Potential effect of nanoformulated iota carrageenan in Aβ1-42 disaggregation: an in vitro, in vivo and in silico study

TL;DR

This study explores how nanoformulated iota carrageenan can break down harmful amyloid plaques linked to Alzheimer's disease, showing promise as a non-toxic treatment.

Contribution

The study introduces nanoformulated iota carrageenan as a novel, biocompatible agent for degrading β-amyloid fibrils in Alzheimer’s disease.

Findings

Nanoformulated iota carrageenan reduced β-amyloid peptide fibrils by 45.5% in fluorescence assays.

The treatment showed neuroprotective effects by promoting neurite growth and reducing neurotoxicity.

In vivo chick embryo studies showed enhanced blood vessel formation after treatment.

Abstract

Alzheimer's disease is the primary contributor to neurodegenerative conditions. These pathologies are identified by the deposition of β-amyloid peptide within brain regions. It develops insoluble fibrils known as senile plaques. These plaques are associated with synaptic dysfunction, neuroinflammation, and progressive cognitive decline. Hence, the degradation and elimination of β-amyloid peptide fibrils from the body are viable therapeutic approaches for managing Alzheimer’s disease.

In the current study, liposomal nanoformulated iota carrageenan was synthesized and characterized using different photophysical tools. The nanoformulated iota carrageenan effectively degraded β-amyloid peptide 1-42, with 45.5 % reduction confirmed by Thioflavin T fluorescence assay. This activity was further supported by turbidity and dynamic light scattering analysis.

The biocompatibility of nanoformulated iota carrageenan and its degraded β-amyloid peptide was determined using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), live/dead cell assay on PC12 cells. Structural disintegration of the β-amyloid peptide fibrils was validated through atomic force microscopy, revealing a significant reduction in fibrillar morphology. In silico studies also evidenced the interaction between the β-amyloid peptide and nanoformulated iota carrageenan. In addition, the neuroprotective potential of nanoformulated iota carrageenan, as evidenced by nanoformulated iota carrageenan-treated β-amyloid peptide, was supported by neurite outgrowth studies. These studies showed that differentiated PC12 cells exhibited larger neurite growth with extensive branching, indicating the reversal of β-amyloid peptide-induced neurotoxicity. CAM assay demonstrated enhanced blood vessel formation in chick embryos treated with nanoformulated iota carrageenan and its β-amyloid peptide-degraded group.

These findings suggest that nanoformulated iota carrageenan holds potential and has nontoxic therapeutic effects for Alzheimer’s disease. Additional in vivo validation is required in future investigations.

Figures

SEM images of (A) CG and (B) nCG; (C) Raman spectra of CG encapsulated in a liposome; (D) the cumulative drug release profile of nCG; (E) the stability of liposomal formulations was plotted by measuring dH at 0, 7, 14, 21 and 28 days; (F) encapsulation efficiency of nCG over 5 weeks

Thioflavin T fluorescence of untreated Aβ1-42 (red) and (black) represents Aβ1-42 treated with CG, Aβ1-42 treated with nCG (green) at different time intervals: (A) day 5, (B) day 6, (C) day 7; particle size distribution of Aβ1-42, Aβ1-42+CG and Aβ1-42+nCG measured by DLS, where, untreated Aβ1-42 is represented as (red) and Aβ1-42 treated by nCG (blue) at (D) day 5 (E) day 6 (F) day 7; (G) degradation at various time intervals; (H) turbidity of Aβ1-42, Aβ1-42+CG and Aβ1-42+nCG were analysed with time

AFM topography images of (A) Aβ1-42 and (B) Aβ1-42 showing reduced Aβ1-42 aggregates after nCG treatment. Cell viability of PC12 cells using the MTT assay: (C) Concentration-dependent effect of CG and nCG; (D) toxicity of Aβ1-42 and ameliorative effect of CG and nCG against Aβ1-42 mediated toxicity (untreated cells served as the control. *p <0.05, p <0.02, *p <0.01, p <0.001 denotes significant differences compared with control)

Fluorescence microscopic image of PC12 cells treated with (A) 2 μg mL-1 CG; (B) 20 μg mL-1 CG; (C) 2 μg mL-1 nCG; (D) 20 μg mL-1 nCG; (E) Aβ1-42; (F) Aβ1-42+CG (5 μg mL-1); (G) Aβ1-42+nCG (5 μg mL-1); (H) untreated PC12 cells as control. The neurite outgrowth of PC12 cell images (I), the sample treated cells on day 3 (J). crystal violet-stained differentiated cells on day 10

Visualization and graphical representation of angiogenesis activity: (A) CAM layer treated with heparin as positive control; (B) untreated control; (C) CAM layer treated with CG (10 μg mL-1); (D) CAM layer treated with CG (100 μg mL-1); (E) CAM layer treated with nCG (10 μg mL-1); (F) CAM layer treated with nCG (100 μg mL-1); (G) total vessel area; (H) total length of the vessels; (I) blood vessel mean thickness; (J) total number of branching points

Visualization and graphical representation of angiogenesis activity: (A) CAM layer treated with a low dose of Aβ1-42; (B) CAM layer treated with high dose of Aβ1-42; (C) CAM layer treated with low dose of Aβ1-42+CG; (D) CAM layer treated with high dose of Aβ1-42+CG; (E) CAM layer exposed to low dose of Aβ1-42+nCG; (F) CAM layer treated with high dose of Aβ1-42+nCG; (G) total vessel area; (H) total vessel length; (I) vessel mean thickness; (J) number of branching points

Molecular interactions of CG with target proteins Aβ1-42 (PDB ID = 2BEG): (A) 2D image; (B) 3D image of CG binding with target Aβ1-42; (C) image of the binding energy results obtained from docking analysis

Tables

Table 1.. Absorption spectra, hydrodynamic diameter and zeta potential of prepared CG and nCG

Samples	λmax / nm	Absorbance	Hydrodynamic diameter, nm	Zeta potential, mV
CG	270	0.194	513	-12.5
nCG	270	0.244	339	-19.6

Full text

Introduction

Amyloidosis is distinguished by the uncontrollable deposition of misfolded protein fibrils in the extracellular matrix region. Alzheimer's disease (AD), Parkinson's disease (PD), prions, and type II diabetes are some of the neurodegenerative diseases caused by amyloidosis [1-3]. Misfolded proteins and fibril deposition are among the most challenging issues facing both human health and the pharmaceutical industry [4]. In AD, progressive neuronal degeneration and memory loss result from the deposition of tau tangles and β-amyloid plaques in brain regions [5]. The amyloid β peptides (Aβ) are formed by the repeated breakdown of amyloid precursor protein (APP) involving γ- and β-secretase enzymes [6]. These Aβ, normally composed of 39-40 amino acids, exist in coiled isoforms under physiological conditions. Following certain structural changes, the predominant isoforms are Aβ1-40 and Aβ1-42 [7,8]. Aβ1-40 is normally considered less toxic, whereas Aβ1-42 exhibits greater aggregation, which contributes to AD pathology [6,9,10]. Excessive accumulation of toxic amyloid aggregates forms senile neuronal plaques, and their highly cytotoxic stable fibrillar nature makes clearance difficult [11,12].

Although numerous drugs have been investigated to suppress amyloid formation, most have shown efficacy in the early stages of aggregation but limited activity against mature fibrils [13]. Additionally, large therapeutic biomolecules and hydrophilic drugs frequently face challenges in targeted delivery due to their low absorption and difficulty crossing the blood-brain barrier (BBB) [14,15]. Nearly 90 % of research found that the BBB remains a critical task for effective drug delivery in neurodegenerative diseases [16,17]. The BBB is a selective biological barrier composed of endothelial cells interconnected by tight junctions that regulate molecular transport into the brain and protect against harmful substances [18]. This selective permeability poses a major challenge for therapeutic delivery, particularly for small and large molecules that are unable to cross the BBB at effective doses [19]. Therefore, establishing biocompatible delivery strategies capable of crossing the BBB and specifically targeting brain cells is essential for developing treatments for AD and PD [20-22].

Marine-derived sulphated polysaccharide offers a promising therapeutic avenue due to their natural abundance and biocompatibility [23,24]. Among others, these algae-derived compounds, such as carrageenan, have attracted considerable attention for their diverse biological properties, including antioxidant, antimicrobial, anticancer, and anticoagulant activities [25,26]. Carrageenans are naturally occurring linear sulphated polysaccharides isolated from red algae, consisting of repeating D-galactose and D-anhydrogalactose units [27]. Iota carrageenan (CG) is distinguished by its anionic nature and its capacity to interact with protein aggregates via electrostatic interactions [28,29]. Due to CG’s high molecular size, limiting tissue penetration and BBB transport, nanoformulations can be an alternative to overcome these challenges [30]. Makshakova et al. [31] demonstrated that the sulphated polysaccharide k-carrageenan and sodium alginate degraded the hen egg white lysozyme amyloid fibrils and caused protein renaturation. Numerous investigations using neuronal cell models and animal systems have been conducted to suppress Aβ1-42 aggregation, providing valuable insights for the design of anti-amyloidogenic therapies [7,32]. This is the first study to investigate the anti-amyloidogenic activity of nanoformulated CG (nCG) against Aβ1-42.

An effective nanotechnology-based drug delivery system offers innovative approaches to nanosized bioactive carriers that are emerging in drug delivery [33,34]. These nanosystems offer numerous benefits, including improved drug delivery, controlled drug release, and increased therapeutic efficacy of encapsulated compounds [35]. Moreover, these nanoscale dimensions allow closer interaction with diseased structures, such as amyloid aggregates, and increase the potential for crossing the BBB [36]. Liposomes, polymeric nanoparticles, micelles, and niosomes are some nanocarriers that can protect therapeutic compounds and improve targeted delivery [37-39]. Encapsulating marine compounds in liposomes, particularly those with amyloid-degrading activity, has not only protected them from enzymatic degradation but also facilitated their passage through the BBB [40].

In this study, we encapsulated iota carrageenan within liposomes to enhance amyloid disaggregation. Here, we utilized liposomes to nanoformulate iota carrageenan and investigated the effect of nCG on amyloid degradation using spectroscopic and imaging techniques, including the Thioflavin T (ThT) fluorescence assay, turbidity measurements, and size measurements by dynamic light scattering (DLS) and atomic force microscopy (AFM). Cytotoxic effect of CG, nCG, Aβ1-42, and Aβ1-42 degraded by CG and nCG was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium (MTT), dual staining assay, and neurite outgrowth on PC12 cells. The in vitro results revealed that nCG can inhibit Aβ1-42 aggregation and reduce the toxic effects of Aβ1-42 aggregates on PC12 cells. In vivo biocompatibility was further evaluated through an angiogenesis study. In addition, the interaction between Aβ1-42 and CG was calculated using in silico approaches.

Experimental

Chemicals used for experiments

Iota-carrageenan, also known as Irish moss; bovine serum albumin (BSA); thioflavin T (a benzothiazole fluorescent dye); and phosphate-buffered saline (PBS) tablets were obtained from Himedia, India. 1,1,1,3,3,3-3-hexafluoro-2-propanol (HFIP) from Sigma. Human Aβ(1-42) lyophilized powder was purchased from Life Technologies. PC12 cell lines were purchased from the NCCS, India. Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS), RPMI-1640 medium, and Nerve growth factor (NGF) were procured from Gibco. An antibiotic-antimycotic solution, MTT bromide, acridine orange (AO), ethidium bromide (EtBr), and dialysis membrane were purchased from Himedia, India, and dimethyl sulfoxide (DMSO) was procured from SRL, India.

Preparation of carrageenan and liposomal nanoformulation

In this approach, the thin-film hydration method was used to formulate liposomal nanoformulations. The detailed protocol was mentioned in previous research by Udayakumar et al. [41]. For this formulation, 20 mg of iota carrageenan was dissolved in 10 mL of demineralized water and magnetically stirred at 60 °C for 30 minutes. After dissolving CG, the liposomal-encapsulated CG was prepared in a 1:1 ratio by sonicating the mixture with a bath sonicator at 40 kHz for 15 min at 25°C. The nanoformulated CG was kept under 4 °C for further analysis.

Characterization of iota carrageenan and nanoformulated iota carrageenan

The UV-Vis absorption spectrophotometer (Shimadzu UV-1900i) and Malvern Nano-ZS-90 zeta sizer were used for characterizations. The surface feature and topography of CG and nCG were investigated using a FEI Quanta FEG200 scanning electron microscope (SEM). For this, the prepared samples were coated on a grease-free glass coverslip. Subsequently, the thin-film-coated CG and nCG samples were dried and subjected to SEM analysis. Further, the functional bonds were studied using Raman spectroscopy (Metrohm).

Drug release profile of nanoformulated iota carrageenan

The release behaviour of nCG was determined by the dialysis bag diffusion method, and the dialysis membrane (2 kDa) was activated [42]. For this study, 3 mL of nCG was loaded into a dialysis membrane bag and submerged in 50 mL of PBS (pH 7.4) under stirring. The release was monitored spectrophotometrically at 270 nm to plot a graph of optical density (OD) to determine the release pattern of nCG from liposomal vesicles.

Stability and encapsulation efficacy of nanoformulated iota carrageenan

The stability of formulations was measured by storing them at room temperature (RT), 4 °C and -20 °C by evaluating the hydrodynamic diameter. The liposome-encapsulated CG was centrifuged at 10,000 rpm for 10 min and the amount of unencapsulated drug was determined from the supernatant by recording OD at 270 nm. To evaluate their long-term encapsulation efficacy, we have stored nCG at RT, 4 and -20 °C. The amount of encapsulation was monitored every seventh day throughout storage, and encapsulation efficiency, % of CG encapsulated in the liposomes, was evaluated using Equation (1) [39]:

$$[eqn]$$

where OD_t_ is the absorbance of total drug and OD_s_ is the absorbance of free drug.

Preparation of Aβ1-42 amyloid fibrils

For preparation of Aβ1-42 amyloid, 0.5 mg of Aβ1-42 was solubilized in 1 mL of 1,1,1,3,3,3-3-hexafluoro-2-propanol (HFIP) and vortexed. The solution was then aliquoted into sterile PCR tubes and dried in a laminar airflow cabinet for 4 h. After drying, the tubes were stored at -20 °C prior to analysis. Aβ1-42 aggregates were reconstituted by treating them with 20 μL DMSO with 180 μL PBS, which served as sample 1 (untreated group), sample 2 was Aβ1-42 with 20 μL DMSO with 170 μL PBS, 10 μL CG and sample 3 was Aβ1-42 with 20 μL DMSO with 170 μL PBS, 10 μL nCG (treated group). Following the method of Metkar et al. [43,44], fibrils were prepared by incubating the samples (25 μM) for 3 days at 37 °C.

Thioflavin T fluorescent assay

The fluorescent-based assay was used to study the disintegration of the Aβ1-42 aggregates. The fluorescence intensity of incubated Aβ1-42, Aβ1-42+CG, and Aβ1-42+nCG samples was analysed. For each measurement, 10 μL from each of the 3 samples was mixed with 3 mL of demineralized water in the corresponding tubes, and 10 μL of ThT (20 μM, pH 7.2) was added to the mixture. Subsequently, treated and untreated Aβ1-42 samples were evaluated for ThT and ThT+BSA fluorescence intensity. Measurements were performed over 3 days using a Jasco-FP8300 spectrofluorometer. The fluorescence intensity of ThT was measured by exciting at 440 nm, and its fluorescence emission spectra was measured between 460 and 600 nm with a slit width of 10 nm. The Aβ1-42 disintegration, %, was assessed using Equation (2):

$$[eqn]$$

The disintegration or degradation between Aβ1-42+CG (CG-treated Aβ1-42) and Aβ1-42+nCG (nCG-treated Aβ1-42) over different time intervals was analysed.

Degradation study using turbidity assay, size profiling, and surface morphology

The ability of CG and nCG to degrade Aβ1-42 fibrils in vitro was determined using a turbidity assay spectrophotometrically (600 nm). In this, 10 μL of treated and untreated samples was added to 3 mL of 10 mM PBS (pH 7.4). The change in aggregation behaviour of treated and untreated samples at various time intervals was determined using a turbidity assay. To measure the size of Aβ1-42 fibrils, in the presence and absence of CG, nCG was recorded at different time points using Zetasizer (DLS), for which 10 μL of incubated samples (Aβ1-42, Aβ1-42+CG, Aβ1-42+nCG) were diluted in 1 mL PBS and allowed to equilibrate for 1 min. The hydrodynamic particle size obtained from DLS measurements was then used to determine fibril size. The disaggregation of amyloid fibrils by nCG was visualized using a Nanosurf C3000 instrument. The Aβ1-42 was incubated with 10 μL of PBS for 24 h at 37 °C to allow degradation. For comparative analysis, Aβ1-42 was treated in the same manner as nCG.

3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium assay/morphological study

The toxicity of the prepared samples was determined using the undifferentiated neuronal cell line PC12 [45]. They were cultured in DMEM medium enriched with 10 % FBS and 1 % antibiotics at 37 °C in a 5 % CO_2_ atmosphere. For this cytotoxicity study, PC12 cells (10^4^ cells per well) were plated in 48-well culture plates and maintained for 24 h at 37 °C. The next day, 85 % of confluence cells were exposed to different concentrations of CG and nCG (2, 5, 10, 15, 20 μg mL^-1^) and Aβ1-42 (20 μg mL^-1^), Aβ1-42+CG, Aβ1-42+nCG (5 μg mL^-1^), respectively. At the end of the 48 h treatment period, 50 μL of MTT was added to each well and incubated for 4 h. The formazan crystals produced by metabolically active cells were solubilized in DMSO, and the absorbance was measured at 570 nm. Cell viability, % was quantified using the protocol established by Girigoswami et al using Equation (3) [45].

$$[eqn]$$

The morphology of the cultured cells was observed using an inverted phase-contrast microscope (Olympus CKX41) at 10× magnification.

Dual cell staining assay

The PC12 cells were cultured on sterile coverslips using DMEM with 1 % antibiotic and 10 % FBS was added. After 24 h, cell adherence to the bottom surface was confirmed using an inverted microscope, followed by treatment with different samples (CG, nCG, Aβ1-42, Aβ1-42+CG, Aβ1-42+nCG). After treatment, live/dead cells were assessed as described in a previous study [46]. The working dye was prepared by mixing acridine orange and ethidium bromide (100 μg mL^-1^), and cells were visualized using a BX51 Olympus fluorescence microscope.

Neurite outgrowth study

Fourth passage PC12 cells were incubated in a T25 flask with DMEM medium. For this differentiation experiment, cells were seeded onto a coverslip in complete RPMI-1640 containing 10 ng mL^-1^ NGF. Then the cells were exposed to CG, nCG, Aβ1-42, Aβ1-42+CG, and Aβ1-42+nCG samples. Neurite outgrowth was analysed by daily monitoring and by capturing morphological changes on days 3 and 10. Finally, Neurite development on day 10 was stained using crystal violet (fixation in 4 % paraformaldehyde (10 to 15 min) and later rinsed with PBS, and 0.1 % crystal violet) [47,48]. The dye-stained cells were observed under a microscope with 10X magnification.

Assessment of angiogenesis using chorioallantoic membrane assay

The 48 h post-fertilized chick embryos were purchased and sterilized with ethanol. Then the eggs were grouped into 5 groups, with 3 eggs per group, including positive and negative controls. A 1 cm square window was made on the top of the air sac to withdraw 3 mL of albumen. The openings on the eggshell membrane were closed and incubated at 60 % constant humidity for one day. The next day, the 72 h completed eggs were exposed to two varying doses of CG, nCG, Aβ1-42, Aβ1-42+CG, and Aβ1-42+nCG. For this treatment, the 0.5 cm diameter of the paper disk was placed on an air sack above the CAM layer, and then the samples were loaded. Again, the window was closed with surgical tape and incubated at 37 °C under 60 % humidity. After 96 h, the eggshells were opened slowly, and angiogenic activity was quantified for further analysis. The total number of vessel development, length, thickness, and branching were analysed using IKOSA software [49].

In silico study

The inhibitory activity of CG against Aβ1-42 was evaluated using Autodock software [41]. For this study, the three-dimensional structure model of Aβ1-42 was obtained from the Protein Data Bank (PDB) using the accession code 2BEG. The protein (Aβ1-42) and compound (CG) were generated using a standard protocol, which involved inserting polar hydrogens, assigning charges, and generating a grid. After performing the docking analysis, each conformation was examined in Discovery Studio to calculate its root mean square deviation (RMSD) and to investigate interactions within the target protein's active binding site.

Statistical analysis

Each experiment was conducted either in duplicate or triplicate. The values obtained are reported as mean ± SEM. One-way ANOVA was used to analyse group differences, and a p-value < 0.05 was considered statistically significant. For the MTT and CAM assays, comparisons were made using Student's t-test. Significance levels are represented as: * - p < 0.05, ** - p < 0.02, *** - p < 0.01 and **** - p < 0.001 in comparison with the untreated control, while α and β represent comparison between the samples.

Results and discussion

Physicochemical properties of the iota carrageenan and encapsulated nanoformulated iota carrageenan

The absorption maximum, along with changes in OD and particle size distribution of the CG and nCG, as analysed using spectrophotometry and DLS measurements, are depicted in Table 1. An absorption peak was observed at 270 nm, corresponding to the characteristic electronic transition of the sulphate ester groups present in CG, confirming the retention of its structural integrity after formulation. The OD increment (Table 1) may be attributed to the entrapment of CG in liposomes, which alters the surrounding microenvironment. The colloidal properties were analysed by DLS, which showed that CG exhibited a hydrodynamic diameter (dH) of 513 nm due to aggregation. The nCG was observed with dH = 339 nm, indicating improved solubility and uniform dispersion. The zeta potential measured for CG was -12.5 mV, and nCG yielded a value of -19.6 mV, indicating higher stability.

The surface structure of CG and nCG was investigated using SEM imaging (Figure 1), where nCG exhibited a quasi-spherical structure with a size of 314.9±4 nm, suggesting successful liposomal encapsulation compared to CG (as illustrated in Figure 1B). Furthermore, Raman spectra of nCG are depicted in Figure 1C.

SEM images of (A) CG and (B) nCG; (C) Raman spectra of CG encapsulated in a liposome; (D) the cumulative drug release profile of nCG; (E) the stability of liposomal formulations was plotted by measuring dH at 0, 7, 14, 21 and 28 days; (F) encapsulation efficiency of nCG over 5 weeks

It revealed distinct peaks at 1645 and 1345 cm^-1^, corresponding to the Amide I and Amide III bands. The band at around 1086 cm^-1^ usually represents nucleic acids. The presence of these characteristic peaks confirms the retention of biomolecular integrity after encapsulation.

Release profile, stability, and encapsulation efficiency of nanoformulated iota carrageenan

Drug release studies are crucial for ensuring the achievement of the intended treatment effect while maintaining safety and formulation stability [50]. The release profile from nCG was fast at the initial stage, peaked at 64 % at 3 h, and then became flat (Figure 1D) [51]. After 3 h, we observed a slow and sustained drug-release profile from nCG, indicating that liposomal encapsulation effectively controlled the diffusion of the loaded CG. Following this, the stability of the vesicle was monitored over 28 days. Samples stored at RT and -20 °C showed some variation in their size on the 7^th^ day. In contrast, the sample stored at 4 °C remained stable throughout the study period, with minimal changes, as shown in Figure 1E. The EE was found to be 73 %, suggesting that the formulation process effectively entrapped a substantial proportion of the compound. The drug leakage analysis over 28 days is depicted in Figure 1F. After 14 days, nearly 26 and 32 % of the compound started to leak from the formulated samples kept at ambient temperature and -20 °C, respectively. In contrast, the efficacy of the 4 °C stored sample was shown to be stable for 28 days. This storage condition preserved the vesicle structural integrity, showing negligible leakage over the same period.

Monitoring Aβ1-42 fibril growth and degradation by iota carrageenan and nanoformulated iota carrageenan

Marine-derived sulphated polysaccharides have shown promising potential for multiple diseases, including the disaggregation of amyloids. Our current findings suggest that the nanoformulated CG exhibits amyloid-clearing potential in both cell and CAM models. Supporting results for this hypothesis are presented in the following sections, with observations recorded at specific intervals: days 5 (120 h), 6 (144 h) and 7 (168 h).

Thioflavin T dye fluorescent assay

Apart from Congo red, ThT is extensively used in amyloid research as a fluorescent marker [52]. In this study, Aβ1-42 degradation by CG and nCG was monitored using the ThT assay. In this study, Bovine serum albumin (BSA) served as the negative control [7,53]. The emission intensity of treated samples (Aβ1-42+CG and Aβ1-42+nCG) was compared to that of untreated (Aβ1-42) and showed a decrease over different time intervals, which was depicted in Figure 2A-C. At day 5, the fluorescence intensity emitted by Aβ1-42 was greater than that of CG and nCG, which interacted with amyloid. CG and nCG-treated amyloid was degraded up to 23.5 to 21.8 %, respectively. The degradation percentages for CG and nCG towards Aβ1-42 samples at day 7 were 29 and 33 %, respectively. Aβ1-42 degradation percentage was observed after day 7, with Aβ1-42 increasing relative to the initial level. CG and nCG-treated Aβ1-42 fibrils were observed with lower fluorescence and were degraded to 40 % and 45.5 %, respectively, as shown in Figure 2G. Similarly, the antiamyloid effect of taurine was analysed using the ThT fluorescence assay [11,54]. Hence, the results proved that the formulated nCG can effectively degrade Aβ1-42 fibrils.

Thioflavin T fluorescence of untreated Aβ1-42 (red) and (black) represents Aβ1-42 treated with CG, Aβ1-42 treated with nCG (green) at different time intervals: (A) day 5, (B) day 6, (C) day 7; particle size distribution of Aβ1-42, Aβ1-42+CG and Aβ1-42+nCG measured by DLS, where, untreated Aβ1-42 is represented as (red) and Aβ1-42 treated by nCG (blue) at (D) day 5 (E) day 6 (F) day 7; (G) degradation at various time intervals; (H) turbidity of Aβ1-42, Aβ1-42+CG and Aβ1-42+nCG were analysed with time

Turbidity assay

Following ThT, the turbidity of Aβ1-42 was assessed. The untreated Aβ1-42 exhibited increased turbidity. Whereas CG and nCG treated with Aβ1-42 revealed a decreased OD at 600 nm. At day 7, Aβ1-42 treated with CG and nCG showed 92 and 99 % degradation, respectively (Figure 2H). Treatment with CG, and especially nCG, showed a clear reduction in absorbance, suggesting the compound's ability to degrade fibrils.

Size assessment by dynamic light scattering

The particle size distribution of Aβ1-42 and Aβ1-42 incubated with CG and nCG was measured using dynamic light scattering (Figure 2D-F). At day 5, the hydrodynamic size of untreated Aβ1-42 amyloid was measured to be 415 nm, as shown in Figure 2D. The Aβ1-42 treated by nCG was recorded at 220 nm. Further, the size of untreated Aβ1-42 on days 6 (Figure 2E) and 7 (Figure 2F) was 429 nm and 616 nm, respectively. The size distribution of Aβ1-42 fibrils increased over time in the absence of treatment. Comparing the nCG treated with Aβ1-42 (untreated), the treated fibril indicated a decreased size distribution. After day 7, the Aβ1-42 treated with nCG was found to be 279 nm, whereas the untreated Aβ1-42 was 616 nm (Figure 2F). Hence, the experiment suggested that Aβ1-42 size increased over time, whereas degradation occurred in Aβ1-42+nCG, indicating the potential effect of nCG. These data support the hypothesis that liposomal encapsulation significantly enhances the degradation potential of CG, with nCG effectively disassembling the Aβ1-42 fibrils. The outcomes from the ThT, turbidity, and DLS assays indicated that nCG was a potential in vitro Aβ1-42-degrading agent. These findings are consistent with our previous study, in which lumbrokinase and serratiopeptidase degraded Aβ1-42 [7,55,56]. Thus, the findings from ThT fluorescence assays, turbidity, and DLS and AFM imaging confirm that nCG effectively degrades Aβ1-42 in vitro.

Atomic force microscope

AFM imaging shows the morphological evidence of amyloid fibril degradation treatment with nCG. After incubating for 24 h, Aβ1-42 (untreated) exhibited the initiation and progressive elongation of amyloid fibrils (Figure 3A). Whereas nCG-treated Aβ1-42 exhibited shorter and smaller aggregates (Figure 3B). The reduction in fibril indicates that nCG may interfere with fibril elongation and promote degradation of preformed fibrils.

*AFM topography images of (A) Aβ1-42 and (B) Aβ1-42 showing reduced Aβ1-42 aggregates after nCG treatment. Cell viability of PC12 cells using the MTT assay: (C) Concentration-dependent effect of CG and nCG; (D) toxicity of Aβ1-42 and ameliorative effect of CG and nCG against Aβ1-42 mediated toxicity (untreated cells served as the control. *p <0.05, **p <0.02, ***p <0.01, ***p <0.001 denotes significant differences compared with control)

In vitro assay of cytological response

Any drug capable of degrading amyloids needs to be thoroughly evaluated for biological safety using both cell lines and animal models. Aβ1-42 degradation often yields fragments of various sizes, which may still pose a cytotoxic risk [57]. Hence, it is essential to analyse the in vitro cellular response to this degradation, particularly those generated by nCG. In this section, we present the in vitro cellular response assessment using the MTT assay, morphological analysis with an inverted microscope, and a dual-cell staining assay. These analyses were conducted for CG, nCG, and intact amyloid Aβ1-42 and the degraded forms of Aβ1-42+CG and Aβ1-42+nCG.

3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium assay

Cell viability in PC12 cells was evaluated using the MTT assay after following treatment with various samples (CG, nCG, Aβ1-42, Aβ1-42+CG, Aβ1-42+nCG) as presented in Figure 3C-D. At a higher dose (20 μg mL^-1^), CG and nCG demonstrated cell viabilities of 65 % and 74.7 %, respectively. Whereas cells treated with Aβ1-42 alone showed decreased cell survival to 15.5 %. Figure 3D represents the ameliorative effects of CG and nCG against Aβ1-42 toxicity. Aβ1-42 degraded by CG and nCG was observed to be 26.9 % and 33.61 %, respectively. Hence, these results indicate that CG and nCG with Aβ1-42 exhibit lower cytotoxicity than untreated Aβ1-42, confirming that degradation mitigates the neurotoxic effects of Aβ1-42. The neuronal toxicity caused by Aβ1-42 has been extensively reported in earlier studies [7], and the observed cell viability results in PC12 cells support these reports and confirm the ameliorative effect of nCG. Statistical analysis further confirms that nCG was observed to differ significantly between the control and treatment groups.

Cell morphology was assessed under an inverted phase contrast microscope (10C magnification) after treatment with the selected concentration of CG, nCG, Aβ1-42, Aβ1-42+CG and Aβ1-42+nCG Figure S1 (Supplementary material). CG and nCG-exposed cells were almost similar to the control group, as depicted in Figures S1A to 1C. The cell density and morphology were normal. Morphological observations of cells also confirm that treatment with Aβ1-42 resulted in increased cell death and the presence of floating cells, whereas Aβ1-42+CG and Aβ1-42+nCG improved the survival of PC12 cells. Therefore, the morphological observations confirm that CG and nCG were not toxic at the selected concentration. nCG decreased the harmful effects of Aβ1-42 on cells.

Live and dead cell staining assay

Viable cells that absorbed acridine orange emitted green fluorescence, while non-viable (dead) cells appeared red under fluorescence microscopy. In this dual staining assay, the untreated control group exhibited complete green fluorescence, indicating healthy cells (Figure 4H). The cells treated with two concentrations of CG and nCG are depicted in Figures 4A to 4D.

Fluorescence microscopic image of PC12 cells treated with (A) 2 μg mL-1 CG; (B) 20 μg mL-1 CG; (C) 2 μg mL-1 nCG; (D) 20 μg mL-1 nCG; (E) Aβ1-42; (F) Aβ1-42+CG (5 μg mL-1); (G) Aβ1-42+nCG (5 μg mL-1); (H) untreated PC12 cells as control. The neurite outgrowth of PC12 cell images (I), the sample treated cells on day 3 (J). crystal violet-stained differentiated cells on day 10

In contrast, cells incubated with Aβ1-42 alone (Figure 4E) showed the maximum number of cell deaths. The Aβ1-42 degraded by CG and nCG showed cell killing of 12 % and 5 % respectively. This assay has been used in several studies to assess anticancer activity [58]. In our present study, CG- and nCG-treated PC12 cells showed a few dead cells. These results further demonstrated the toxicity of Aβ1-42 and, at the same time, that nCG reduced this toxicity. Aβ1-42 significantly decreased cell viability up to (12±7 %) in comparison with the control. Hence, the visual image from this study further supports the biocompatibility of CG and nCG, corroborating the results of the MTT study.

Neurite outgrowth study

The neurite developing activity of the samples (CG, nCG, Aβ1-42, Aβ1-42+CG, Aβ1-42+nCG) for neurite outgrowth was assessed in NGF-differentiated PC12 cells. NGF (10 ng mL^-1^) on cells exhibited typical neuronal morphology, with elongated neurites, as shown in Figures 4I and 4J. At day 3, the initial stage of treatment across all groups showed cell differentiation, as shown in Figure 4I. Treatment with CG and nCG samples at 20 μg mL^-1^ for 10 days resulted in an effective enhancement of neurite outgrowth compared to NGF alone-treated Figure 4J. This analysis revealed an increase in average neurite growth. The nCG-treated cells exhibited more extensive branching than cells in all other groups, indicating a neuritogenic effect. By day 10, the NGF-treated cells exhibited multiple branches and longer neurites.

To evaluate neuroprotective potential, differentiated cells were treated with Aβ1-42, which reduced the neurite outgrowth and induced cellular shrinkage Figure 4I-J. Whereas the Aβ1-42 cotreated with CG and nCG considerably ameliorated Aβ-induced neurite damage, restoring neurite length and morphology compared to other groups. Overall, the results indicate that the compound promotes neurite outgrowth and offers neuroprotection against Aβ toxicity in differentiated cells. Therefore, the findings revealed that the cells treated with NGF alone developed long neurite differentiation. At the same time, cells treated with nCG showed enhanced differentiation, and no toxic effects were observed in the differentiated cells. Treatment with Aβ1-42 reduced differentiation, supporting its neurotoxic properties. Co-treatment of CG and nCG with Aβ1-42 was associated with fewer toxic effects.

In vivo study

The blood vessels were measured using IKOSA software, as shown in Figures 5 and 6. The negative control without treatment developed a normal vascular network, whereas the CAM layer treated with nCG at two concentrations (10 and 100 μg mL^-1^) exhibited an increased number of vessels compared with the negative and positive controls.

Visualization and graphical representation of angiogenesis activity: (A) CAM layer treated with heparin as positive control; (B) untreated control; (C) CAM layer treated with CG (10 μg mL-1); (D) CAM layer treated with CG (100 μg mL-1); (E) CAM layer treated with nCG (10 μg mL-1); (F) CAM layer treated with nCG (100 μg mL-1); (G) total vessel area; (H) total length of the vessels; (I) blood vessel mean thickness; (J) total number of branching points

Visualization and graphical representation of angiogenesis activity: (A) CAM layer treated with a low dose of Aβ1-42; (B) CAM layer treated with high dose of Aβ1-42; (C) CAM layer treated with low dose of Aβ1-42+CG; (D) CAM layer treated with high dose of Aβ1-42+CG; (E) CAM layer exposed to low dose of Aβ1-42+nCG; (F) CAM layer treated with high dose of Aβ1-42+nCG; (G) total vessel area; (H) total vessel length; (I) vessel mean thickness; (J) number of branching points

The data displayed in the insets of Figure 5A show that the positive control with heparin effectively enhanced angiogenesis. Figures 5A to 5J show that the blood vessel development after treatment was statistically significant between CG and nCG. nCG-treated chick eggs showed higher levels of blood vessels compared to the heparin and CG-treated samples. nCG-treated samples exhibited significantly greater angiogenesis than the negative control. The total vessel area, length, thickness, and number of branches are presented in Figures 5G to 5J. The angiogenesis activity of Aβ1-42 co-treated with the sample groups was depicted in Figure 6.

Total area

The total vascular area is often used as a reliable parameter to evaluate changes in neovascularization. As shown in Figure 5, the control group observed normal blood vessels. In the treatment groups, both CG and nCG (10 μg mL^-1^) showed greater vessel area. Whereas the CAM layer treated with Aβ1-42 exhibited a lesser blood vessel area, indicating the toxic properties of amyloid aggregates. The Aβ1-42+CG- and Aβ1-42+nCG-treated CAM layer facilitated vascular remodelling (Figures 6C-6F).

Vessel total length

Vessel elongation is a major step in angiogenesis, contributing to the growth and integration of the vascular network. The formation of new branches and the extension of existing vessels directly affect the overall vessel length. As shown in Figure 5, CG and nCG exhibited larger vessels than the other groups. The CAM layer treated with CG at low doses (10 and 100 μg mL^-1^) and nCG at 100 μg mL^-1^ developed longer vessels than the control. nCG treated with a high dose showed a significant difference from CG. The total vessel length of Aβ1-42, Aβ1-42 co-incubated with CG and nCG treated CAM layer was observed with large vessels (Figure 6A to 6F).

Vessel mean thickness

Variations in average vessel thickness are closely associated with angiogenesis progression. The layer treated with CG, nCG, and heparin developed comparable vessel thickness Figure 5. Aβ1-42-treated CAM layer observed with thin vessel formation due to the toxic effect, and the blood vessels were ruptured Figure 6B. The vessel thickness across these groups may be explained by the possibility that these agents primarily influenced vessel area and length rather than diameter.

Branching points

The number of branching points serves as a key indicator of angiogenesis. Figure 5J shows that nCG-treated eggs had more branching points than the control group. Figures 5E and 5F show the nCG-treated CAM layer. The nCG treated with a low dose was significantly higher than the CG and the control. The Aβ1-42+nCG-administered CAM layer showed a significant difference relative to the control groups (Figure 6J).

In silico study

The interaction mechanism of CG with the target protein Aβ1-42 was investigated using molecular docking analysis. The simulation predicted an RMSD value of -12.175 kJ mol^-1^ and an inhibition affinity (KI) of 7.32 mM. Results revealed that our compound iota-carrageenan is associated with Aβ1-42 (PDB ID = 2BEG) through alkyl interaction at Leu34 and establishes conventional hydrogen bonding with key residues Ile32 and Asp23 (Figure 7).

Molecular interactions of CG with target proteins Aβ1-42 (PDB ID = 2BEG): (A) 2D image; (B) 3D image of CG binding with target Aβ1-42; (C) image of the binding energy results obtained from docking analysis

The protective role of KCG-derived pentasaccharide against Aβ25-35-induced neurotoxicity in human neuroblastoma cell lines was further studied. They observed that KCP-treated cells increased cell viability, reduced apoptosis, and downregulated cleaved caspase-3 expression. Hence, this suggests that carrageenan can be a therapeutic candidate for AD [59]. Similarly, the K-CG obtained from Hypnea musciformis showed neuroprotective, anticancer, and antimicrobial effects by reducing proliferation and apoptosis, thereby mitigating the toxic effect [60]. Sun et al. [61] demonstrated neuroprotective effects by reducing Aβ levels through suppression of microglial-mediated inflammation and by improving cognitive function in an AD mouse model. Hence, based on these studies, we can understand that carrageenan from a marine source can be considered an effective candidate for neuroprotective effects. The antiamyloidogenic effects of iota carrageenan have not yet been investigated in either a cell line or an animal model. This research work was used to enhance the activity of the compound we have nanoformulated. We designed a liposomal formulation of iota carrageenan with enhanced biocompatibility for targeting Aβ1-42 disaggregation.

Conclusions

Protein misfolding and aggregation remain major issues in biomedical research and treatment development in amyloidosis. One of the most significant obstacles faced by drugs is the BBB, which prevents roughly 90 % of possible therapeutic medicines from entering the brain. Although various compounds face considerable difficulties crossing the BBB due to their size and physicochemical properties, this underscores the critical need for alternative techniques that can cross the BBB and react at the disease site. Among other things, sulphated polysaccharides such as CG possess inherent biocompatibility and stability, which makes them promising candidates for anti-amyloid and neuroprotective applications. This study revealed nCG can effectively disaggregate Aβ1-42, indicating promise as a safe and effective treatment alternative. These findings encourage further research into marine polysaccharide-based treatments for amyloidosis. Future studies should focus on in vivo pharmacokinetic investigations and long-term safety analyses.

Supplementary material

Additional data are available at https://pub.iapchem.org/ojs/index.php/admet/article/view/3122, or from the corresponding author on request.