Evaluating stability and bioactivity of Rehmannia-derived nanovesicles during storage

Plant-derived nanovesicles (PDNVs) have garnered growing attention in the biomedical field owing to their abundance in plant-derived ribonucleic acids (RNA), proteins, lipids and metabolites. The question about the preservation of PDNVs is a crucial and unavoidable concern in both experiments’ settings and their potential clinical application. The objective of this research was to examine the impact of varying storage temperatures on the stability and bioactivity of Rehmannia-derived nanovesicles (RDNVs). The results showed that RDNVs aggregated after 2 weeks of storage period at 4 °C, and the particle size of some RDNVs gradually increased with time, along with the increase of solution potential. After 2 months of storage, all RDNVs exhibited varying levels of aggregation irrespective of storage temperature. The bioactivities of nanovesicles under different temperature storage conditions revealed a gradual decline in cell proliferation inhibition bioactivity over time, significantly lower than that of freshly prepared RDNVs. In contrast, the preservation of anti-migratory activity in RDNVs was found to be more effective when subjected to rapid freezing in liquid nitrogen followed by storage at − 80 °C, as opposed to direct storage at − 80 °C. These findings suggest that temperature alone may not be sufficient in safeguarding the activity and stability of RDNVs, highlighting the necessity for the development of novel protective agents for PDNVs.


Isolation of RDNVs
We actively washed fresh rehmannia (Jiaozuo, China) with running water, followed by three additional washes with distilled water.Subsequently, we chopped it and juiced it with cold phosphate-buffered saline (PBS) using a juicer.After the juicing process with a ratio of 1 g of rehmannia to 2 mL of PBS, we filtered out the residue and subjected the remaining liquid to low-speed centrifugation.We centrifuged it first at 2000 g for 10 min, followed by 5000 g for 20 min, and finally 10,000 g for 1 h to remove debris and residue, respectively.To prevent the difficult resuspension caused by nanovesicle aggregation, we used centrifugation at 100,000 g for 30 min to collect nanovesicles 19 .After that, we subjected the obtained nanovesicles to two additional washes using 100,000 g for 30 min to eliminate impurities from the solution.After decontamination using a 0.22 μm filter membrane, we utilized them for downstream experiments.

Bicinchoninic acid (BCA) protein quantification of RDNVs
For BCA protein quantification of RDNVs, different volumes (0, 1, 2, 4, 8, 12, 16, 20 μL) of the same concentration (0.5 mg/mL) of bovine serum albumin (BSA) were used to formulate a 20 μL reaction system with PBS, respectively.Sample wells to be tested were added with 2 μL of sample and 18 μL of PBS.Then, 200 μL of 50:1 configuration of BCA reagent (Beyotime, Shanghai) solution of solution A and solution B were added to each system.After incubation at 37 °C for 30 min, the absorbance (OD) values were detected at 562 nm using an microplate reader (SpectraMax iD3).A standard curve was plotted based on the values obtained and substituted to calculate the concentration of the samples.

Storage of RDNVs
The nanovesicles quantified by BCA were diluted to 1 mg/mL with PBS at 4 °C and divided into (1) liquid nitrogen quick-freezing and then transferred to − 80 °C group, (2) direct storage at − 80 °C group, (3) direct storage at − 20 °C group, and (4) direct storage at 4 °C group.To prevent repeated freezing and thawing, each sterile freezing tube was labelled in advance as to its purpose and storage time, and was only removed when used for bioactivity assessment and testing.

Potential and particle size distribution assessment of RDNVs
Prior to potential and particle size testing, frozen RDNVs were thawed rapidly in a 37 °C water bath and diluted for measurement immediately after the samples were thawed and fully resuspension.100 μL of 1 mg/mL RDNVs was taken and diluted using 900 μL of PBS liquid and the potential and particle size distribution of the solutions were tested using a dynamic light scatterometry (Anton Paar, Austria) under the same conditions using quartz potentiostat dishes and normal PVC dishes respectively.

Morphological assessment of RDNVs
RDNVs were observed under transmission electron microscope (FEI TecnaiG2, Japan).Firstly, after the solution was fully resuspended, 5 μL of RDNVs was taken on a clean PVC membrane and mixed thoroughly with 5 μL of 4% paraformaldehyde.Then, a 400-mesh copper mesh was covered and fixed for 30 min.Subsequently, the mixed liquid was removed and washed three times with distilled water.The residual liquid was blotted out using filter paper and stained with 2% phosphotungstic acid solution (pH = 7) for 2 min.The staining liquid was removed and washed 3 times with distilled water.Finally, the residual liquid was blotted out using filter paper and dried at room temperature for more than 30 min for observation.

Cell viability assay
Cells were seeded in 96-well plates at a density of 2000 cells per well.The solution was configured into four concentrations of 50 μg/mL, 25 μg/mL, 12.5 μg/mL and 6.25 μg/mL using complete medium.The intervention was carried out by adding the appropriate concentration of drug at 24 h after cell implantation.At 48 h after addition of RDNVs, the medium was aspirated and CCK8 solution (Invitrogen) diluted in a 1:9 ratio was added.A complete blank group was set up and 100 μL of liquid was added to each well and incubated for 1 h.The absorbance (OD) values were measured at 450 nm using an microplate reader(SpectraMax iD3).

Scratch assay
Cells were grown in 12-well plates with 800,000 cells per well.After the cells had grown to approximately 90%, a "cross" scratch was created on the cell monolayer using a 200 μL pipette tip.Post-scratch interventions were performed using serum-free medium configured with 50 μg/mL RDNVs.Photographs were taken at 0 h at the beginning of the scratch intervention and again when the scratch healed in the blank control group (approximately 30 h).The images were captured at the upper and lower intersections of the "cross" scratch.The blank area was analyzed using ImageJ software.

Statistical analysis
Numerical results were expressed as mean ± standard deviation.Comparisons between groups were made using the appropriate t-test for comparing numerical data between two groups, while ANOVA was applied to compare three or more groups.Multiple comparisons were used to determine significant differences between specific groups when significant differences existed.The level of significance was set at p < 0.05.

Statement
The plant material, Rehmannia, used in this study has been identified and authenticated.It was purchased from Wen County, Jiaozuo, Henan Province, China, and originated from a farm where it was legally cultivated and harvested for commercial purposes.Experimental research and field studies on plants (either cultivated or wild), including the collection of plant material, were carried out in compliance with relevant institutional, national, and international guidelines and legislation.The LN4 cells line was derived from the oral squamous cell carcinoma Cal-27 cell line, which was purchased from ATCC.The detailed construction process is described in our previous work 18 .The LN4 cells line exhibits enhanced lymph node metastasis capabilities, with STR analysis confirming the retention of Cal-27 cell fingerprinting information.

Effect of different preservation temperatures on the morphology of RDNVs
We investigated the effects of different preservation conditions on the physicochemical characteristics of RDNVs.Firstly, we visualized the morphology of RDNVs.The results showed that at the beginning, the vesicles were more dispersed(Fig.1A).However, after 2 weeks of storage at 4 °C, a significant fusion aggregation of RDNVs was observed (Fig. 1B1-B3).We also observed the occurrence of aggregation and fusion under the − 20 °C storage conditions (Fig. 1C1-C3).However, the effects were less pronounced under the − 80 °C and after transferring to − 80 °C following rapid freezing in liquid nitrogen (Fig. 1D1-D3, E1-E3).And the degree of fusion gradually increased.After one month, the RDNVs showed significant aggregation under the storage conditions of 4 °C and − 20 °C (Fig. 1B2, C2), while the dispersion remained relatively unaffected under the − 80 °C storage conditions and after transferring to − 80 °C following rapid freezing in liquid nitrogen (Fig. 1D2, E2).However, at 2 months, we observed aggregation and fusion of RDNVs regardless of the storage temperature (Fig. 1B3, C3, D3, E3).

Effect of different preservation temperatures on the particle size distribution of RDNVs
Subsequently, we evaluated the dynamic distribution of the particle size of RDNVs under different preservation temperature conditions using dynamic light scattering.We observed that the average particle size of the RDNVs was about 200 nm initially (Fig. 2A), and after 2 weeks of storage at 4 °C, some of the RDNVs had undergone aggregation or fusion (Fig. 2B1).At 30 days, the size distribution peak of the vesicles under the 4 °C storage condition showed a tendency towards lower dispersion, indicating a more pronounced increase in size (Fig. 2B2).After storage at − 20 °C for 14 and 30 days, there was an increasing trend in the size of RDNVs (Fig. 2C1, 2).However, the size of RDNVs stored at − 80 °C and after transferred to − 80 °C storage condition following flash-freezing in liquid nitrogen showed no significant changes in size within 1 month (Fig. 2D1-D2, and 2E1-E2).However, after two months of storage, all these vesicles started to show noticeable aggregation (Fig. 2D3 and E3), with the most severe aggregation observed under the 4 °C storage condition (Fig. 2B3).This result is consistent with the observations made under electron microscopy, where RDNVs also exhibited fusion and aggregation after storage.

Effect of different preservation temperatures on the potential distribution of RDNVs
The negative charges on the surface of the lipid bilayer may mutually repel each other, which could be a key factor in maintaining the stability of the solution 20 .However, due to the presence of not only lipids but also proteins and other components in this vesicular structure 19 , long-term storage may affect the changes in the solution potential of RDNVs.As such, we further evaluated the dynamic potential distribution of the RDNVs under different preservation temperature conditions.We observed that initially the potential of the RDNVs was around − 12 mV (Fig. 3A).At the end of 2 weeks of storage, the zeta potentials of RDNVs solutions in all subgroups increased (Fig. 3B1, C1, D1), except for that were transferred to − 80 °C after liquid nitrogen rapid freezing group, which did not show significant changes (Fig. 3E1).When evaluated at the next 1-and 2-month time points, particle potentials under 4 °C storage conditions changed significantly from negative to positive potentials, with a potential increase of about 14 mV (Fig. 3B1-B3).Overall, the potential distribution of RDNVs under − 20 °C storage conditions was more stable, with only a slight upregulation of approximately 4 mV after Vol:.( 1234567890 two months of storage (Fig. 3C1-C3).Compared to transferring to − 80 °C after liquid nitrogen flash-freezing, RDNVs stored at − 80 °C showed an earlier upregulation of potential (Fig. 3D1-D3).However, there was no significant difference in potential between RDNVs stored for 1-2 months after being transferred to − 80 °C after liquid nitrogen rapid freezing and those directly stored at − 80 °C (Fig. 3E1-E3).

Effect of different preservation temperatures on the ability of RDNVs to inhibit tumor cell proliferation
Currently, there have been studies characterizing the physical properties of PDNVs under different temperature storage conditions 11,12 .However, there has been a lack of systematic tracking and evaluation of the bioactivity of these PDNVs.Subsequently, we evaluated the effect of RDNVs on LN4 cell viability under different preservation temperature conditions.At the initial isolated nanovesicles at a concentration of 50 μg/mL, 50% of cell viability could be inhibited after 48 h of action, while some inhibitory effect on LN4 cell viability was also observed at a concentration of 6.25 μg/mL (Fig. 4A).Under the storage condition of 4 °C, except for RDNVs with a concentration of 50 μg/mL, which maintained their proliferative activity in LN4 cells after 60 days of storage, all other concentrations became ineffective (Fig. 4C).Under the storage condition of − 20 °C, RDNVs could still inhibit the proliferation activity of LN4 cells to varying degrees after 30 days of storage, but the efficacy decreased compared to freshly RDNVs (Fig. 4B).Surprisingly, storing RDNVs directly at − 80 °C for 14 days significantly reduced their anti-proliferative activity (Fig. 4A).On the other hand, when RDNVs were rapid freezing in liquid nitrogen and then transferred to − 80 °C, they showed better preservation of RDNVs' bioactivity compared to other conditions (Fig. 4A-C).However, none of these conditions could ideally preserve the anti-proliferative activity of RDNVs.

Effect of different preservation temperatures on the ability of RDNVs to inhibit tumor cell migration
Furthermore, we also assessed the impact of different storage conditions on the ability of RDNVs to inhibit LN4 cell migration.We observed that fresh RDNVs at a concentration of 50 μg/mL could reduce cell motility by approximately 75%.However, under 4 °C storage conditions, the ability to inhibit cell movement decreased by almost half after 60 days (Fig. 5A-C).Under − 20 °C storage conditions, the ability of RDNVs to inhibit LN4 cell migration decreased over time (Fig. 5A, C, and D).Within a storage time of 14 days, RDNVs maintained their bioactivity in rapid freezing in liquid and transferred to − 80 °C storage conditions, performing at levels comparable to fresh samples.After 2 months of storage, the bioactivity of the vesicles remained present under

Discussion
PDNVs have shown various advantages in the field of disease treatment, such as good biocompatibility, low toxicity, drug loading capacity, and easy modifiability 4,21 .They have shown great potential in areas such as tissue regeneration and anti-tumor effects, and are expected to inject new power into the development of cosmetics and drugs 1, 22 .Rehmannia is a commonly used plant with extensive medicinal value, and it has been shown to have potential in areas such as cognitive improvement 23 , anti-tumor effects 24 , and tissue protection 25 .Rehmanniaderived nanovesicles have been found can mitigate acute lung injury induced by lipopolysaccharides and restore intestinal microbiota balance 17 .Our previous research discovered the potential of RDNVs in inhibiting squamous cell carcinoma, but this bioactivity varies among nanovesicles derived from Rehmannia purchased in the same region but during different seasons.This suggests the need for proper preservation of RDNVs and also poses a challenge for batch stability of RDNVs.Common methods for preserving PDNVs include low-temperature storage (such as freezing or refrigeration), freeze-drying method, and the addition of protective agents.Most of the literature uses − 80 °C as a storage condition for plant-derived nanovesicles.Other articles have used rapid freezing with liquid nitrogen or rapid freezing with liquid nitrogen followed by freeze-drying to preserve PDNVs, as rapid freezing with liquid nitrogen is expected to reduce ice crystal formation 9 .Leng et al. evaluated the stability of blueberry-derived nanovesicles stored at different temperatures (including 4 °C, − 20 °C, and − 80 °C) and found that short-term storage at 4 °C and long-term storage at − 80 °C better maintained the stability of the vesicles, although the study tracked them for 30 days 12 .In our study, we extended the time to 2 months initially and found that prolonged preservation by cryopreservation may not be good enough to maintain nanovesicles stability.
In this study, we tracked the changes in particle size, morphology, and potential of RDNVs at different storage temperatures and over different time periods.We found that regardless of the storage temperature, RDNVs experienced reduced potential and phenomena such as fusion and aggregation after approximately 2 months of storage.This is similar to conclusions drawn from previous studies.In this study, we introduced a group that underwent rapid freezing with liquid nitrogen followed by storage at − 80 °C, which is a storage condition used in some studies.We found that this storage condition appeared to better maintain vesicle stability in the short term, suggesting that direct rapid freezing with liquid nitrogen and continuous storage in liquid nitrogen may yield better results.However, it seems difficult to reach a definitive conclusion about which temperature is more suitable for storing a particular type of vesicle to maintain its stability for a long time.In Zeng et al. 's study, it was found that nanovesicles derived from aloe vera could be well maintained at − 20 °C, and they speculated that this may be due to the presence of certain components in the nanovesicles from aloe vera that have better stabilizing effects 19 .On the other hand, in Ge et al. 's study, they found that nanovesicles derived from Brucea javanica did not show significant changes in size and particle size after one year of storage at − 80 °C26 .These studies suggest that there may be inherent differences in the stability of PDNVs themselves.The storage conditions of nanovesicles from different plant sources may vary.When studying the storage conditions of nanovesicles, the effects on vesicle activity and stability should be considered simultaneously.On the one hand, the storage conditions should maintain the structural integrity and functional activity of the nanovesicles to ensure the effective release of the active ingredients at the time of use.On the other hand, the storage conditions should prevent irreversible changes in the composition and properties of the nanovesicles and maintain their consistency and reproducibility.
In this study, we also evaluated the anti-proliferative and anti-migratory activities of RDNVs against oral squamous cell carcinoma cells at different storage temperatures.We found that, apart from the storage condition of liquid nitrogen snap-freezing followed by transfer to − 80 °C, which preserved the anti-proliferative activity of RDNVs for 2 weeks, other temperature conditions failed to maintain the anti-proliferative activity of RDNVs.However, the anti-migratory ability was not significantly compromised as the anti-proliferative activity.Storage conditions at -20 °C, − 80 °C, and rapid freezing with liquid nitrogen followed by storage at − 80 °C all maintained good activity at 2 months, with the best results observed for the latter condition.This differential result may be attributed to the fact that RDNVs are nanovesicles containing multiple components, and different active ingredients in the vesicles may contribute to their anti-proliferative and migration-inhibiting effects.www.nature.com/scientificreports/Also, this difference in bioactivity may be due to the fact that after fusion and aggregation of PDNVs, their morphology and stability can influence the uptake pathway by which they enter the cell.For example, nanoparticles with a size of 150-200 nm are mainly taken up by endocytosis mediated by clathrin or caveolin mediated endocytosis, whereas nanovesicles with a size of 250 nm-3 μm are taken up by cells in the form of macropinocytosis and phagocytosis 27 .The different uptake efficiencies would further lead to changes in the bioavailability of the contents of the RDNVs, which in turn would affect the biological activities they exerts.
Therefore, future research can explore more precise and controllable storage conditions 5 .This includes optimizing parameters such as storage temperature, humidity, as well as introducing new protective agents or preservatives 28 .These protective agents can safeguard the structure and functionality of nanovesicles during storage and mitigate adverse effects from the external environment.They provide additional protection to maintain the stability and activity of nanovesicles.Common protective agents include sucrose, protein stabilizers, and antioxidants 29 .When researching protective agents for PDNVs, the impact of the agents on the nanovesicles and their compatibility with them must be considered.The protective agents should interact with the nanovesicles to preserve their structural and functional integrity, prevent aggregation or degradation during storage.Additionally, the protective agents should exhibit good biocompatibility to ensure no adverse effects on human or biological environments upon application.In recent years, studies have evaluated the effectiveness of protective agents in preserving the stability of PDNVs.Kim et al. assessed the effectiveness of 1,3-butanediol or TMO in protecting the stability of nanovesicles derived from Dendrobium leaves 11 .They set four temperatures (− 20 °C, 4 °C, 25 °C, and 45 °C) and stored the nanovesicles for four weeks, comparing the stability of fresh nanovesicles with those protected by the agents.They found that TMO provided better protection for nanovesicles derived from Dendrobium leaves when stored at 4 °C compared to 1,3-butanediol.This suggests that protective agents may serve to safeguard PDNVs.However, the study only evaluated stability and did not address whether the protective agents have any additional impact on the activity of PDNVs during long-term protection or whether they can maintain the sustained bioactivity of PDNVs.
Using some natural products as protective agents may be a promising direction 30 .For example, certain polysaccharides like trehalose have been found to improve the stability and preservation of cell-derived extracellular vesicles 31 .In a study on the isolation of nanovesicles from tobacco, it was found that the addition of trehalose reduced their aggregation during the isolation process.This raises the question of whether it could potentially enhance the stability of vesicles during the storage process, which is worth investigating 32 .Additionally, natural antioxidants such as vitamin C and glutathione hold potential for protecting nanovesicles.Furthermore, the effects of protective agents under different storage conditions should be investigated.For instance, the suitability of protective agents may differ between cryopreservation and lyophilization.Therefore, it is necessary to evaluate the effectiveness and compatibility of different protective agents under various storage conditions.
In future research, a systematic evaluation of the effectiveness of various protective agents in different applications of PDNVs and their interactions with different storage conditions can be conducted.This will help determine the optimal protective agents and storage methods to maintain the bioactivity and stability of PDNVs for better utilization in drug delivery and other biomedical applications.

Conclusion
This study aims to investigate the effects of different storage temperatures on the stability and bioactivity of RDNVs.We found that regardless of the storage temperature, RDNVs undergo fusion aggregation and a decrease in potential after long-term preservation.Furthermore, we discovered that different storage temperatures have varying effects on the anti-proliferation and anti-migration activities of RDNVs.Conditions involving rapid freezing in liquid nitrogen followed by storage at − 80 °C showed better preservation of RDNV bioactivity in the short term.These results indicate that relying solely on low-temperature refrigeration may not be sufficient to effectively protect the activity and stability of RDNVs, necessitating the urgent development of new PDNVs protectants.These protectants should maintain the structure and function of vesicles during storage while exerting no additional impact on vesicles activity, thus better preserving vesicles activity and providing assurance for the future industrialization of PDNVs.

Figure 2 .
Figure 2. Particle size distributions of RDNVs under different preservation temperature conditions and preservation time.(A) The particle size distributions of fresh RDNVs.(B1-B3) The particle size distributions of RDNVs under 4 °C storage conditions at day 14, day 30, and day 60.(C1-C3) The particle size distributions of RDNVs under − 20 °C storage conditions at day 14, day 30, and day 60.(D1-D3) The particle size distributions of RDNVs under − 80 °C storage conditions at day 14, day 30, and day 60.(E1-E3) The particle size distributions of RDNVs under − 80 °C following rapid freezing in liquid nitrogen storage conditions at day 14, day 30, and day 60.(RDNVs, Rehmannia derived nanovesicles; LN, liquid nitrogen.)

Figure 4 .
Figure 4. Temporal changes in the inhibitory effect of RDNVs on cell proliferation under different temperature storage conditions.(A) Changes in the inhibitory effect of RDNVs on cell proliferation at different concentrations after 14 days of storage at different temperatures.(B) Changes in the inhibitory effect of RDNVs on cell proliferation at different concentrations after 30 days of storage at different temperatures.(C) Changes in the inhibitory effect of RDNVs on cell proliferation at different concentrations after 60 days of storage at different temperatures.

Figure 5 .
Figure 5. Dynamic changes in the effects of RDNVs on the motility of LN4 cells under different storage temperatures.(A) Effects of fresh RDNVs on the motility of LN4 cells.(B) Effects of RDNVs on cell motility after storage at 4 °C for 14, 30, and 60 days.(C) Relative inhibition of LN4 cell movement by RDNVs compared to the control group after storage at different temperatures for different durations.(D) Effects of RDNVs on cell motility after storage at − 20 °C for 14, 30, and 60 days.(E) Effects of RDNVs on cell motility after storage at − 80 °C for 14, 30, and 60 days.(F) Effects of RDNVs on cell motility after storage at − 80 °C following rapid freezing in liquid nitrogen for 14, 30, and 60 days.