Novel ROS-Responsive Marine Biomaterial Fucoidan Nanocarriers with AIE Effect and Chemodynamic Therapy

Chemodynamic therapy (CDT) has been widely used in the treatment of many kinds of tumors, which can effectively induce tumor cell apoptosis by using produced reactive oxygen species (ROS). In this paper, ROS-sensitive multifunctional marine biomaterial natural polysaccharide nanoparticles (CT/PTX) were designed. Aggregation-induced emission (AIE) molecules tetraphenylethylene (TPE) labeled and caffeic acid (CA) modied fucoidan (FUC) amphiphilic carrier material (CA-FUC-TK-TPE, CFTT) was fabricated, in which the thioketal bond was used as the linkage arm between TPE and fucoidan chain, giving the CFTT material ROS sensitivity. In addition, amphiphilic carrier material (FUC-TK-VE, FTVE) composed of thioketal-linked vitamin E and fucoidan was synthesized. The mixed carrier material CFTT and FTVE self-assembled in water to form nanoparticles (CT/PTX ) loaded with PTX and Fe 3+ . CT/PTX nanoparticles could induce ROS oxidative stress in tumor sites through the CDT effect induced by Fe 3+ . The CDT effect was combined with the chemotherapeutic drug PTX to achieve tumor inhibition. In vitro cell studies have proved that CT/PTX nanoparticles have excellent cell permeability and tumor cytotoxicity. In vivo antitumor experiments conrmed effective antitumor activity and reduced side effects.


Introduction
Since 2001, a series of luminescent materials with aggregation-induced luminescence (AIE) properties have been developed [1][2][3]. Compared with the aggregation-caused quenching effect (ACQ), aggregationinduced luminescent uorophores (AIEgens) have almost no radiation in dilute solution, but have high radiation in the aggregated state due to limited intramolecular motion [4,5]. AIEgens have the advantages of high uorescence e ciency, excellent optical stability, high signal-to-noise ratio, large Stokes shift, and resistance to photobleaching [6,7]. As a type of newly developed probe, AIEgens have a wide range of medical applications [8]. Tetrastyrene (TPE) and its derivatives are the most studied AIE compounds [9].
Reactive oxygen species (ROS) play an important role in biological processes. High levels of ROS can cause oxidative damage to cellular biomolecules such as lipids, proteins, and nucleic acid molecules, leading to tumor cell death [10]. Chemodynamic therapy (CDT) is a new therapeutic strategy, which uses biochemical reactions (such as Fenton reaction and Fenton-like reaction) to produce ROS for the killing of tumor cells [11][12][13]. Iron-mediated Fenton reaction could convert hydrogen peroxide (H 2 O 2 ) into highly cytotoxic hydroxyl radicals (•OH) [14].
Tumor cells proliferate abnormally fast and have a high metabolic rate [15]. The cells are in a state of oxidative stress, and the mitochondria are prone to produce excessive ROS [16]. High ROS levels have been widely used in the development of stimulus-responsive drug delivery systems. Compared with widely used intrinsic signals such as pH and GSH, ROS in tumor tissues has higher speci city [17][18][19][20].
When nanoparticles with ROS-responsive thioketal bond arrive at the tumor site with a high ROS level, the thioketal bond breaks and the nanoparticles disintegrate to release the loaded drugs, thus achieving a responsive anti-tumor effect [21,22].
In recent years, more and more natural polysaccharides from the ocean have been developed and used in the construction and in vivo transport of nanomedicines [23]. Fucoidan is a kind of sulfurated polysaccharide, mainly composed of fucose, which can be extracted from different kinds of brown algae [24]. Fucoidan are already used in food, dietary supplements, and cosmetics [25]. Fucoidan has many biological activities, such as anti-virus, anti-in ammatory, immunomodulatory, and (anti-)angiogenic potential [26,27]. In addition, through different mechanisms and cancer-related pathways, fucoidan was reported to have potential anticancer effects [28][29][30]. Because of its good hydrophilicity and biocompatibility, fucoidan has been studied and applied in drug delivery system [31].
In this study, according to the tumor microenvironment with a high ROS level, multi-functional nanoparticles based on natural marine fucoidan were designed. Caffeic acid-fucoidan-thioketaltetraphenylethylene (CA-FUC-TK-TPE, CFTT) was successfully synthesized. In order to improve the stability of nanoparticles, adjust the particle size to the ideal range, and improve the drug loading and encapsulation e ciency, fucoidan-thioketal-vitamin E (FUC-TK-VE, FTVE) was designed and synthesized.
CFTT and FTVE were mixed according to the mass ratio of 4:1 to form the composite carrier material. Through the dialysis method, paclitaxel (PTX) and the appropriate amount of FeCl 3 ·6H 2 O were loaded into the nano-micelles formed by the composite carrier material to obtain CT/PTX. The PTX was encapsulated into the hydrophobic core of the nanomicelles; while ferric ions were chelated by CA groups through the non-covalent coordination interactions. Under the enhanced permeability and retention (EPR) effect, CT/PTX in the circulation could accumulate to the tumor site [32,33]. After the CT/PTX

Synthesis of CFTT
The synthetic routes of CFTT were revealed in Fig. 1A.
First, 0.187 mM of TK was dissolved in THF, then oxaloyl chloride (0.187 mM) was slowly added at 0 ℃ [34]. After stirring at room temperature for 3 h, TK with one end of carboxyl group activated was obtained, and the system was described as solution 1. An appropriate amount of triethylamine was dropped into the THF solution of TPE-OH (0.187 mM) to obtain solution 2. Under the ice bath condition, solution 2 was slowly added into solution 1 and stirred at 55 ℃ for 6 h without light. At the end of the reaction, THF and unreacted oxaloyl chloride and triethylamine were removed using a rotary evaporator. Single-substituted TK-TPE puri ed products were obtained by column chromatography.
The puri ed TK-TPE (0.187 mM) was dissolved in formamide, then 1.5 eq of EDC and 1.5 eq of DMAP were added to activate the carboxyl group of TK-TPE at 35 ℃ for 2 h. After activation, the formamide solution of FUC was added and stirred at 35 ℃ for 48 h without light. After 48 h, the reaction solution was dialysed in deionized water for 12 h, and the deionized water was changed every 2 h. FUC-TK-TPE (FTT) was obtained by lyophilization after dialysis.
0.187 mM of CA was dissolved in formamide, then 1.2 eq of EDC and HOBT were added to activate the carboxyl group for 3 h. After the activation, the formamide solution of FTT was added into the activated CA reaction solution, and the reaction was stirred at room temperature without light for 48 h. After the reaction, CA-FUC-TK-TPE (CFTT) was obtained by dialysis and freeze-drying. 1 H-NMR was used to verify the structure of CFTT.

Synthesis of FTVE
The synthetic route of FTVE was shown in Fig. 1B.
After one end of the carboxyl group of TK was activated by the acyl chloride method, THF solution containing 80.54 mg of D-α-tocopherol (VE) was slowly added under ice bath condition, and a small amount of triethylamine was injected at the same time. After stirring at 55 ℃ for 6 h, the reaction solution was rotated to remove volatile components. Pure VE-TK was obtained by column chromatography. After activation at room temperature for 2 h, the formamide solution of FUC was added and reacted at 35 ℃ for 36 h. Finally, FUC-TK-VE (FTVE) was obtained through dialysis and lyophilization. 1 H-NMR was used to verify the structure of FTVE.
2.4. Preparation of CT/PTX nanoparticles 8 mg of CFTT and 2 mg of FTVE were dissolved in 4 mL of formamide, and then PTX (1 mg/mL) was slowly and successively added into the carrier material solution. After the solution was mixed evenly by ultrasound, the solution was dialysed in the 2000 Da dialysis bag for 10 h, and appropriate FeCl 3 ·6H 2 O was added for further dialysis. After dialysis, CT/PTX was obtained through 0.22µm membrane ltration.

Characterizations
The particle size and polydispersity index (PDI) of CT/PTX were determined by Delsa Nano C Particle Analyzer [35]. TEM was used to observe the morphology of CT/PTX nanoparticles. The content of PTX was determined by HPLC at 227 nm [36]. The DL% were calculated as follows: DL% = weight of drugs in nanoparticles / weight of nanoparticles × 100% 2.6. In vitro ROS-responsive assay 1 mL of concentrated CT/PTX nanoparticles solution was packed into dialysis bags of 2000 Da respectively. These dialysis bags were put into centrifuge tubes containing 47 mL PBS (pH 7.4, containing 0.5% Tween 80) with different concentrations of H 2 O 2 (0, 0.1, 1, and 10 mM). The samples were incubated on a thermostatic water bath shaker at 37°C. At different time points speci ed, 1.0 mL of the release medium was collected, and 1.0 mL of fresh release medium was subsequently added to keep the medium volume constant. The concentration of PTX in the collected release medium was determined by HPLC.
In order to prove that CT/PTX nanoparticles could induce Fenton reaction to produce ROS, methylene blue (MB) was used to study Fenton catalytic activity of CT/PTX nanoparticles in the presence of H 2 O 2 [11]

In vitro cytotoxicity assays
FT/PTX and CT/PTX nanoparticles were prepared by the dialysis method. Different from the preparation of CT/PTX, the carrier material of FT/PTX is a mixture of FTT and FTVE, and the mass ratio of FTT to FTVE is 4:1. The cytotoxicity of CT/PTX and FT/PTX nanoparticles against A549 was determined by MTT assay [37,38]. A549 cells were respectively plated in 96-well plates. After incubation for 24 h, the old culture medium was discarded, and a fresh complete culture medium containing different preparations (Free PTX, FT/PTX, CT/PTX) was added. After incubation for 24 h or 48 h, the cells were incubated with MTT solution for 4 h. Then, DMSO was added to dissolve the violet formazan crystals. Finally, the absorbance was measured at 490 nm using a microplate reader.

Cellular uptake and distribution
A549 cells and B16F10 cells were incubated in a 24-well plate for 24 h. When the cells adhered to the wall, CT/PTX at a dose of 10 µg mL − 1 (PTX) were added and incubated for 1 h. After washing and xing, cell images were obtained with an inverted uorescent microscope to observe the cellular uptake.

ROS detection assay
The ROS generation capacities of Free TPE-OH, FT/PTX and CT/PTX were evaluated by DCFDA, which is non-uorescent however transformed into DCF with green uorescence in the presence of ROS [39,40].
The A549 cells were incubated in a 6-well plate and treated with FT/PTX and CT/PTX (Fe 3+ concentration: 8 µg/mL) for 5 h. After incubation, an appropriate amount of DCFDA working solution was used to detect the ROS generation.

In vivo distribution and imaging
0.2 mL of Free DiR, FT/DiR and CT/DiR were delivered into nude mice bearing A549 tumors by caudal vein injection when the tumor had grown to an appropriate size. At different time points (2, 4, 8, 12, 24 h), and in vivo uorescence imaging system was used to monitor the distribution of uorescence in the body of the tumor-bearing nude mice.

In vivo antitumor e ciency and histological analysis
The nude mice bearing A549 tumors were randomly divided into four groups, including saline, Free PTX, FT/PTX, and CT/PTX. The A549 tumor-bearing nude mice were intravenously injected with different PTX preparations (PTX dosage: 10 mg/kg) every three days. The tumor volume and bodyweight of the nude mice were measured and recorded before each administration. After the nude mice were humanely sacri ced, major organs and tumors were collected and preserved in 4% paraformaldehyde solution quickly. Hematoxylin and eosin (H&E) staining assay was conducted to study the anti-tumor effect of different PTX preparations. The expression of bcl-2 and MMP-9 in tumor tissues was investigated by immunohistochemistry assay to explore the mechanism of tumor cell apoptosis.

Characterization of CFTT and FTVE
The 1 H-NMR spectrum of CFTT was shown in Fig. 2A. In the 1 H-NMR spectrum of FUC, the signal peak at 1.23 ppm is the -CH 3 signal peak of FUC. In the FTT spectrum, the peaks at a (δ6.70 ppm) and b (δ6.45 ppm) were the characteristic peaks of the proton from the TPE-O benzene ring; the peak at c (δ2.74 ppm) corresponded to the -CH 2 -of TK, and the typical signal of FUC was observed at 1.2 ppm. The above results indicated that TPE-OH was successfully connected to the FUC chain through the TK bond. In the 1 H-NMR spectrum of CFTT, in addition to the FTT-related characteristic peaks, new characteristic peaks appeared in the range of 6.1 6.3 ppm were the signals of CA, con rmed that CFTT was successfully obtained.
The 1 H-NMR spectrum of FTVE was shown in Fig. 2B. FTVE presented chemical shifts located at approximately 8.3 ppm (a) and 2.7 ppm (b), which were typical signals of D-α-tocopherol (the 'H' of benzene ring) and TK (-CH 2 -), respectively. The above results all proved that FTVE was successfully synthesized.

Characterization of CT/PTX nanoparticles
The particle hydrodynamic diameter distribution, Zeta potential and the TEM image of CT/PTX were shown in Fig. 3. The particle size of CT/PTX nanoparticles was 150.3 ± 12.0 nm, and the polydispersity index (PDI) was 0.098 ± 0.029, indicating the excellent uniformity of the nanoparticles. TEM images (Fig.  3B) showed that the nanoparticles were spherical. The DL% of PTX in the nanoparticles was 5.80 ± 0.91%.

In vitro ROS-responsive assay
The in vitro PTX release characteristics of CT/PTX nanomicelles at different H 2 O 2 concentrations were shown in Fig. 4A. When the concentration of H 2 O 2 was 0 mM, almost no ROS was produced. The coreshell structure of CT/PTX is relatively stable, and the PTX release is slow, which is bene cial to avoid the release of PTX in normal tissues and reduce the side effects. It could be observed that with the increase of H 2 O 2 concentration in the release medium, the cumulative release of PTX also increased gradually.
These results indicated that the PTX release of CT/PTX nanomicelles was concentration (ROS) dependent.
As shown in Fig. 4B, when CT/PTX nanoparticles and H 2 O 2 were incubated with MB for 3h, the absorbance of MB decreased signi cantly, indicating the production of •OH indirectly. However, no signi cant decrease in absorbance of MB was found when MB was treated with H 2 O 2 alone. The results showed that Fe 3+ in CT/PTX nanoparticles could be used as Fenton reaction catalyst-like to produce ROS.

In vitro cytotoxicity assays
The toxicity of Free PTX, FT/PTX and CT/PTX to A549 cells was investigated (Fig. 5A,B). It could be observed that the activity of A549 cells changed with the increase of PTX concentration in different preparations. Compared with the FT/PTX group, the CT/PTX group showed stronger cell inhibition after 48 h incubation, indicating that CT/PTX produced cytotoxic ROS through the CDT effect. The free PTX group showed strong cytotoxicity, which may be attributed to the inhibitory effect of organic solvents added in the preparation of Free PTX.
The toxicity of CFTT/FTVE material to A549 cells was shown in Fig. 5C. When the treatment time was 24 h, the inhibitory effect of CFTT/FTVE material on A549 cells was not obvious. After 48 h treatment, with the increase of the concentration of CFTT/FTVE material, the cell survival rate decreased slightly and showed low toxicity, which may be attributed to the potential anti-tumor effect of fucoidan.

Cellular uptake and distribution
The cellular uptake of CT/PTX in the A549 cells and B16F10 cells were observed with an inverted uorescence microscope. According to Fig. 6, after incubation with the cells for 1 h, the blue uorescence of the CT/PTX appeared in the cytoplasm, suggesting that the nanoparticles could be rapidly endocytosed.

ROS detection assay
The stronger the green uorescence intensity in A549 cells treated with DCFDA, the more ROS were generated in the cells. According to Fig. 7, compared with the control and FT/PTX group, the CT/PTX group had the strongest green uorescence intensity, indicating that CT/PTX nanoparticles produced a large amount of intracellular ROS under the effect of CDT.

In vivo distribution and imaging
In vivo real-time uorescence imaging of Free DiR, FT/DiR, and CT/DiR was shown in Fig. 8A. In the Free DiR group, DiR was mainly distributed in the liver, and there was almost no DiR accumulation in the tumor site. Compared with the Free DiR group, the DiR uorescence intensity of FT/DiR and CT/DiR group increased gradually at the tumor site over time, indicating accumulation of nanoparticles at the tumor site.
Fluorescent imaging of isolated tissues (Fig. 8B) showed that DiR uorescence was observed in the FT/DiR and CT/DiR groups at the tumor site, indicating that nanoparticles could accumulate at the tumor site effectively through the EPR effect, which was consistent with the results of in vivo imaging in nude mice.

In vivo safety evaluation
It was worth noting that there was no signi cant difference in the change of body weight (Fig. 9A) and no obvious tissue and organ damage (Fig. 9B) of nude mice within a short period of administration, indicating that the designed multi-functional CT/PTX nanoparticles based on marine polysaccharides had satisfactory safety.

In vivo antitumor e ciency and histological analysis
As shown in Fig. 10A, the tumor inhibition effect of the Free PTX group was unsatisfactory. Different from the tumor site accumulation characteristics and blood circulation stability of nanoparticles, Free PTX rarely accumulated at the tumor site, leading to poor therapeutic effect. Results showed that the antitumor effect of CT/PTX was more obvious than that of FT/PTX, which indicated that treatment with PTX and CDT was effective.
The results of the H&E staining assay of tumors was shown in Fig. 10B. Tumor cells in the saline group were closely arranged and normal in shape. The morphology of tumor cells in the PTX group was not signi cantly changed, but the density of tumor cells was slightly decreased compared with the saline group. In the CT/PTX group, loose cells, abnormal cell morphology, and damaged nuclei were observed, indicating its surprising anti-tumor effect in vivo.
The high expression of bcl-2 can slow down the apoptosis of tumor cells and promote the development of drug resistance. In immunohistochemistry, when bcl-2 was positively expressed in tumor cells, brownish yellow granules appeared in the cytoplasm. As observed in Fig. 10C, the obvious brown color appeared in the tumor cytoplasm of the saline and Free PTX group, suggesting the strong expression of bcl-2. The brown-yellow color of tumor cytoplasm was the weakest in the CT/PTX group, indicating that the expression of bcl-2 in tumor cells was rapidly down-regulated after administration.
MMP-9 promotes the growth and metastasis of tumors. When MMP-9 is positively expressed in tumor cells, the cytoplasm and membrane of tumor cells appear brownish yellow granules. In Fig. 10D, compared with the saline and Free PTX groups, the tumor cells in the CT/PTX group showed a weaker brown-yellow color, which indirectly re ected less MMP-9 positive expression, indicating that the CT/PTX group effectively inhibited the invasion and metastasis of the tumor.

Conclusion
We designed and constructed a type of novel ROS-responsive nanomedicine based on marine fucoidan by combining PTX chemotherapy and CDT. The formed CT/PTX nanoparticles have the appropriate particle size, spherical structure, surface charge and drug loading capacity. The ROSsensitivity of CT/PTX was demonstrated by an in vitro release assay. In vitro cell uptake experiments showed that the CT/PTX could be rapidly endocytosed. MTT assays showed that CT/PTX nanoparticles had an obvious inhibitory effect on the tumor cell. ROS detection experiments indicated that CT/PTX nanoparticles could form ROS waterfall after entering into tumor cells. In vivo studies have shown that CT/PTX nanoparticles exhibited reduced side effects, enhanced tumor accumulation and improved anticancer activity. Overall, this study provides a promising strategy for developing marine polysaccharide-based nanomedicines with satisfactory anticancer effects and security.

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