Protocol to produce plant-based hybrid nanovesicles from fresh turmeric and pepper using polyethylene glycol

Summary

In addition to proteins, microRNAs, and lipids, plant-derived exosome-like nanovesicles (ENVs) are also enriched with host plant bioactives. Both curcumin and piperine are water insoluble, lack bioavailability, and are extracted by non-ecofriendly solvents. Herein, we present an eco-friendly protocol for co-isolating both curcumin and piperine in the form of hybrid ENVs. We describe steps for sample pre-processing, combined homogenization of plant materials, filtration, and differential centrifugation. We then detail procedures for polyethylene glycol-based fusion and precipitation of hybrid ENVs.

For complete details on the use and execution of this protocol, please refer to Kumar et al.¹

Graphical abstract

Highlights

• •Protocol for the purification of both curcumin and piperine in hybrid nanovesicles
• •One-pot preparation of hybrid nanovesicles (TPENVs) from edible plants
• •Obtain synergistic bioactives from different plant species in one protocol

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

In addition to proteins, microRNAs, and lipids, plant-derived exosome-like nanovesicles (ENVs) are also enriched with host plant bioactives. Both curcumin and piperine are water insoluble, lack bioavailability, and are extracted by non-ecofriendly solvents. Herein, we present an eco-friendly protocol for co-isolating both curcumin and piperine in the form of hybrid ENVs. We describe steps for sample pre-processing, combined homogenization of plant materials, filtration, and differential centrifugation. We then detail procedures for polyethylene glycol-based fusion and precipitation of hybrid ENVs.

Before you begin

Plant derived exosome-like nanovesicles (ENVs) are similar to mammalian exosomes in structure and function. However, in addition to proteins, microRNAs and lipids, plant ENVs are also enriched with host plant bioactives. These include bioactives such as curcumin,² quercetin,³ sulforaphane⁴ etc., which are known for their potential health benefits due to their anti-inflammatory and anti-oxidant properties. Preparation of designer/hybrid nanovesicles (hNV) through membrane fusion between vesicles of different kinds, such as exosomes and liposomes, is an emerging technology. These hNVs acquire novel attributes such as superior targeting capacity, higher biocompatibility and low immunogenicity.⁵ Adopting this membrane fusion principle in plant ENVs will enable the production of hybrid ENVs, which, in particular, will aid in the co-isolation of two or more bioactives from different host plants with synergistic therapeutic effects. In this study, we developed a simple protocol using polyethylene glycol (PEG)-based fusion and precipitation, for the isolation of hybrid ENVs containing two synergistic bioactives, curcumin and piperine, from Turmeric (Curcuma longa) and Pepper (Piper nigrum) plants.

Differential centrifugation followed by ultracentrifugation is a conventional method used for mammalian exosome isolation, which has also been adopted to isolate plant ENVs with utmost purity.⁶ However, this procedure is non-scalable and ultracentrifuges are expensive. To overcome these limitations, molecular crowders such as polyethylene glycol (PEG) have been employed to replace ultracentrifugation in exosome isolation. Taking clues from this, we have recently demonstrated the ability of PEG 6000 to precipitate ginger ENVs.⁷ Here, we show that the PEG precipitation method is also effective in the isolation of hybrid ENVs from turmeric and pepper (TPENV). This is possible due to the in situ PEG mediated fusion of individual turmeric and pepper ENVs resulting in hybrid ENVs (TPENV). Such hybrid TPENVs derived from turmeric and pepper contains both curcumin and its bioenhancer, piperine. In order to compare the relative yield of ENVs and associated bioactives in hybrid versus standalone ENVs, it is advisable to isolate ENVs turmeric alone or pepper alone parallelly.

The isolated TPENVs containing curcumin and piperine can be characterized by dynamic light scattering (DLS) and scanning/transmission electron microscopy (SEM/TEM) for their nanosized nature and surface morphology. Techniques like fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) are the standard methods to confirm the hybrid nature of vesicles by prior labeling of individual nanovesicles with tractable fluorescent dyes.⁸ However, in the case of hybrid TPENVs, simpler techniques such as UV-visible spectroscopy and fluorescent microscopy can be employed to determine the hybrid nature of TPENVs by exploiting the inherent fluorescent properties of curcumin. Furthermore, high-performance liquid chromatography (HPLC) is utilized to validate the presence of both curcumin and piperine in TPENVs with appropriate standards.

Note: Fresh turmeric rhizome and/or pepper seeds are needed for ENV isolation. The actual curcumin content in turmeric varies between 2% to 5% based on the geological origin of the turmeric rhizome.⁹ To obtain high curcumin yield in hybrid TPENVs, it is ideal to choose the turmeric variety with the highest curcumin content. Alternatively, curcumin content from multiple local varieties can be assessed by conventional Soxhlet extraction method and further quantification by HPLC method, prior to hybrid ENV isolation.¹⁰

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
Curcuma longa (turmeric)	Local sources	Madras variety
Piper nigrum (pepper)	Local sources	N/A
Chemicals, peptides, and recombinant proteins
PEG 6000	Sigma-Aldrich	Cat#81260
Hydrochloric acid 37%	Merck	Cat#1930010521
Curcumin	Sigma-Aldrich	Cat#08511
Piperine	Sigma-Aldrich	Cat#75047
Other
Blender	Preethi Diamond, 750 W	214/002 TNE2008022303
Nylon mesh (100 μm pore size)	Local brand	Local brand
Centrifuge	Centurion Scientific Limited	Cat# K241R
Rotor	Centurion Scientific Limited	Cat# BRK5206
Falcon	Tarsons	Cat# 54601
pH meter	Hanna Instruments	HI5222
Magnetic stirrer	Tarsons-Spinot	Cat#6030
Tube rotator horizontal	BIOBEE TECH	Code: 8479 8200
Qsonica ultrasonicator	Solid state cooling systems	Q800R3-110

Step-by-step method details

This section describes the pre-processing of turmeric and pepper samples followed by a detailed hybrid ENV isolation protocol (Figure 1). For comparison, standalone turmeric and pepper ENV isolation are also performed. The last section of the protocol includes the characterization of hybrid and standalone ENVs through size and zeta potential, scanning electron microscopy (SEM), thin layer chromatography (TLC), and fluorescent imaging. Curcumin and piperine quantification in ENVs are done using HPLC and absorption spectroscopy.

Figure 1.

Overall workflow for hybrid TPENV isolation

Day 1

Part 1: Sample collection and pre-processing

Timing: 1 h for turmeric peeling; 12–16 h for pepper soaking

This part of protocol is presented in Figure 2.

Note: In the preparation of hybrid nanovesicles, the amount of individual membrane vesicles to be hybridized is often determined by the actual lipid concentration of pure vesicles.¹¹ Such a method cannot be adopted for hybrid ENV preparation due to the complexity of the starting material being individual plant materials. However, a rational alternative for this issue is to combine individual plant materials, which contain equal lipid content. The lipid content of the plant can be derived using the proximate composition of plants. Nevertheless, this could differ in plants from different geological origins which need to be taken into account before hybrid ENV isolation. In the case of TPENV preparation, the proximate composition of Indian varieties of turmeric and pepper plants is summarized in Table 1.

Figure 2.

Sample collection and pre-processing

This figure outlines the initial weight of samples taken and the pre-processing performed before ENV isolation. All the raw materials used were taken at different weights, normalized to their initial lipid content (A and C). Further, in TPENVs, the weight of turmeric and pepper was adjusted to achieve an equal contribution of lipids (B) from each plant material.

Table 1.

Proximate analysis of turmeric rhizome and pepper (in percentage)

Plant	Moisture	Protein	Lipid	Carbohydrate	Ash	Fiber	Reference
Turmeric	84.25	1.20	1.08	9.10	0.66	0.72	12
Pepper	13.18	10.39	2.74	36.22	4.58	33.16	12

Taking turmeric and pepper plants with equal lipid concentrations also aids in the direct comparison of total ENV concentration and relative curcumin/piperine content in hybrid TPENV versus standalone TENV or PENV. Once the preparation of hybrid TPENVs is established, the ratio between individual plant materials can be varied to obtain higher curcumin/piperine yield as desired. Herein, based on the proximate composition of Indian turmeric and pepper, they are taken in a specific weight ratio to achieve a 1:1 lipid equivalent from each plant (Table 2). In other words, 93.3 g of turmeric, 36.6 g of pepper, 46.4 g and 18.3 g of turmeric and pepper respectively were used in the preparation of standalone TENV, PENV, and hybrid TPENV, respectively which corresponds to the total lipid content of 1 g in each ENV preparation.

• 1.
  Pre-processing of turmeric rhizome:

  • a.
    Take fresh turmeric, surface sterilize it with 70% ethanol, wash it with sterile distilled water thrice, and scrape the peel out.
  • b.
    Use 93 g and 46.4 g of fresh peeled turmeric for TENV and hybrid TPENV isolation, respectively.
• 2.
  Pre-processing for pepper seeds:

  • a.
    Wash the pepper seeds with sterile distilled water and discard the floating hollow seeds present.
  • b.
    Soak 36.6 g and 18.3 g of pepper for PENV and hybrid TPENV, respectively, in 3 times (w/v) distilled water for 12–16 h.
  • c.
    Decant the water as much as possible and use the soaked pepper immediately for PENV and hybrid TPENV isolation.

CRITICAL: For the pre-processing of pepper, remove the soaked water to avoid contamination, and this step can also aid in the removal of traces of anti-nutritional factors present.

Table 2.

Weight of plant material for ENV isolation

ENV type	TENV (turmeric alone)	TPENV (turmeric + Pepper)	PENV (pepper alone)
Weight of plant sample (in g)	93	46.4 + 18.3	36.6
Lipid content (in g)	1	0.5 + 0.5	1

Part 2: Homogenization and filtration

Timing: 60 min

Homogenization of samples and filtration of homogenate was done based on the published protocol⁷ with minor modifications necessary for hybrid TPENV isolation.

This part of the protocol is presented in Figure 3.

• 3.Take 93 g of Turmeric to homogenize for TENV isolation.
• 4.Take 36.6 g of soaked pepper to homogenize for PENV isolation.
• 5.Take 46.4 g of Turmeric along with 18.3 g of soaked pepper to co-homogenize for hybrid TPENV isolation.
• 6.Blend the samples along with sterile distilled water (corresponding to 1.5 to 2 times the weight of the sample taken) in a mixer grinder (750-watt power, maximum rpm 18,500 under no load conditions and 11,000 rpm with load) at medium speed settings for 5 min with 30 s on/off cycles.
• 7.Keep the volume of extract constant between Turmeric, Turmeric and/or pepper extract. Here, we have made up the extract volume to 200 mL.
• 8.Filter the homogenate obtained, through nylon mesh (100 μm pore size).
• 9.Transfer the filtrate into falcon tubes for further steps.

CRITICAL: Take care to perform all of these steps in the aseptic environment to avoid contamination. The use of autoclaved distilled water can also help in minimizing contamination. During homogenization, an adequate volume of water needs to be added (not less than two times the weight of the sample taken) and blended to get optimal yield. However, the addition of excess water should also be avoided, as it would result in a proportional increase of PEG 6000 to be added in further steps. 30-s on and off cycles have to be followed without fail to avoid heat generation. Alternatively, cold distilled water can be used for the same process. Filtration can be done with nylon mesh ≤ 100 μm. Avoid storing the homogenate and proceed for sequential differential centrifugation immediately. While homogenizing, care must be taken to avoid cross-contamination between samples if the same container is used for different ENV preparations, especially between pepper ENVs and turmeric ENVs. Due to the hydrophobic nature of curcumin, jars in which TENVs are purified often contain curcumin stains bound to the walls, and it is hard to clean. One option is to prepare PENVs first, followed by homogenizing TPENVs sample. Alternatively, dedicated jars can be used for each ENV purification routinely.

Figure 3.

Homogenization and filtration of samples

Turmeric and/or pepper samples were blended and filtered using nylon mesh. The obtained filtrate was collected for further steps.

Part 3: Differential centrifugation

Timing: 70 min

This step is performed to sequentially eliminate the fibers, cellular debris, apoptotic bodies, cell organelles, and micro vesicles from the homogenate to obtain the soluble S10 extract containing ENVs.

This part of the protocol is presented in Figure 4.

• 10.Subject the filtered homogenate to sequential centrifugation, i.e., 2000 g for 10 min at 4°C – the supernatant obtained is termed as S2, discard the pellet and collect S2.
• 11.Centrifuge S2 supernatant at 6000 g for 20 min at 4°C – the supernatant obtained is termed as S6, discard the pellet and collect S6.
• 12.Centrifuge S6 supernatant at 10,000 g for 40 min at 4°C – the supernatant obtained is termed as S10, discard the pellet. S10 supernatant obtained is further taken for pH adjustment step.

CRITICAL: Store S10 extracts in amber tubes or brown containers (or wrapped in aluminum foil), as curcumin is photosensitive. Samples like pepper, which is rich in lipid content, may often develop a white fat layer on the top of the supernatant at 10,000 g step. This layer can be eliminated by filtering the supernatant with nylon mesh again prior to PEG precipitation.

Figure 4.

Sequential differential centrifugation of the extract

(A) 2000 g for 10 min at 4°C.

(B) 6000 g for 20 min at 4°C.

(C) 10,000 g for 40 min at 4°C.

Pause point: Though it is highly recommended to proceed with the PEG precipitation step immediately after S10 extract preparation, under unavoidable circumstances, S10 extract can be stored at 4°C for a maximum of 24 h in a dark and air-tight container. Longer storage of S10 extract often leads to contamination, fermentation, and curcumin degradation.

Part 4: pH adjustment step

Timing: 15 min

The pH of S10 extract from TENV, PENV, and TPENV varies between 6.5 and 6.8. Acidification has been shown to increase the yields of exosomes and ENVs in earlier reports.¹³ Hence, we have adopted the strategy of acidifying the S10 extract to increase yields of ENVs.

This part of the protocol is presented in Figure 5.

• 13.Adjust the pH of S10 extract to 5 ± 0.1, using 6N hydrochloric acid. Add the acid to S10 drop by drop making sure the pH is changed in a controlled manner while stirring the S10 continuously using a magnetic stirrer.
• 14.On successful pH adjustment, S10 is further taken for the PEG precipitation step.

CRITICAL: Constant stirring using a magnetic stirrer is crucial for accurate pH measurement due to the suspension/colloidal nature of the S10 extract. The pH adjustment needs to be done under a fume hood as a safety measure to avoid inhaling fumes from concentrated HCl. Alternatively, dilute HCl (1 N) can be used for pH adjustments but it may significantly dilute the S10 extracts. Care should be taken not to over acidify the extracts with excessive addition of HCl while adjusting the pH.

Figure 5.

Acidification of S10 extracts

Part 5: Membrane fusion coupled precipitation of hybrid ENVs by PEG 6000

Timing: 12 h

PEG 6000 is a molecular crowding agent known to selectively precipitate exosomes as well as ENVs.¹⁴ Further, PEG is also employed for facilitating membrane fusion to form hybrid ENVs between exosomes and liposomes.^5,15 Hence, in this step, two simultaneous events are thought to occur. A) PEG-mediated membrane fusion between individual turmeric and pepper ENVs present in the co-homogenate, resulting in hybrid TPENVs and B) Precipitation of hybrid ENVs by the molecular crowding action of PEG 6000.⁵

This part of the protocol is presented in Figure 6.

• 15.Take 45 mL of S10 extract in a falcon tube. Add PEG 6000 to a final concentration of 10% (w/v) (5 g) and make the volume up to 50 mL using sterile distilled water.
• 16.Dissolve the PEG 6000 by rotating the falcon tube in a horizontal tube rotator for 10 min.
• 17.Keep the falcon tubes at 4°C for 8–12 h under constant end-to-end rotation before the precipitation step.

Note: Fluorescent labeling of ENVs with lipophilic dyes such as Dil (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) can aid in tracking their fate under in vitro applications. However, post-production labeling of the purified ENVs with fluorescent dyes requires the removal of unbound dyes via an additional ultracentrifugation step. Here, we suggest the addition of Dil before PEG precipitation to a final concentration of 0.5 μM, so that unbound dyes are removed in the spent supernatant. If any such dyes are added, allow the dye to mix well with the S10 extract (5–7 min) using a tube rotator before the addition of PEG.

Figure 6.

Addition of PEG to ENVs

PEG 6000 is added to pH-adjusted S10 extracts to achieve a final concentration of 10% (w/v), to induce the fusion of vesicles and further precipitation of ENVs.

Day 2

Part 6: ENV precipitation and resuspension

Timing: 60 min

This section explains the precipitation of ENVs by centrifugation after incubation with PEG 6000, followed by ENV resuspension and storage. This part of the protocol is presented in Figure 7.

• 18.For ENV precipitation, centrifuge the S10 extract at 10,000 g for 30 min at 4°C.
• 19.After centrifugation, discard the supernatant, gently invert the tubes over a tissue towel for 2 min to drain excess liquid, and resuspend the ENV pellet either with sterile distilled water or PBS. Make up all the ENVs suspension to equal volumes.

Note: A sufficient volume of water is used to resuspend ENV pellets to achieve a uniform suspension without any visual aggregation. For ENVs isolated in this study with 1 g lipid equivalent of TPENV, a final suspension volume of 5 mL provides uniform suspension. If visual aggregates are seen upon storage, mild sonication can be employed using a water bath sonicator at 70% amplitude for no more than 2 min.

CRITICAL: Incidence of contamination after reconstitution of ENVs can be reduced by the addition of antibiotics recommended for mammalian cell culture (penicillin/streptomycin and/ or amphotericin), as per the manufacturer’s recommendation. Alternatively, dilute suspension of ENVs in PBS can be placed in a sterile tissue culture dish and subjected to germicidal UV exposure for a short time (< 10 min) before in vitro experiments. However, this step can also significantly influence UV-sensitive secondary metabolites present in ENVs, including curcumin.

Figure 7.

ENV precipitation and resuspension

ENVs were isolated using PEG 6000-based precipitation and the final ENV pellet obtained was suspended in sterile double distilled water. The yellow tinge of the pellets in TENV and TPENV but not in PENV indicates the presence of curcumin in these ENVs, which can also be observed in resuspended TENVs and TPENVs.

Isolated TPENVs can be stored at 4°C for up to 2–4 weeks without visible aggregation. TENV/TPENVs should be stored in amber vials to reduce the incidence of curcumin photodegradation. For longer storage purposes at lower temperatures (−80°C), 25 mM trehalose can be used as a cryoprotectant. Trehalose, a non-reducing sugar increases the suspension stability,¹⁶ inhibits aggregation,¹⁷ and cryodamage of ENVs.

Expected outcomes

Two earlier reports have purified ENVs from turmeric rhizomes via the differential ultracentrifugation protocol and have demonstrated the presence of curcumin.^2,18 In our published study,¹ given the synergistic role of piperine in precluding curcumin biotransformation in vivo, we established a protocol for the preparation of hybrid nanovesicles from turmeric and pepper co-homogenate containing both curcumin and piperine. The preparation of hybrid nanovesicles from plant homogenates was achieved by exploiting the dual properties of PEG 6000 as a molecular crowder (to precipitate ENVs) and to induce membrane fusion between individual ENVs from turmeric and pepper present in co-homogenate.¹⁹ In addition to being a cost-effective method for isolating hybrid TPENVs containing both curcumin and piperine, we also serendipitously noted a 6.2-fold higher yield of curcumin in hybrid TPENVs compared to stand-alone TENVs.

After isolation, ENVs were characterized for their size, stability (Zeta potential), and morphology using Malvern size & zeta analyzer, and scanning electron microscopy (SEM). The hybrid nature of TPENVs was confirmed using thin-layer chromatography (TLC), prior fluorescent labeling of ENVs in S10 extract followed by microscopic analysis, and high-performance lipid chromatography (HPLC). The details of characterization and functionality studies and the outcomes can be assessed in our publication.¹

Characterization of TENVs, PENVs, and TPENVs

The nano-sized range of ENVs was confirmed by measuring their size, polydispersity index, and zeta potential using a zeta sizer (Figure 8). All ENVs were within the 200–300 nm size range, with TPENVs exhibiting a narrower size distribution (210 nm) compared to TENVs (297 nm) and PENV (234 nm), while the polydispersity index (PDI) remained largely unaffected (Figure 8A). The zeta potential of ENVs varied between −25 mV to −27 mV without significant difference between the three ENVs (Figure 8B). Scanning electron microscopy (SEM) analysis confirmed their nearly spherical morphology and size in agreement with the results obtained from the Zeta sizer¹ (Figure 8C).

Figure 8.

Biophysical characterizations of TENV, PENV, and TPENV

(A) The size and zeta potential values of TENV, PENV, and TPENVs were measured using a Malvern zeta sizer.

(B) Average size, PDI, and zeta potential values from triplicate batches of ENVs shown. Error bars indicate mean ± SD. ∗p < 0.05.

(C) Morphological analysis of ENVs visualized through SEM. Scale bar-1 μm.

Hybrid nature of TPENVs

Thin layer chromatography (TLC) demonstrated that TPENVs possessed a lipid profile that overlaps with both TENVs and PENVs¹ (Figure 9A). This observation strongly suggests that TPENVs are of a hybrid nature, combining characteristics from both parent vesicle types. Typically, fluorescence resonance energy transfer (FRET) is employed to investigate the membrane fusion between different vesicle subtypes. This method is possible when the individual nanovesicle populations are labeled with FRET compatible dyes before fusion.

Figure 9.

Hybrid nature of TPENVs

(A) TLC separation of lipids from TENV, TPENV and PENVs.

(B) Schematic of fluorescence labeling of TENVs and PENVs.

(C) Immunofluorescence image showing the presence of both curcumin (green) and PENV-derived lipids (in red) in TPENVs.

Unfortunately, this approach could not be used for plant hybrid ENVs due to the complex nature of starting material used for homogenization and the in-situ assembly of hybrid TPENVs during PEG precipitation in S10 extract. However, the intrinsic fluorescence properties of curcumin per se at 425 nm, can be exploited for confirmation of hybrid TPENVs. To demonstrate the formation of hybrid TPENVs, (Figure 9B) we combined S10 aqueous extracts from pepper (containing DIL fluorescent dye) and turmeric, followed by incubation with PEG 6000. This approach yielded hybrid TPENVs exhibiting detectable fluorescence of both curcumin (in green, from turmeric ENVs) and DIL (in red, from pepper ENVs), conclusively confirming the creation of hybrid nanovesicles facilitated by PEG 6000.

In contrast, standalone TENVs and PENVs exhibited either curcumin or DIL fluorescence, respectively, but not both (Figure 9C). These findings underline the hybrid nature of TPENVs and highlight the utility of curcumin fluorescence for the visualization of hybrid ENVs containing curcumin. In summary, our study employing TLC and fluorescence microscopy techniques demonstrated the hybrid nature of TPENVs, shedding light on their unique composition and potential applications.

Co-presence of curcumin and piperine in TPENVs

To quantify curcumin and/or piperine content in isolated ENVs, methanolic extracts were subjected to HPLC¹ (Figure 10). Standard curcumin and piperine showed λ_max at 425 nm & 342 nm, respectively, with a retention time of 5.2 and 5.8 min. TPENVs showed peaks at both 5.2 and 5.8 confirming the presence of both curcumin and piperine while TENVs and PENVs showed either curcumin or piperine peaks, respectively. Notably, TPENVs contained approximately 6.2 times more curcumin compared to TENVs. The total piperine content in PENV and TPENVs was 49.23 ± 3.50 and 58.25 ± 1.29 mg/kg of pepper, respectively.

Figure 10.

Co-presence of curcumin and piperine in TPENVs

(A) HPLC chromatograms of curcumin and piperine standards at 425 and 342 nm.

(B–D) showing HPLC chromatograms of TENV, TPENV, and PENV methanolic extracts respectively.

(E and F) Total yield of curcumin and piperine in TENV, TPENV, and PENV samples calculated based on calibration curve plotted from standards. The curcumin content is with reference to the wet weight of turmeric rhizome and the piperine content is with reference to the dry weight of pepper seeds. Error bar denotes mean ± SD from three independent batches ∗∗∗p < 0.001.

Limitations

Despite the cost-effectiveness and scalability, the PEG-based hybrid ENV isolation method has its limitations. The purity of the final hybrid TPENVs remains a major concern. Unlike differential centrifugation followed by density gradient centrifugation which yields ENVs with utmost purity, PEG is known to precipitate proteins and other macromolecular complexes. This can lead to unintended contamination of hybrid ENVs with proteins, residual PEG, and other macromolecular complexes derived from both turmeric and pepper S10 extracts. This can affect the downstream applications and hinder accurate characterizations. Alternatively, PEG isolated hybrid ENVs can be further purified via sucrose density centrifugation, ultrafiltration or size exclusion chromatography to obtain ultra-pure vesicles.^20,21 Similarly, the size distribution of isolated ENVs can be variable, leading to a heterogeneous mixture of vesicles with a high polydispersity index (PDI). This variation can introduce inconsistency in research and applications where uniformity is essential. Residual PEG can also interact with proteins in the sample, potentially altering their cargo or causing protein aggregation, and can precipitate along ENVs. Natural variability is another concern; The composition of plant-derived ENVs can vary based on factors such as plant species, variety, geological conditions, and growth conditions. This inherent variability can complicate standardization efforts impacting reproducibility. Storage of ENVs is challenging to maintain its stability and functionality. Repeated freeze and thaw cycles can damage ENVs by causing ice crystal formations.

Troubleshooting

Problem 1

Low yield of TPENVs or curcumin content.

Potential solution (step 1)

• •Ensure that you are starting with an adequate amount of plant tissue. Mature turmeric rhizomes have to be chosen for TPENV isolation.
• •It is always advisable to get fresh turmeric rhizome, free of fungal or bacterial contamination and with no axillary or terminal buds developed on it. Curcumin can undergo photodegradation upon exposure to visible light. Hence, TPENVs should be isolated/purified under minimum light conditions.
• •Complete homogenization is important to achieve higher yields of TPENVs. The homogenization time can be increased to 10 min. However, pre-cooled distilled water needs to be used, to prevent heat generation.
• •The choice of an appropriate blender jar (best suitable capacity) based on the amount of sample is critical. The sample volume should be between 50% and 70% of the jar capacity to achieve complete homogenization. It is imperative to follow the same homogenization conditions (time and number of cycles) strictly between biological replicates to achieve consistent results.

Problem 2

ENV preparation is contaminated with other cellular debris or macrovesicles or aggregates as seen in SEM.

Potential solution (step 3)

• •Use a pre-filtration step (e.g., 0.45 μm or 1 μm filter) for S10 extract to remove larger particles and debris before PEG precipitation. Preferably use polyether sulphone (PES) membrane filters which have low protein and DNA binding affinity. PES also exhibit higher chemical resistance and are hydrophilic, with minimal non-specific binding to hydrophobic curcumin.
• •Increase the number and length of pre-centrifugation steps by performing 10,000 g steps twice for 40–60 min, each.

Problem 3

ENVs are difficult to resuspend after the PEG precipitation and visible aggregates are present making it difficult to obtain a homogeneous sample.

Potential solution (step 6)

• •Mild sonication of samples can help to disrupt the aggregates and reform the vesicles. This can be achieved through a water bath sonicator set at medium power for 2–5 min.
• •Alternatively, nano extrusion through appropriate pore size filters (0.45 μM for TPENVs) using a mini-extruder (Sigma Aldrich) can be implemented to avoid the aggregates and achieve uniform sized vesicles.
• •PEG isolated hybrid ENVs can be further purified via sucrose density centrifugation, ultrafiltration or size exclusion chromatography to get rid of aggregates.²²

Problem 4

Batch-to-batch variation in ENV yield, with inconsistent results.

Potential solution (step 1)

• •Standardize your isolation protocol by carefully following the same procedures and using the same reagents each time. Monitor and maintain consistent parameters such as incubation times, temperatures, and centrifugation speeds.
• •Pay attention to the quality and consistency of the source material (e.g., plant tissue). The variety and source of turmeric and pepper used should be kept constant to achieve the same results.
• •The quality of the starting material plays a crucial role in deciding the quality of the final product. While selecting turmeric rhizomes, ensure that the rhizomes are fresh, firm, and appear juicy when chopped. Avoid slimy, rotting, or dried rhizomes. For pepper, choose pepper seeds (round and black) that are mature, well-dehydrated, and devoid of fungal contamination. Over-soaking of pepper may be avoided as this may avoid further contamination.
• •Fresh samples are ideal for isolation. Do not store turmeric rhizome for more than a week at 4°C. If not in immediate use, Turmeric can be stored for up to 2–3 weeks at 25°C–30°C. Prolonged storage dehydrates the turmeric leading to loss of water content.
• •The quality of PEG 6000 used is important (> 99% purity). PEG 6000 bought from different vendors often shows different results in terms of the extent of other contaminants and fusion efficiency.

Problem 5

ENV preparation contains a significant amount of protein contaminants.

Potential solution (step 3, step 5)

• •Increase the number and length of pre-centrifugation steps by performing 10,000 g steps twice for 40–60 min, each.
• •PEG 6000 concentration can be reduced up to 8% to minimize protein co-precipitation without affecting TPENV yield and PEG precipitation can be performed at 8000 g instead of 10,000 g.⁷
• •Consider using sucrose density gradient centrifugation, ultrafiltration or size-exclusion chromatography to further purify ENVs from protein contaminants.

Problem 6

Presence of residual PEG interfering with the TPENV characterization and downstream experiments.

Potential solution (characterization)

• •PEG is water soluble. Hence, Dialysis of TPENVs in sterile water or PBS can remove residual PEG. Verify the removal of PEG through techniques such as the Barium iodide staining method on SDS-PAGE²³ or Fourier-transform infrared spectroscopy (FTIR).

Problem 7

Bacterial or Fungal contamination of ENVs upon storage.

Potential solution (step 6)

• •Store ENVs along with 0.25–1 μg/mL of Anti-bacterial/Anti-fungal agents, (Penicillin streptomycin or Amphotericin) immediately after isolation. Storing ENVs in small single-use aliquots can help minimize the risk of contamination and preserve the integrity of the remaining samples.
• •Alternatively, for cell culture experiments, ENVs can be diluted in culture media and exposed to germicidal UV light for < 5 min for sterilization. However, UV exposure can also affect curcumin and other phytochemicals present in ENVs.

Problem 8

Curcumin content/composition is decreased upon storage.

Potential solution (step 6)

• •Curcumin is photosensitive, it has to be stored in amber tubes, brown bottles, or tubes covered with aluminum foil. Light exposure can lead to either the photodegradation of curcumin or a change in its isoforms.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Gopinath M Sundaram (gopinath@cftri.res.in).

Technical contact

Requests for further details on technical aspects should be directed to the technical contact, Meghana N. Kumar (meghanankjnv@gmail.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The protocol did not generate new unique code.