Resveratrol and prednisolone loaded into human serum albumin nanoparticles for the alleviation of rheumatoid arthritis symptoms: an in vitro and in vivo study
Publicado en línea: 28 jun 2024
Páginas: 16 - 25
Recibido: 15 dic 2023
Aceptado: 24 abr 2024
DOI: https://doi.org/10.2478/msp-2024-0005
Palabras clave
© 2024 Yongcai Song et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Rheumatoid arthritis (RA) is a chronic autoimmune-disease-causing inflammation, joint pain, and joint destruction, severely affecting the quality of life of millions worldwide. Current treatments like NSAIDs and DMARDs have limitations and adverse effects. Hence, innovative therapies are crucial [1–3].
Recent advancements in nanomedicine have opened up promising avenues for enhancing drug delivery and augmenting the therapeutic efficacy of various medications [4, 5]. Among the candidates showing promise, Resveratrol and Prednisolone, known for their potent anti-inflammatory and immunomodulatory properties, have emerged as potential agents for RA management [6, 7]. Resveratrol is a natural polyphenol found in various dietary sources, including grapes, red wine, and peanuts [6]. It is known for its antioxidant and anti-inflammatory properties. Extensive research has shown that resveratrol can inhibit the activity of inflammatory enzymes and cytokines associated with RA, thus mitigating the inflammatory processes responsible for joint damage and pain [8–10]. Furthermore, resveratrol can enhance the activity of the body’s endogenous antioxidant systems, providing a multi-faceted approach to combat the oxidative stress often associated with RA [11]. Prednisolone, on the other hand, is a synthetic glucocorticoid used extensively in RA treatment for its potent immunosuppressive and anti-inflammatory effects. It functions by suppressing the immune system’s inappropriate response and the release of pro-inflammatory mediators, providing rapid relief from RA symptoms [12–14]. However, its longterm use is associated with serious side effects such as osteoporosis, weight gain, and hypertension, underscoring the importance of developing more targeted delivery systems to limit its systemic exposure [15].
Nevertheless, the clinical utility of these drugs is constrained by challenges related to bioavailability, systemic side effects, and rapid clearance from the body [16]. Albumin nanoparticles (ALNPs) have gained prominence as an ideal drug delivery system due to their biocompatibility, nonimmunogenicity, and versatility in encapsulating a wide range of therapeutic compounds [16, 17]. The aim of the current research is to load ALNPs with both prednisolone and resveratrol and assess their ability to modulate inflammatory responses for treating RA. This is the first study developing a carrier system for prednisolone and resveratrol using ALNPs to treat RA.
Prednisolone-loaded ALNPs (PREALNPs) and resveratrol-loaded ALNPs (RESALNPs) were prepared using a method as described before [18]. Briefly, 500 mg of bovine serum albumin (BSA) was added to 5 ml of Milli-Q and mixed with a magnetic stirrer for 15 minutes at room temperature. Next, 20 ml of ethanol containing 2 wt.% prednisolone or 2 wt.% resveratrol was slowly added to the BSA solution and thoroughly mixed until the solution changed color. Then, glutaraldehyde solution was added to the mixture at 8 v/v% and mixed overnight. The solution was centrifuged at 15,000 rpm for 45 minutes, and the pellet was dispersed in Milli-Q water, followed by sonication (5 minutes) and lyophilization (48 hours). The nanocarrier powder was kept at 4°C until further use.
PREALNPs and RESALNPs were first lyophilized and then coated with gold for 250 seconds, and then they were analyzed under 25 kV accelerating high voltage.
Various characteristics of PREALNPs and RESALNPs, including particle size and zeta potential utilizing dynamic light scattering (Zetasizer Nano ZS90, Malvern Instruments, UK)
The viability of MG63 cells cultured with different concentrations of PREALNPs and RESAL-NPs was assessed using an MTT assay kit (Abcam, USA). Briefly, 7,000 cells were cultured in 96 well-plates with DMEM (Invitrogen, USA) culture media containing 10% FBS (Invitrogen, USA), 1% antibiotics (Sigma Aldrich, USA), and different concentrations of nanoparticles (25
The impact of PREALNPs and RESALNPs on the release of key inflammatory cytokines such as IL6, IL-1
The evaluation of resveratrol and prednisolone release from PREALNPs and RESALNPs involved the dissolution of 100 mg of each nanocarrier sample in 15 ml of PBS, followed by a 14-day incubation period. At various time intervals, 0.5 ml of PBS was sampled, and the absorbance was measured at 305 nm and 245 nm for resveratrol and prednisolone, respectively. Subsequently, an equivalent volume of fresh PBS was introduced into the release medium. The obtained optical density data points were then applied to the standard curves for resveratrol and prednisolone in PBS, enabling the calculation of cumulative drug release.
In order to induce a rheumatoid joint model, 5
After sacrificing the animals on day 21, the hind paw joint tissues were harvested, and the tissue levels of TNF-
Data was analyzed using Graphpad prism via student’s t-test and one-way ANOVA methods. All experiments were repeated at least three times.
Results showed that PREALNPs, RESALNPs, and drug-free ALNPs had smooth surfaces with a narrow size distribution. The particles were almost round, and no signs of aggregation were observed. Particle size measurement showed that PREALNPs, RESALNPs, and drug-free ALNPs had around 396.88 ± 76.41 nm, 392.49 ± 97.31 nm, and 338.02 ± 77.75 nm of mean particle size, respectively.
Results (Table 1) showed that the hydrodynamic size and zeta potential of RESALNPs, PRE-ALNPs, and drug-free ALNPs were not significantly different between groups, with p-value > 0.05. The size measured by the DLS was bigger than the size measured by SEM imaging.
DLS assessment results
| Sample name | Hydrodynamic size | Zeta potential |
|---|---|---|
| RESALNPs | 504.067 ± 30.38 nm | −26.05 ± 6.60 mV |
| PREALNPs | 499.91 ± 55.68 nm | −24.93 ± 9.94 mV |
| ALNPs | 536.90 ± 42.30 nm | −26.33 ± 3.21 mV |
Results (Figure 2) showed that at 25
Results (Figure 3) showed that at all the studied concentrations, the anti-inflammatory activity of ALNPs was not significantly different from the control group, signifying the negligible immunomodulatory potential of ALNPs. At all concentrations, the immunomodulatory activity of PREALNPs was significantly higher than that of other groups. In addition, the immunomodulatory activity of RESALNPs was significantly higher than that of ALNPs and control groups.
Fig. 1.
SEM images of (A) RESALNPs, (B) PREALNPs, and (C) drug-free ALNPs. Right panel shows the size distribution of each nanocarrier
Fig. 2.
MTT assay with MG-63 cells cultured with RESALNPs, PREALNPs, and ALNPs during 7-day culture. * shows p-value < 0.05
Fig. 3.
Anti-inflammatory assay results with RESAL-NPs and PREALNPs compared with ALNPs and cells cultured with the normal media as the control group, * shows p-value < 0.05
Results (Figure 4) showed that resveratrol and prednisolone were released from PREALNPs and RESALNPs in a controlled manner that sustained for over 14 days. Drug release initiated by a burst release phase that continued with a slow release phase. At the end of 14th day, the cumulative release of resveratrol and prednisolone were around 68.99 ± 3.83% and 72.15 ± 4.08%, respectively.
Fig. 4.
Release of resveratrol and prednisolone from ALNPs during the course of 14 days in PBS
The histopathological findings (Figure 5) from the control group revealed a range of issues in the joints, including swelling, deterioration of cartilage and bone, the formation of pannus, synovial tissue overgrowth, and inflammation, all of which were confirmed by the presence of inflammatory cell infiltration. However, in the RESALNPs and PRE-ALNPs groups, there was a noticeable improvement in paw inflammation and joint swelling. Surprisingly, the Hybrid group exhibited a significant reduction in paw swelling when compared to the other groups. Within the RESALNPs and PRE-ALNPs groups, there were clear signs of relief from severe paw inflammation and joint swelling. Conversely, the Hybrid group displayed a remarkable decrease in the rate of paw swelling compared to the other groups. In the control group, we also observed substantial damage to the hyaline cartilage and significant alterations in the bone structure, including severe bone degeneration. In the control group, there was evidence of synovial hyperplasia and a significant presence of inflammatory cells, as well as the formation of pannus tissue, which ultimately led to severe damage to the cartilage. Notably, a consistent density of cartilage tissue was not detected, and the formation of pannus tissue contributed to the degradation of articular cartilage and the progression of bone lesions. In contrast, animals in the Hybrid group displayed a protective layer of smooth hyaline cartilage covering the bone surface in both treatment groups. Furthermore, we observed slight changes in the cartilage and bone layers, including an increase in cartilage thickness and a reduction in the penetration of inflammatory cells.
Fig. 5.
Hematoxylin and Eosin (H&E) and Masson’s trichrome staining images of cartilage tissue in different groups
Results (Figure 6) showed that the tissue concentrations of IL-6 and TNF-
Fig. 6.
Tissue concentrations of TNF-
Nanocarriers represent a promising avenue for advancing the treatment of rheumatoid arthritis, offering a solution to the challenges associated with drug delivery in this chronic autoimmune disease [19, 20]. In the current study, resveratrol and prednisolone were administered using ALNPs to improve their bioactivity and therapeutic functions. The developed nanocarriers had a small size and narrow size distribution. The small size of ALNPs enables them to penetrate specific tissues, overcome tissue barriers, and improve drug bioavailability [20, 21]. DLS assay showed that the hydrodynamic size of all nanocarriers was slightly bigger than the size measured by the SEM images. This size reduction could be due to the lyophilization process that eliminates water layers around particles. The zeta potential of ALNPs, much like that of other nanoparticles, serves as a gauge of their surface charge. Generally, ALNPs bear a negative zeta potential, signifying an overall negative electric charge on their surface. This stems from the ionization of specific functional groups within the albumin molecules comprising the nanoparticles. The magnitude of this zeta potential plays a pivotal role in shaping the nanoparticles’ stability and conduct in various biological and environmental settings. A higher absolute zeta potential often signifies increased electrostatic repulsion between nanoparticles, ultimately leading to enhanced dispersion and stability. For ALNPs, their negative zeta potential serves to deter aggregation and bolster their performance in applications such as drug delivery [22, 23]. The biocompatibility of our developed nanocarriers was validated by our MTT assay results, which is in accordance with the results of previous studies [17, 24]. The boost in cell viability in response to culture with RESAL-NPs can be attributed to the ability of resveratrol to enhance cell metabolic activity. Anti-inflammatory potential is crucial for any treatment strategy intended for the treatment of rheumatoid arthritis. Our results showed that both RESALNPs and PREALNPs had significantly higher immunomodulatory function compared with ALNPs and control groups. Prednisolone and resveratrol exhibit anti-inflammatory activity through different mechanisms. Prednisolone, a synthetic corticosteroid, suppresses inflammation by binding to glucocorticoid receptors, which leads to the inhibition of pro-inflammatory gene expression and the reduction of immune responses [25]. In contrast, resveratrol, a natural polyphenol, exerts its anti-inflammatory effects by modulating various signaling pathways, including the inhibition of inflammatory mediators like NF-κB and the activation of anti-inflammatory pathways [26]. Both compounds can mitigate inflammation, but they do so through distinct molecular mechanisms. Sustained release from ALNPs could be due to the swelling, biodegradation, or diffusion mechanisms [27]. However, in the in vivo settings, the combinations of these mechanisms may take place. Our in vivo studies showed that the healing activity of the Hybrid group was highest among experimental groups and was associated with a significant reduction in tissue concentrations of pro-inflammatory cytokines. It could be that the synergistic anti-inflammatory activity of PREALNPs and RESALNPs may have caused the observed healing functions. Resveratrol activates sirtuin 1 (SIRT1), a deacetylase enzyme that can modulate inflammation and immune responses [28]. Resveratrol’s antioxidant properties further protect against oxidative stress, which plays a role in rheumatoid arthritis pathogenesis. On the other hand, prednisolone, being a corticosteroid, operates by attaching to glucocorticoid receptors found within cells. This attachment hinders the activation of pro-inflammatory transcription factors, such as NF-κB and AP-1, which results in the inhibition of pro-inflammatory gene expression [29, 30]. As a consequence, it reduces the generation of inflammatory cytokines and immune responses. When used together, prednisolone and resveratrol may offer a more comprehensive approach to rheumatoid arthritis management. Prednisolone provides potent and rapid immune suppression, while resveratrol enhances anti-inflammatory effects and counteracts oxidative stress.
In summary, our current research has successfully harnessed the therapeutic potential of resveratrol and prednisolone by encapsulating them within ALNPs, marking a significant step forward in the quest for an effective treatment for rheumatoid arthritis. The resulting nanocarriers proved to be both non-toxic and adept at modulating inflammatory responses, laying a solid foundation for their application in targeted drug delivery. The compelling outcomes of our in vivo study underscore the tangible benefits of our approach, as animals treated with resveratrol and prednisolone-loaded ALNPs exhibited markedly improved alleviation of hind paw joint issues. This promising observation not only speaks to the efficacy of our developed nanocarriers but also suggests a potential breakthrough in enhancing the therapeutic outcomes for rheumatoid arthritis sufferers. Complementing the in vivo findings, the results from the ELISA assay provide valuable insights into the underlying mechanisms of our treatment. The implication that our developed therapy may have contributed to the reduction of inflammatory reactions at the injury site further solidifies the potential clinical relevance of our approach. Moving forward, our research will refine ALNPs encapsulating resveratrol and prednisolone for enhanced rheumatoid arthritis treatment. We will optimize the nanocarrier design, explore new drug combinations, and conduct thorough preclinical validation. These efforts aim to ensure safety, efficacy, and long-term benefits in animal models. Following successful preclinical studies, we will progress to clinical trials, starting with safety assessments in Phase I and efficacy evaluations in Phase II. Our ultimate goal is to deliver personalized medicine, offering tailored treatments that significantly improve the lives of rheumatoid arthritis patients.