its Limitation in Anticancer Application. Front Pharmacol. 2020;11:558741.
- Lamb R, Harrison H, Hulit J, Smith D L, Lisanti M P, Sotgia F. Mitochondria as new therapeutic targets for eradicating cancer stem cells: Quantitative proteomics and functional validation via MCT1/2 inhibition. Oncotarget. 2014;5:11029-37.
- Singh Y, Viswanadham K, Pawar V K, Meher J, Jajoriya AK, Omer A, Jaiswal S, Dewangan J, Bora HK, Singh P, Rath SK, Lal J, Mishra DP, Chourasia MK. Induction of Mitochondrial Cell Death and Reversal of Anticancer Drug Resistance via Nanocarriers Composed of a Triphenylphosphonium Derivative of Tocopheryl Polyethylene Glycol Succinate. Mol Pharm. 2019;16:3744-3759.
- Chan DC. Mitochondrial Dynamics and Its Involvement in Disease. Annu Rev Pathol. 2020;15:235-259.
- Wang H, Zhang F, Wen H, Shi W, Huang Q, Huang Y, Xie J, Li P, Chen J, Qin L, Zhou Y. Tumor- and mitochondria-targeted nanoparticles eradicate drug resistant lung cancer through mitochondrial pathway of apoptosis. J Nanobiotechnology. 2020;18:8.
- De Francesco EM, Ózsvári B, Sotgia F, Lisanti MP. Dodecyl-TPP Targets Mitochondria and Potently Eradicates Cancer Stem Cells (CSCs): Synergy With FDA-Approved Drugs and Natural Compounds (Vitamin C and Berberine). Front Oncol. 2019;9:615.
- Lee Y H, Park H I, Chang W S,Choi J S. Triphenylphosphonium-conjugated glycol chitosan microspheres for mitochondria-targeted drug delivery. Int J Biol Macromol. 2021;167:35-45.
- Alexis F, Pridgen E, Molnar L K, Farokhzad OC. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharm. 2008;5:505-15.
- Wang B, He X, Zhang Z, Zhao Y, Feng W. Metabolism of nanomaterials in vivo: blood circulation and organ clearance. Acc Chem Res. 2013;46:761-9.
- Poon W, Zhang YN, Ouyang B, Kingston BR, Wu J LY, Wilhelm S, Chan W CW. Elimination Pathways of Nanoparticles. ACS Nano. 2019;13:5785-5798.
- Tan Y, Zhu Y, Zhao Y, Wen L, Meng T, Liu X, Yang X, Dai S, Yuan H, Hu F. Mitochondrial alkaline pH-responsive drug release mediated by Celastrol loaded glycolipid-like micelles for cancer therapy. Biomaterials. 2018;154:169-181.
- Duan F, Jin W, Zhang T, Zhang F, Gong L, Liu X, Deng X, Gao W. Self-Activated Cascade Biocatalysis of Glucose Oxidase-Polycation-Iron Nanoconjugates Augments Cancer Immunotherapy. ACS Appl Mater Interfaces. 2022. (http:// doi: 10.1021/acsami.2c04894)
- Yu H, Jin F, Liu D, Shu G, Wang X, Qi J, Sun M, Yang P, Jiang S, Ying X, Du Y. ROS-responsive nano-drug delivery system combining mitochondria-targeting ceria nanoparticles with atorvastatin for acute kidney injury. Theranostics. 2020;10:2342-2357.
- Lou H, Ji A, Qu C, Liu H, Jiang L, Chen H, Cheng Z. A Small-Molecule Based Organic Nanoparticle for Photothermal Therapy and Near-Infrared-IIb Imaging. ACS Appl Mater Interfaces. 2022;14:35454-35465.
- Babij NR, McCusker EO, Whiteker GT, Canturk B, Choy N, Creemer LC, Amicis CVD, Hewlett NM, Johnson PL, Knobelsdorf JA, Li F, Lorsbach BA, Nugent BM, Ryan SJ, Smith MR, Yang Q. NMR Chemical Shifts of Trace Impurities: Industrially Preferred Solvents Used in Process and Green Chemistry. Organic Process Research & Development. 2016;20:661-667.
- Hillaireau H, Couvreur P. Nanocarriers' entry into the cell: relevance to drug delivery. Cell Mol Life Sci. 2009;66:2873-96.
- Wang H, Shi W, Zeng D, Huang Q, Xie J, Wen H, Li J, Yu X, Qin L, Zhou Y. pH-activated, mitochondria-targeted, and redox-responsive delivery of paclitaxel nanomicelles to overcome drug resistance and suppress metastasis in lung cancer. J Nanobiotechnology. 2021;19:152.
- Contreras-Ruiz L, de la Fuente M, Párraga JE, López-García A, Fernández I, Seijo B, Sánchez A, Calonge M, Diebold Y. Intracellular trafficking of hyaluronic acid-chitosan oligomer-based nanoparticles in cultured human ocular surface cells. Mol Vis. 2011;17:279-90.
- Torchilin VP, Rammohan R, Weissig V, Levchenko TS. TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors. Proc Natl Acad Sci USA. 2001;98:8786-91.
- Christian AE, Haynes P, Phillips MC, Rothblat GH. Use of cyclodextrins for manipulating cellular cholesterol content. J Lipid Res. 1997;38:2264-72.
- Petrov AM, Naumenko NV, Uzinskaya KV, Giniatullin AR, Urazaev A K, Zefirov A L. Increased non-quantal release of acetylcholine after inhibition of endocytosis by methyl-β-cyclodextrin: the role of vesicular acetylcholine transporter. Neuroscience. 2011;186:1-12.
- Wang LH, Rothberg KG, Anderson RG. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J Cell Biol. 1993;123:1107-17.
- Perisa D, Rohrer L, Kaech A, von Eckardstein A. Itinerary of high density lipoproteins in endothelial cells. Biochim Biophys Acta. 2016;1861:98-107.
- Sreedurgalakshmi K, Srikar R, Harikrishnan K, Srinivasan L, Rajkumari R. Cetuximab-siRNA Conjugate Linked Through Cationized Gelatin Knocks Down KRAS G12C Mutation in NSCLC Sensitizing the Cells Toward Gefitinib. Technol Cancer Res Treat. 2021;20:15330338211041453.
- Yamada S. Catabolism of chondroitin sulfate. Cell Mol Biol Lett. 2015;20:196-212.
- Prabhakar V, Sasisekharan R. The biosynthesis and catabolism of galactosaminoglycans. Adv Pharmacol. 2006;53:69-115.
- Li J, Wei Y, Yang X, Wu W, Zhang M, Li M, Hu Z, Liu Y, Wang N, Yu X. Rational Construction of a Mitochondrial Targeting, Fluorescent Self-Reporting Drug-Delivery Platform for Combined Enhancement of Endogenous ROS Responsiveness. ACS Appl Mater Interfaces. 2020;12:32432-32445.
- Chang Y, Chen K, Chen L. Mitochondrial ROS1 Increases Mitochondrial Fission and Respiration in Oral Squamous Cancer Carcinoma. Cancers (Basel). 2020;12:
- Zhao R, Ning X, Wang M, Yu A, Wang Y. A multifunctional nano-delivery system enhances the chemo-co-phototherapy of tumor multidrug resistance via mitochondrial-targeting and inhibiting P-glycoprotein-mediated efflux. J Mater Chem B. 2021;9:9174-9182.
- Mao Z, Xia W, Wang J, Chen T, Zeng Q, Xu B, Li W, Chen X, Xu S. Perfluorooctane sulfonate induces apoptosis in lung cancer A549 cells through reactive oxygen species-mediated mitochondrion-dependent pathway. J Appl Toxicol. 2013;33:1268-76.
- Cai M, Liang W, Wang K, Yin D, Fu T, Zhu R, Qu C, Dong X, Ni J, Yin X. Aperture Modulation of Isoreticular Metal Organic Frameworks for Targeted Antitumor Drug Delivery. ACS Appl Mater Interfaces. 2022.(http:// doi: 10.1021/acsami.2c07450)
- Zhao T, Fu Y, Sun H, Liu X. Ligustrazine suppresses neuron apoptosis via the Bax/Bcl-2 and caspase-3 pathway in PC12 cells and in rats with vascular dementia. IUBMB Life. 2018;70:60-70.
Scheme 1 and 2 are available in the Supplementary Files section.
No competing interests reported.
- SupplementaryInformation8.21.docx
- Scheme1.png
Scheme 1. Scheme of the synthesis of TPP-TPGS and CS-FA.
- Scheme2.png
Scheme 2. Scheme of the structures and projected mechanism of action of CS-FA/TT/PLGA@cela NPs to treat breast cancer. a Fabrication scheme of CS-FA/TT/PLGA@cela NPs. b Scheme of the transport pathway after intravenous injection of CS-FA/TT/PLGA@cela NPs.
