INTRODUCTION:

The 2022 Global Cancer Statistics Report shows that breast cancer is the leading cause of cancer death among women,¹ followed by cervical cancer which ranks fourth.²

While treatments for these cancers have been developed from surgery to chemotherapy, these therapeutic approaches are usually associated with side effects³, and off-target effects on normal tissues.^4,5 This calls for the need to develop new targeted therapies that ensure maximum efficacy with minimum systemic toxicity. Fucoidan is an anticancer potential bioactive sulphated polysaccharide. It exerts its antitumor actions by means of induction apoptosis, disruption of cell cycle, and activation of natural killer cells.⁶ It offers minimal drug resistance, almost negligible side effects, with high biological activity, thereby acting as an attractive lead for the treatments of cancer.⁷However, the therapeutic application of fucoidan is often limited by its poor bioavailability and inability to selectively target tumor cells.

To overcome these challenges, nanoparticle-based drug delivery system (NDDS) have come into prominence due to their capability for tumor accumulation via enhanced permeability and retention (EPR) effects.^8,9 Particularly, the use of folic acid receptors (FRs), which are overexpressed in many cancer cells, provides a mechanism for active targeting.¹⁰ The drug delivery technique has great potential for directing cancer therapy by combining specific ligands and encapsulated nanoparticles.¹¹ Folic acid, being small in size and non-immunogenic with high binding affinity to FRs, has widely been used to functionalize nanoparticles for cancer therapy.Its stability over a wide range of pH and temperature further extends its usability in targeted drug delivery.^12,¹³

Although significant progress has been made in NDDS using folate receptors, there remains a critical gap in the effective integration of biologically active polysaccharides like fucoidan within such systems. Prior research has largely focused on synthetic polymers or single-component drug systems, leaving a paucity of studies exploring multi-component systems that combine fucoidan’s anticancer activity with FR-mediated targeting.^14,15,15This innovative idea has been applied to creating polysaccharide drug conjugates for the drug delivery system.¹⁶^,17

This study has hypothesized that encapsulation of fucoidan, derived from Sargassum plagiophyllum, with a k-carrageenan matrix functionalized with folate (carrageenan folate), would enhance its targeted delivery to cancer cells. The developed system is likely to increase the accumulation of fucoidan in tumor tissues due to FR-mediated active targeting and EPR-driven passive targeting, reducing off-target effects. This study will develop a completely new folate-functionalized nanocapsule platform that meets the unmet medical needs of effective and selective polysaccharide delivery with high bioactivity. The expected outcome can thus lay a foundation for extended translational research in cancer treatment by using polysaccharide-based therapeutics.

MATERIALS AND METHODS:

Materials:

S. plagiophyllum was obtained from Yogyakarta, Indonesia. All chemicals, including ethanol, HCl, BaCl₂, Na₂SO₄, fucose, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCl), DMSO, folic acid, phosphate buffers pH 2, 7 and 8.5, 4-dimethylamino pyridine (DMAP), k-carrageenan, doxorubicin, microtetrazolium (MTT), and phosphate buffer saline (PBS) were pure grade provided by Sigma Aldrich and Merck.

Fucoidan extraction and isolation:

First, 300mL of ethanol was used to extract 50mg of S. plagiophyllum for 24hours at room temperature and an additional hour at 70°C. The prospective residue was dried overnight once the extraction process was complete and collected by centrifugation. Ethanol extraction and incubation conditions were selected based on the maximization of recovery of bioactive fucoidan with regard to its chemical integrity. The residue was extracted in the microwave for 5 minutes at 750 W and exposed to radiation after being suspended in 300mL of a 0.1M HCl solution. The suspension underwent a second centrifugation for 15min at 3,000rpm. It is essential to understand that a 1:1 volume ratio of the filtrate to absolute ethanol was used.¹⁸ Microwave extraction at optimized power (750 W) is effective in disrupting algal matrices without degradation of the polysaccharides. Fucoidan extract was purified using a DEAE Sephadex column with a gradient of 0.5M – 2.5 M NaCl as an eluent.¹⁹ The carbohydrate and sulfate contents were determined using the phenol-sulfuric acid, and BaCl₂-gelatin methods, respectively, with fucose and Na₂SO₄ serving as reference. The fucoidan was characterized using an FTIR (Bruker Tensor II, resolution 4 cm^-1, wavenumber range 400-4000 cm^-1) and HPLC (Agilent 1260, Zorbax SB-Aq column, 30°C, mobile phase: acetonitrile:water, 85:15, flow rate 1 mL/min).

Carrageenan folate (Cf) synthesis:

Carrageenan folate was produced through the reaction of folic acid and k-carrageenan. Folic acid and EDCl were linked at the beginning of the reaction, and DMAP and k-carrageenan were addressed in the second stage. The reactions involved mixtures from both the first and second stages.²⁰ An FTIR and UV-Vis spectrometers were used to characterize the products.

Synthesis of F-CNPs and F-CfNPs:

The fucoidan extract was dissolved in DMSO, and a 1:2 k-carrageenan mixture was added. DMSO was chosen for the ability to solubilize both k-carrageenan and folic acid. The combination was then treated with ultrasonic energy to produce F-CNPs. F-CfNPs were made using the same procedure with carrageenan folate in place of k-carrageenan. Ultrasonication has ensured nanoparticle formation, disruption of aggregates, and improved interaction of polymers.

Characterization and release of F-CNPs and F-CfNPs:

The characterization of F-CNPs and F-CfNPs using FTIR (functional groups) and Dynamic Light Scattering (DLS) Zetasizer Nano ZS90 (Malvern Instruments) was performed to identify their polydispersity index (PDI), particle size, and zeta potential (z). Transmission electron microscopy (TEM) JEOL JEM-2100 describes their morphology.²¹ Release studies of F-CNPs and F-CfNPs were performed through a dialysis method at pH 2, 7, and 8.5 using a semipermeable cellulose membrane and PBS as an outer membrane solution. Through periodic sampling of one ml aliquots at a time, the release was monitored for 24hours. The amount of the active ingredient in the released fucoidan extract was calculated based on its absorbance.

Anticancer evaluation:

HeLa and MCF.7 cancer cells were grown in 96-well plates to an 80% confluent state before being incubated for 24hours at 37°C with a flow of 5% CO₂. The cell media were then taken out. Samples of F-CNPs and F-CfNPs in a concentration range were put into each well. 10mL of MTT was then added to the cells after an additional 24h of incubation. MTT cells were added and incubated for 4 hours before receiving 100mL of 10% SDS in 0.01 N HCl.²² The maximum setting of 595nm on an ELISA reader (Thermo Fisher Multiskan Sky) was used to compute absorbance. Doxorubicin was used as a reference.

RESULT:

Yield and characterization of fucoidan:

The extraction and purification of fucoidan from S. plagiophyllum yielded 13.40%, with a fucose content of 84.08mg 100g^-1and sulfate contents of 10.62%. These values indicate a high-quality fucoidan extract suitable for biofunctional applications. The FTIR spectra (Figure 1) confirmed the functionsl groups characteristic of fucoidan, including O-H stretching (3250 cm^-1), S=O stretching (1203-1240 cm^-1), and C-O-S bending (876-927 cm^-1) (Table 2). Monosaccharides analysis by HPLC (Table 1) revealed the presence of fucose, glucose, mannose, xylose and galactose, glucuronic and galacturonic acid, consistent with typical fucoidan compositions.

Figure 1. FTIR Spectra of fucoidan

Table 1. Retention time of monosaccharides of fucoidan from Sargassum plagiophyllum

Retention time (min) of samples	Retention time (min) of standards	Monosaccharides
7.95	8.31	Galacturonic acid and glucuronic acid
12.59	12.50	Glucose
13.86	14.08	Mannose, xylose, galactose
15.71	15.65	Fucose

Table 2. List of fucoidan-specific infrared (IR) vibrational modes

Frequency	Functional groups	Bond
3250	O-H	Stretching
2934	>CH₂	C-H Stretching
1632	C=C	C-H Stretching
1203-1240	S=O	Stretching
1019-1076	C-O-C	-
876-927	C-O-S^-	Bending

Characterization of nanoencapsulation fucoidan:

UV-Vis (Figure 2) confirmed the conjugation of folic acid, as evidenced by the characteristic peaks of folic acid at 280nm and 360nm, hence confirming the successful synthesis of carrageenan folate. Carbonyl ester peaks at 1632 cm^-1were identified in FTIR spectra of k-carrageenan folate, further supporting esterification (Figure 3). The typical peak of folate acid is associated with the presence of carbonyl bonds in the molecular structure of folate acid. k-carrageenan exhibit prominent peaks at 1161 cm^-1 due to the S=O sulfate esters, 1105-1053 cm^-1 due to C-O-C bonds and 963 cm^-1 due to CH₂ stretching for carrageenan.

Figure 2. UV-Vis spectrum of k-carrageenan, carrageenan folate, and folic acid

Figure 3. FTIR Spectra of k-carrageenan-folate

The physicochemical properties:

The encapsulated nanocarriers, F-CNPs and F-CfNPs, exhibited favorable physicochemical characteristics (Table 3): Particle Size: The particle size was significantly reduced to 84, 2±12.1nm for F-CNPs and 93.2±10.7nm for F-CfNPs, compared to unencapsulated fucoidan at 221.4±12.4nm.

Polydispersity index (PDI): a lower PDI was observed for F-CNPs, 0.143 compared with F-CfNPs, 0.369, indicating a more homogeneous nanoparticle distribution.

Zeta potential: Both nanocarriers presented negative zeta potentials (F-CNPs: 35.4±5.4mV; F-CfNPs: 38,1±4.3 mV), which indicated good colloidal stability.

Table 3. Physicochemical properties of F-CNPs and F-CfNPs

Type	PDI	Size (nm)	Zeta potential (mV)
k-carrageenan	0.361	267.5 ± 13.8	-10.1 ± 9.0
Cf	0.417	279.6 ± 17.4	-18.0 ± 6.3
Fucoidan	0.283	221.4 ± 12.4	-13,3 ± 8.7
F-CNPs	0.143	84.2 ± 12.1	-35.4 ± 5.4
F-CfNPs	0.369	93.2 ± 10.7	-38.1 ± 4.3

In Vivo Stability and Therapeutic Efficacy: Small size and uniform distribution will facilitate deep penetration into the tumor tissues with an enhanced retention effect for both F-CNPs and F-CfNPs. A highly negative zeta potential maintains the colloidal stability in biological systems with a reduced degree of aggregation and enhances circulation time. Such physicochemical properties are likely to enhance in vivo targeting and efficacy.

Figure 4. TEM image of (A) F-CNPs and (B) F-CfNPs

TEM imaging (Figure 4) showed near-spherical morphologies for both F-CNPs and F-CfNPs, thus justifying their potential for cellular uptake.

Release study:

The LE of the fucoidan in F-CNPs and F-CfNPs was 92.34% and 95.41%, respectively. The LA value for F-CNPs and F-CfNPs was 46.17% and 47.70%, respectively, showing high encapsulation efficiency. The in vitro release profile (Figure 5) indicated sustained released over 7 hours at optimum release at pH 8.5, mimicking the tumor microenvironment. F-CfNPs exhibited superior release kinetics to F-CNPs at all pH values, which might be due to increased solubility of carrageenan folate.

Figure 5. Fucoidan bioactive component release from F-CNPs, and F-CfNPs

Anticancer activity:

MTT assay revealed an improved anticancer efficacy of the nanocapsulated formulations against HeLa and MCF.7 cell as shown in Figure 6.

IC₅₀Values:

HeLa Cells:

· F-CNPs: 33.13±4.53mg ml^-1

· F-CfNPs: 21.14±3.59mg ml^-¹(p<0.05 compared to F-CNPs)

· Doxorubicin: 3.16±3.56mg ml^-1

MCF.7 Cells:

· F-CNPs: 30.56±3.86mg ml^-1

· F-CfNPs: 24.92±3.83mg ml^-1 (p < 0.05 compared to F-CNPs)

· Doxorubicin: 15.79±2.84mg ml^-1

Figure 6. Anticancer activity against (a) HeLa cells and (b) MCF.7 cells

DISCUSSION:

The extracted fucoidan yield was greater than that produced by the microwave technique from Ascophylum nodosum,²³ Fucus vesiculosus,²⁴ and Spiraea thurnbergii²⁵ but lower than that derived from A nodosum.²⁶ The yield of the fucoidan extract can be decreased by high temperature, prolonged extraction times, high microwave power,²⁷ and the different solubilities of the polysaccharides in the solvents.²⁸ Monosaccharide content of fucoidan (S. plagiophyllum) which was extracted with 0.1 M HCl using microwave extraction, namely fucose, mannose, xylose, galactose, glucuronic acid and galacturonic acid (Table 1). The retention times of mannose, xylose and galactose appear in one peak with the same retention time, this is due to the limitations of the column which can not separate monosaccharides that have close retention times. The monosaccharide content of the results of this study is almost the same as the monosaccharide content of S. plagiophyllum from India which was extracted with HCl for 24 hours, namely fucose, galactose, mannose, and xylose²⁹. Fucoidan exhibited eight absorbance peaks around 3248 cm¹- 882 cm¹, according to the FTIR data (Fig. 1). Similar to the fucoidan commercially produced by U. pinnatifida, Sargassum turbinarioides and Sargassum ilicifolium displayed eight peaks in their FTIR data (3416 - 833 cm¹).³⁰ The FTIR data indicate the existence of sulfate groups with wavelength values between 1203-1240 cm¹, corresponding to the sulfate group’s -S=O stretching vibration, was proposed to provide a reliable gauge of the sulfate esters quantities in polysaccharides. The signal at 882 cm^-1identifies the sulfate fucose.

Carrageenan folate was produced from the esterification reaction between k-carrageenan and folic acid catalyzed by EDCl and DMAP. The reaction between folic acid and EDCl activates the carboxyl group, releasing protons to form folic anion and protonated EDCl. To create O-acylisourea compounds, the folic anion will attack the carbodiimide EDCl. These compounds will then react with the nucleophile DMAP to produce ester intermediates that are very reactive to the -OH group of k-carrageenan, ultimately forming carrageenan folate.³¹

Carrageenan exhibits a final UV absorption at 230 nm because it lacks an auxochrome or chromophore group. The UV-Vis spectrometer detected two bands of folic acid absorption at 300 and 350nm; another report found the bands at 304 nm and 390nm.³² Meanwhile the n → π* transition occurred at 350nm as opposed to the π → π* transition at 300nm. The compounds created when k-carrageenan was esterified with folic acid had an absorption band at 230, 300, and 360 nm, indicating the formation of carrageenan folate. Folic acid and k-carrageenan esterification products displayed an absorption band at 300 nm due to the folic acid in π → π* transition. This observation indicates that carrageenan folate was produced (Fig 2). From the results of UV-Vis and FTIR spectra (Fig 3), it can be concluded that k-carrageenan folate compounds have been formed.

Encapsulation of fucoidan-carrageenan and fucoidan-carrageenan folate was successful. Prior research suggested that fucoidan, in combination with other natural polysaccharides, like carrageenan, may interact synergistically through van der Waals forces, electrostatic interactions, and hydrogen bonding as a result of the existence of charged hydroxyl and sulfate groups.^33,34 The physiochemical characteristics of F-CNPs and F-CfNPs were compared to k-carrageenan, Cf, and fucoidan to differentiate those properties before and after nanoencapsulation. F-CNPs and F-CfNPs achieved useful results with particles smaller than 100 nm (Table 3), this is following the expected size in the field of nanomedicine³⁵. The F-CNPs in this study were smaller than the chitosan-fucoidan encapsulation from a previous study (234.73nm).³⁶

The release at pH 2 was the lowest for both nanocapsules. The poor release at acidic pH may be due to particle aggregation caused by reduced electrostatic repellency. In F-CNPs and F-CfNPs, the k-carrageenan and carrageenan folate sites are electronegative. Alkaline circumstances cause nanocapsule repulsion between particles, causing the pores of the nanocapsule particles to widen and enabling them to release the biologically active components. In contrast, when the environment is acidic, particle interactions with hydrogen pull the trigger, causing the tiny pores to expand and particles to open.²⁰

According to earlier research, fucoidan has anticancer properties in various cancer cells in vivo and vitro^6,37 F-CNPs, and F-CfNPs can be distinguished by the presence or absence of folic acid on cancer cells as a preferred target of FRs. Breast and cervical cancer cells have alpha receptors for folate (FRa).³⁸ The ligands of folate-drug conjugates are connected to the drugs by endocytosis when they target folate-based receptors and infiltrate the cell cytoplasm. The complex is internalized, allowing the nanoparticle’s cargo to be efficiently delivered into the cancer cells. This specificity reduces off-target effects and enhances the therapeutic efficacy.³⁹

Clinically, F-CfNPs are a targeted therapy with reduced systemic toxicity, suitable for combination treatments and applicable to other FR-positive cancers, such as ovarian and lung cancers. The synergy of fucoidan and carrageenan improves the stability of nanoparticles, the efficiency of encapsulation, and pH-responsive drug release for optimized activities in tumor microenvironments. However, the enhanced efficacy of of F-CfNPs is still much lower compared to that induced by doxorubicin. Future studies should therefore be directed toward further optimizations in drug loading, performance, and safety assessment using in vivo models, with application in broader cancer models. This study hence uncovers the potential of F-CfNPs toward precise and effective cancer therapy.

CONFLICT OF INTEREST:

There are no conflicts of interest among the writers in this inquiry.

CONCLUSION:

In summary, Fucoidan extracted from S. plagiophyllum demonstrated strong in-vitro anticancer activity against in-vitro MCF.7 and HeLa cell lines. κ- carrageenan and Cf were used as carriers in DDS to increase the fucoidan extract’s bioactivity. HeLa and MCF.7 cell lines exhibited more inhibitory activity when exposed to F-CfNPs than to F-CNPs. These encouraging findings suggest that folate ions present in fucoidan nanocapsules can increase the inhibitory action in vitro of cancer cells by concentrating on FRs

ACKNOWLEDGEMENT:

The authors wish to thank The Ministry of Research and Technology, Republic of Indonesia, for supporting this research under the Flagship University Research: Penelitian Pasca Sarjana - Penelitian Disertasi FY 2022 Research Programme (Postgraduate Doctoral Dissertation Research) with contract number: 782/UN3.15/PT/2022.

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Received on 22.05.2024 Revised on 19.10.2024

Accepted on 01.01.2025 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2501-2507.

DOI: 10.52711/0974-360X.2025.00357

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