Stability and time-dependent behavior of Aloin A and Aloin B in aqueous solutionsThe therapeutic potential of Aloins, despite reported benefits in numerous studies, is constrained by their poor stability and rapid degradation in aqueous solutions. Prior research has investigated the stability of Aloins and other anthraquinones across diverse pH and temperature conditions, revealing that dehydration could notably enhance their stability during storage19. Nevertheless, there is limited information regarding the stability of Aloin in aqueous solutions over time. To address this gap, we followed by RP-HPLC analysis, the disappearance of the peak relating to the metabolite over time (Fig. 2). AloA and AloB (purity ≥ 98%) were dissolved in PBS at pH 7.4 with 0.5% DMSO and kept in the dark, both at RT and at 37 °C, and analysed over 48 h. In agreement with the data reported20, through LC–MS analysis we observe for both metabolites an interconversion of AloA (tR = 14.9 min) into AloB (tR = 15.3 min) or vice versa after 24 h (see Figs. S1, S2 in the Supporting Information). Furthermore, the formation of the 10-OH derivative (tR = 13.5 and 14.1 min) as the main product was observed by HPLC–MS analysis after only a few hours, for both Aloins (Figs. S3, S4 in the supporting Information). We found a persistence of AloA and AloB lower than 40% already after 12 h, which is reduced to values lower than 20% after 24 h.Figure 2Stability of Aloin A (Δ) and Aloin B (◊) at 37 °C in PBS at pH 7.4: % Aloin remaining in the 0–48 h range.These findings collectively indicate that the validity of experiments in aqueous solutions is only guaranteed within a brief timeframe, typically a couple of hours and that enhancing the stability of these compounds in water is critical for their therapeutic application.Exploring the role of Aloin A and Aloin B in modulating Aβ peptide amyloid aggregationAlzheimer’s disease (AD) stands as the prevailing type of age-related dementia, marked by a gradual decline in memory and cognitive functions21. A prominent pathological feature of the disease is the existence of extracellular aggregates of amyloid β (Aβ) peptides in senile plaques22. Within the AD brain, aberrant processing of the amyloid precursor protein (APP) by β- and λ-secretases can lead to high levels of Aβ peptides, ultimately accumulating in neuronal tissues and contributing to cell death23. Therefore, targeting the inhibition of Aβ self-assembly emerges as a promising therapeutic strategy for AD treatment8 and numerous compounds, including those derived from natural sources, have been evaluated for their anti-aggregating properties24,25,26,27,28. Despite exhibiting neuroprotective properties, the impact of Aloins on Aβ peptide amyloid aggregation remains unexplored. To address this gap, we investigated the influence of AloA and AloB on amyloid growth using Thioflavin T (ThT) fluorescence assays at a 1:1 Ligand:peptide molar ratio (Fig. 3). Interestingly, within a two-hour timeframe, during which Aloin molecules maintain stability in aqueous solution, their effect on Aβ aggregation is negligible. However, at longer durations, potentially involving Aloin degradation byproducts, AloB (see Fig. 3 panel A, blue curve) significantly reduces the total fiber formed, evident in the decrease in Imax value (see Table 1). Aloin A under these conditions (see Fig. 3 panel A, red curve) doesn’t affect any kinetic parameters significantly (see Table 1). At higher concentrations, AloB (see Fig. 3 panel B, blue curve) exhibits no concentration-dependent behavior, with a reduction in amyloid aggregates similar to that observed at a 1:1 ratio. Conversely, AloA (see Fig. 3 panel B, red curve) in a 2:1 molar ratio significantly diminishes the amount of Aβ fiber formed, indicated by a notable decrease in Imax (Table 1). Our findings evidence that both AloA and AloB are unable to prevent Aβ fiber formation and suggest that this mechanism is likely not involved in the neuroprotective properties of Aloins.Figure 3(A) Kinetics of fiber formation measured by ThT fluorescent emission for 10 μM Aβ1-40 (black line) and in presence of Aloin A at 1:1 ratio (red line) or Aloin B 1:1 ratio (blue line). (B) Kinetics of fiber formation measured by ThT fluorescent emission for Aβ1-40 10 μM (black line) and in presence of Aloin A at 1:2 ratio (red line) or Aloin B 1:2 ratio (blue line). All the experiments were conducted in phosphate buffer 10 mM, 100 mM NaCl, pH 7.4 at 37 °C. Traces are the average of three independent experiments.Table 1 Kinetic parameters derived from ThT curves.Aloin A and Aloin B are 20S proteasome inhibitorsThe proteasome, a 2500 kDa proteolytic molecular machine, encompasses various enzymatic activities (proteolytic, ATPase, de-ubiquitinating) collaboratively aiming for protein degradation29. In eukaryotes, it consists of the 20S proteasome core particle, a cylinder-shaped multimeric protein complex capped on both ends by the 19S complex, responsible for substrate recognition, unfolding, and translocation into the 20S proteasome’s lumen30. This 700 kDa, cylinder-shaped protease comprises 28 protein subunits arranged in four stacked rings, each consisting of seven subunits31,32. Mammalian proteasomes exhibit five different peptidase activities: chymotrypsin-like (ChT-L), trypsin-like (T-L), peptidylglutamyl-peptide hydrolyzing (PGPH), branched-chain amino acid preferring (BrAAP), and small neutral amino acid preferring (SNAAP). These activities cleave bonds on the carboxyl side of hydrophobic, basic, acidic, branched chain, and small neutral amino acids, respectively33. According to proteolysis models, subunits in the 19S particle recognize polyubiquitin chains of four or more residues on the target protein; other subunits cleave the polyubiquitin chain for recycling. Target proteins undergo unfolding and are directed into the 20S proteolytic core for cleavage into small peptides. These peptides are released from the proteasome and degraded into amino acids by cytosolic exopeptidases. Several preclinical studies have documented enhanced proteasome expression and activity across various cancer types34,35,36,37,38. The underlying cause of augmented proteasome activity remains unclear, although it is likely associated with stressful conditions within the tumor microenvironment. The inhibition of proteasome activity has hence the potential to disturb the balance between tumor suppressors and oncoproteins, thereby decreasing cancer progression39,40,41. While the anticancer properties of Aloins targeting various mechanisms are established, their impact on the proteasome remains unexplored. To address this issue, Aloins effects on proteasome 20S activity were assessed using a fluorogenic peptide to measure ChT-L peptidase activities, as detailed elsewhere42. The fluorescence intensity over time was plotted, with the slope indicating the inhibition potency. In these assays, the equimolar mixture of AloA and AloB (AloAB) and the pure epimers AloA and AloB were tested in the same range of concentration (1–750 µM). Increasing concentrations of Alo AB exhibited a more pronounced inhibitory effect compared to equivalent concentrations of pure isomers AloA and AloB (Fig. 4, panel A and B). This behavior can be explained by considering that, because the structures of the two molecules differ, they may occupy distinct allosteric sites that work in concert to increase the overall effect. This hypothesis, however, should be confirmed by further structural studies of ligand-target complexes. Encouraging and reproducible data were observed with Alo AB and human 20S proteasome, displaying a significant decrease in chymotryptic activity at concentrations exceeding 5 µM. The IC50 value for Alo AB (28 µM ± 7) was calculated as described in the experimental section, and the normalized concentration–response plot is shown in Fig. 4, panel C. These findings indicate that the combination of the two Aloins could serve as a potent proteasome inhibitor, potentially possessing antiproliferative properties.Figure 4(A) Normalized activity of human 20S proteasome compared to the control, in an equimolar mixture (AloAB): concentration range 1 × 10–6–5 × 10–4 M. (B) Normalized activity of human 20S proteasome compared to the control of individually isomer AloA and AloB. (C) Nonlinear fit of the concentration–response plot for the inhibition of ChT-L residual activities of human 20S proteasome in the presence of increasing concentrations of AloAB.CDs-PNM/AloAB adduct preparation and characterizationCarbonized polymer dots (CDs), composed of an inner carbonized core covered by a polymer chain shell, have received significant interest in the field of nanotechnology for biomedical application including drug delivery. Recently, we developed a novel one-pot synthetic strategy to produce biocompatible, luminescent and photothermally responsive carbon polymer dots starting from poly(N-isopropylacrylamide) (PNM)43,44. The carbonized polymer dots-PNM (CDs–PNM) were obtained by simple heating of PNM (200 °C, 4 h), without using solvents and additives, through a condensation/aromatization mechanism. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) analyses and dynamic light scattering measurements evidenced the spherical shape and nano-dimensions of the CDs–PNM (diameter around 100–200 nm with a carbon-core of 8 ± 2 nm)43,44. We demonstrated that the CDs–PNM possess drug loading/release properties controlled by a lower critical solution temperature (LCST) behavior, that induces a coil-to-globule conformational transition of the polymer chains. Practically, at room temperature (25 °C) the CDs-PNM adopt a hydrophilic coil-structure permitting an effective drug loading process while at higher temperature (37 °C) the transition to a hydrophobic globule-structure induces an efficiently drug release effect. This mechanism was largely demonstrated for various cargo molecules as curcumin43,45, methylene blue46 cytarabine45 and doxorubicin47. Basing on the demonstrated drug loading versatility and drug delivery potentialities45, we decided to investigate the CDs–PNM as a novel nanocarrier for AloAB. The loading of AloAB in the CD–PNM nanocarrier could results in an increased stability of this noticeably unstable natural product and improved intracellular vehiculation due to the demonstrated cellular uptake of the CDs-PNM43. The aqueous dispersion of CDs-PNM/Alo AB was prepared as described in the experimental section. The AloAB loading capacity percentage (LC%) was calculated to be about 33.3% and drug release at 37 °C is ascribable to the coil-to-globule conformational transition, as previously reported for other CDs-PNM/drug adducts. The CDs-PNM/AloAB adduct formation was confirmed by spectroscopic and DLS measurements. In details, the UV–Vis absorption spectrum of CDs-PNM/AloAB showed the typical AloAB absorption bands at 268 nm, 300 nm and 366 nm, red-shifted compared to the absorption bands of AloAB in water (268 nm, 296 nm and 355 nm) (Fig. 5A). The n–π* broad-band at 360 nm and the π–π* at 271 nm confirmed the presence of the CDs core in the CDs-PNM/AloAB adduct. Similarly, the Circular Dichroism (CD) measurements supported the CDs-PNM/AloAB nanostructures formation through the presence of diagnostic signals in the range 230–310 nm (Fig. 5B), typical of AloB and AloA (Fig. S5 of Supporting Information). No CD signal was recorded for the CDs-PNM water dispersion.Figure 5CDs-PNM/AloAB spectroscopical measurements: (A) optical absorption spectra of CDs–PNM 10 µg/µL (blue-line), Aloin-loaded CDs–PNM (black line) in water and AloAB in water (red line) and (B) CDs spectra of CDs-PNM/AloAB and CDs-PNM.DLS measurements for CDs–PNM/AloAB in water at pH 7.0 and temperature of 25 °C showed a main population with mean hydrodynamic diameter centered at 246 ± 10.1 nm, greater than the hydrodynamic size obtained for CDs–PNM (101 ± 9 nm). The Z-potential investigation showed the presence of negatively charged (− 13.5 ± 0.35 mV) nanostructures. According to a previous work,44 our findings indicate that CDs-PNM nanostructures exhibit a protective action against the degradation of AloAB in water. The optical absorption changes, monitored over time (0–15 days) for AloAB and CDs-PNM/AloAB water solutions stored at room temperature and in the dark (Fig. 6A,B), show that the absorption of AloAB at 355 nm decreases by 12% after 15 days whereas the corresponding band of CDs-PNM/AloAB at 360 nm decreases by 6% after 16 days. At the same time, the increase of the absorbance band at 450 nm, related to AloAB degradation, was 5 times lower in the presence of CDs-PNM furthering supporting the stabilization effect.Figure 6Optical absorption changes of (A) Alo B and (B) CDs-PNM/AloAB in water, after storage in the dark at room temperature.Antiproliferative activity of Aloins and Aloins-loaded CD nanoparticlesThe isolation of the two epimers AloA and AloB from the Aloe Vera leaves allows the investigation of the biological activities of the two compounds, separately. Among the recognized properties of Aloe Vera, we chose to study the antiproliferative activities of AloA and AloB, on human neuroblastoma cell line SH-SY5Y. Based on previous data18,48,49 we exposed the cells to concentrations ranging from 50 to 400 µM, and we tested the antiproliferative effect of the treatments with the Incucyte SX1 Live-Cell Analysis System. After 48 h of exposure, using the Basic Analyzer Software, we obtained a readout of cytotoxicity over time in a label-free manner and automatically measured within the cellular incubator on living cells (Fig. 7A,B). The growth curves revealed a similar trend for all the treatments applied. Indeed, as expected, untreated cells showed exponential cellular growth over time, starting from 1, which was arbitrarily assigned to the initial cell seeding density, to about 1.5 after 48 h of incubation. These values correspond to the phase area confluence normalized to Time 0. On the contrary, a dose–response effect was observed for both Alo A and Alo B. In particular, exposure to 50 and 100 µM of single compounds, did not affect significantly the normal rate of growth, while 200 µM and 400 µM concentrations, were able to reduce the rate of growth and cellular confluence to 0.7 after 48 h of treatment, as also indicated in Fig. 7. Results were further validated through MTT assays (Fig. 7C), ensuring consistency in the measurement of cell viability under identical conditions. In summary, the data implies that while separating the two epimers may be advantageous for evaluating a robust structure–activity relationship, taking also into account the influence of principal metabolites like emodin50, the tested biological activity does not indicate any discernible differences between Aloin A and B. The antiproliferative activity observed for AloA and AloB was then compared to AloAB which showed a similar extent of effect in the dose–response experiment (Fig. S6 of Supporting information) as confirmed by the IC50 values calculated for each curve (AloA:213 ± 33.3; AloB:198.7 ± 31; AloAB:218.9 ± 38.9). To further validate the obtained results, we used HeLa as a different model of a cancer cell line. Data revealed a more resistant behavior of these cells to Aloin compared to SH-SY5Y (Fig. S7 of supporting information). Accordingly, under the same conditions, the proteasome inhibitor, bortezomib, showed higher efficacy on neuroblastoma cells compared to HeLa (see IC50 values in Fig. S8 of Supporting information), suggesting that neuroblastoma cells can be more sensitive to compounds with proteasome inhibitory activity. Given that the epimers AloA and AloB exhibited similar effects on neuronal cells and to improve the slight antiproliferative activities of the compounds, we loaded AloAB onto carbon-based polymer dots (CPDs-PNM), which are known to be safe for cells43 and suitable carriers for drugs with poor water-solubility and bioavailability. We compared the antiproliferative effect of CPDs-PNM/AloAB with the activity of AloAB alone, on human neuroblastoma cells using a lower range of concentrations (5–100 μM). As shown in Fig. 8, AloAB loaded in CDs-PNM, showed greater antiproliferative activity, compared to the free compound. Specifically, at 100 μM concentration, 65% of cell viability was observed by MTT assay after 48 h of treatment. The higher cytotoxic effect of the CDs-PNM/AloAB may be related to the preservation of the entrapped Aloin from degradation and/or more effective cellular uptake already demonstrated for the luminescent CDs-PNM on neuroblastoma cells44.Figure 7Antiproliferative response in neuroblastoma SH-SY5Y cells to increasing concentrations (50–400 μM) of AloA (A) and AloB (B) as quantified by the Incucyte SX1 Live-Cell Analysis System. The readout of cellular growth was assessed every 6 h over 48 h. Values for each timepoint represent means ± SEM of 3 replicates and are normalized to control wells. (C) MTT analysis of the neuroblastoma cell lines treated with Alo A and Alo B. Cell viability was assessed after 48 h of treatment. Bars represent means ± SEM of three independent experiments with n = 3 each. ****P < 0.0001, versus Ctrl by one-way ANOVA + Dunnett’s test. (D) Representative optical images of human neuroblastoma cells after 48 h of exposure with AloA or AloB (50–400 μM).Figure 8MTT analysis of the neuroblastoma cell lines treated with CDPs-PNM (0.2 mg/mL) alone or loaded with increasing concentrations of AloAB (5–100 μM). Cell viability was assessed after 48 h of treatment. Bars represent means ± SEM of three independent experiments with n = 3 each. ****P < 0.0001, *P < 0.05 versus Ctrl by one-way ANOVA + Tukey test.
