Statement of Novelty

  • This study aimed to select a better specific solvent for KL-derived lignin nanoparticle (LNP) synthesis.

  • Here, methanol, THF, ethylene glycol, DMSO, and 70% ethanol were used as organic solvents for lignin to self-assemble and nanoparticle formation.

  • Following adequate characterization, the chosen LNP was used to investigate its role as a drug delivery system for enhancing the bioavailability of the drug curcumin.

  • It’s effect on plant growth using Vigna radiata was also examined to explore the prepared LNP’s application in the agricultural sector if any.

Introduction

The pulp and paper industries are the most environmentally hazardous, particularly in countries such as India [1]. A nation’s economy depends mainly on a tenable balance between industrialization and sustainable management of industrial waste generated. Nowadays, it is inconceivable to imagine life without paper; thus, the demand for paper is increasing by 5–6% yearly [2]. Although most developed countries have transitioned to digital media and electronic communication, the demand for paper is growing daily due to its use as packaging materials and sanitary products [3]. Although the SARS-CoV-2 pandemic has remarkably affected the international market, more than 399 million metric tons of paper have been produced globally. By 2030, paper consumption is predicted to increase rapidly and reach 461 million metric tons [4].

India’s yearly paper production is approximately 10.11 million tons, accounting for 2.6% of the global output [2]. Furthermore, with an annual growth rate of more than 10%, India has one of the fastest-growing pulp and paper markets [1]. The pulp and paper industry’s economic benefits have made it a crucial worldwide sector. However, the sector is among the top five polluters of precious water bodies, generating around 100 million kg of pollutants [5, 6]. Every year, enormous quantities of toxic wastewater are discharged into aquatic ecosystems [7].

Kraft lignin (KL), phenol derivatives, and inorganic chemicals are the most prevalent contaminants in the black liquor of pulp and paper mill effluents, with an annual production of kraft lignin alone of over 170 billion metric tons [8, 9]. Kraft lignin is the primary energy source in kraft mills [10]. However, the exercise of lignin for energy revival alone is not economically feasible, as the energy demand of a regular biorefinery requires only around 40% of the produced lignin [9, 11]. KL-containing effluents harm aquatic life, such as respiratory stress, liver damage, and genotoxicity [4, 12]. Due to its natural rigidity, heterogeneity, and complexity, the exploitation of lignin is challenging. Alternatively, the successful utilization of lignin can expand the range of renewable resources in producing chemicals, fuels, and materials. Adding together, value-added lignin products can augment the economic competitiveness of many biofuels and biochemicals mainly produced from cellulose and hemicelluloses [13,14,15,16]. Therefore, nowadays, the focus on lignin has vastly increased and is well documented. However, the use of nanoscale lignin remained shadowy until recently. The physical and chemical features of the largest renewable source of aromatics in the biosphere in the form of lignin nanoparticles can help enhance the applicability more sophisticatedly. Lignin has been discovered to be an excellent precursor for producing carbon nanostructures due to its high carbon-rich chemical structure and ease of chemical modification. The synthesis of carbon nanostructures from lignin has received little attention [17,18,19]. However, the preparation of nanoparticles can represent a potent means for lignin valorization as it combines straightforward methodologies with high application potential.

Nowadays, it appears that nanotechnology can serve as a hopeful solution in various fields, including the health sector because it has proved that it can perform a crucial role in developing advanced therapeutic and diagnostic strategies. Biopolymer-based nanoparticles, produced from living organisms like silk and chitosan, are gaining attention in drug delivery systems due to their biocompatibility, biodegradability, and low immunogenicity [20]. These nanoparticles improve drug stability, bioavailability, targeted delivery, tissue engineering, and gene delivery [21]. These systems have the potential to protect the drug from degradation, improve its solubility and stability, and improve its absorption and distribution in the body [22, 23]. Lignin-based nanoparticles have shown potential in drug delivery, tissue engineering, and bioimaging with their controlled structures, improved antioxidant activity, and better miscibility with polymers. They have also been used in oral drug delivery systems [24].

Among the various herbal drugs used in India, Curcumin is the most acceptable one as an alternative medicine. Curcumin is a naturally occurring polyphenolic compound found in turmeric (Curcuma longa). Curcumin’s anti-inflammatory properties have been shown to help treat various inflammatory conditions, including arthritis, colitis, and asthma. Furthermore, it has been shown to improve cardiovascular health, metabolic disorders, and cognitive function [25]. Additionally, some studies have indicated that curcumin might have neuroprotective and antidepressant qualities [26,27,28]. Despite its numerous potential therapeutic properties, its clinical use has been limited due to its poor bioavailability, rapid systemic clearance and rapid metabolism. Therefore, achieving therapeutic doses of curcumin in the body is complex and necessitates the development of a drug delivery system capable of improving bioavailability and efficacy [29].

Apart from its use as a drug delivery system, the recent advancement in the preparation of lignin nanoparticles for the controlled release of pesticides, herbicides, and fertilizers is also catching attention [30]. They can be important as sustainable biomaterials for nano-enabled agricultural applications [31]. As reported, lignin nanoparticles at specific concentrations benefitted Zea mays [32] and Glycine max without bioactive compounds [33].

This study aimed to select a better specific solvent for KL-derived lignin nanoparticle (LNP) synthesis. Here, methanol, THF, ethylene glycol, DMSO, and 70% ethanol were used as organic solvents for lignin to self-assemble and nanoparticle formation. Following adequate characterization, the chosen LNP was used to investigate its role as a drug delivery system for enhancing the bioavailability of the drug curcumin. It’s effect on plant growth using Vigna radiata was also examined to explore the prepared LNP’s application in the agricultural sector if any.

Experimental Section

Reagents and Materials

Kraft lignin (Product Code-1,001,124,159, Lot No- MKBH3445V) having 5% impurities, Methanol (HPLC grade), and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) were purchased from Sigma Aldrich. THF, ethylene glycol, DMSO, nitric acid, and other essential reagents were purchased from Merck. HiMedia supplied the dialysis bag (pore size 5000 Da). In all experiments, milli-Q-water was utilized. Filtration was accomplished using a vacuumed filter arrangement with polycarbonate filters and millipore filter paper.

Lignin Nanoparticles Synthesis with Different Solvents

The lignin nanoparticles were prepared with KL using different solvents at a time, such as with HPCL grade methanol, THF, Ethylene glycol, DMSO, 70% ethanol and designated as LNPMet, LNPTHF, LNPEG, LNPDMSO and LNPEth respectively we followed the methods as reported by Dia et al., 2017 with some modification in relating to the ratio of organic solvent and acid used [34]. In brief, 2 gm of kraft lignin was dissolved in 200 ml (10 mg/ml concentration) each of five different organic solvents under constant stirring at 800 rpm for 1 h at room temperature and filtered using a 0.45 μm syringe filter. To this, about 200 ml of 0.025 M HNO3 was added at the rate of 0.5 ml/min, maintaining a 1:1 (v/v) ratio with organic solvent, and kept at a constant stirring of 800 rpm for 4 h. The solution was then subjected to dialysis against deionized distilled water for three days by changing the water thrice daily. The resulting product was washed thrice with deionized water and dried in a hot air oven at 50 °C for eight hours.

Characterization of Prepared Lignin Nanoparticles

The size and morphological characterization of both free and drug-loaded nanoparticles were examined using dynamic light scattering (DLS), Field emission scanning electron microscope (FESEM), and X-ray diffraction analysis (XRD) [35, 36]. For Physico-chemical Characterization, the nanoparticles were examined using zeta potential, Energy dispersive X-ray (EDAX), Fourier transform infrared spectra (FTIR) and Thermogravimetry/Differential Thermal Analysis (TG/DTA) [37].

Dynamic light scattering was used to characterize the hydrodynamic size distribution of lignin nanoparticles using a Malvern metasizer Nano series—Nano ZS Zen 3600. A DLS suspension was made by dissolving LNP in Milli Q water at a concentration of 0.1 gm/L and ultrasonicated it to obtain a well-dispersed suspension. A Malvern zetasizer analyzed Zeta Potential. Both morphology and size of the samples were studied by FESEM using a Zeiss Sigma VP. EDAX was also studied using a Zeiss Sigma VP. For this, the samples were coated with platinum and gold in a ratio of 1:1 using plasma technology with the help of Quorum Sputter coater (SC7680).

Further XRD was used to determine the apparent degree of crystallinity of LNP samples from Panalytical X’Pert PRO (15°–60°) at a scanning rate of 6° per min. To comprehend the chemical interactions of functional groups at the interface, the FTIR spectra of lignin nanoparticles were analyzed with the Perkin Elmer Spectrum 100 FT-IR Spectrometer. The thermal stability of the samples was investigated using thermo gravimetric analysis with PERKIN ELMER Pyris series Diamond TG/DTA. Samples weighing 10 mg were heated from 0 to 1000°C at a rate of 20 ℃/minute.

Preparation of LNP as DDS Using Curcumin as a Model drug

For Curcumin Loaded LNP (LNP-C): After selecting the solvent the same protocol was used to prepare lignin nanoparticles as a drug delivery system for curcumin as a model drug. Curcumin was added at 2 µg/ml concentration before adding HNO3 to the solution.

Drug Loading and In-Vitro Drug Release Profile

Curcumin entrapment efficiency inside LNP-C was determined [38]. In brief, 10 mg of LNP-Cs were dissolved in 10 ml of ethylene glycol, and the solution was sonicated at 40% amplitude for 30-second intervals for up to 15 min. The resulting solution was centrifuged for 10 min at 10,000 rpm at 25 ± 2 °C. The supernatant was collected, and the Curcumin content was determined spectrophotometrically at an absorption maximum of 425 nm. The Entrapment efficiency [EE] was calculated by dividing the amount of curcumin in the nanoparticles by that used in the loading process. Similarly, the loading capacity [LC] was determined by dividing the weight of curcumin obtained from nanoparticles divided by the weight of nanoparticles.

$$\% {\text{EE}} = \frac{{{\text{Practical Drug Load}}}}{{{\text{Theoretical Drug Load}}}}~ \times 100\%$$
(1)
$$\% {\text{LC}} = \frac{{{\text{Total curcumin obtained from LNPC}}}}{{{\text{Weight of the nanoparticles}}}}~ \times 100\% ~$$
(2)

The swelling index of nanoparticles was investigated for 24 h at pH 1.2 and 7.4. In pre-weighed micro-centrifuge tubes, about 10 mg of LNP-C were dispersed (2 mL) in phosphate-buffered saline. The samples were centrifuged at 13,000 rpm at 25 °C for min at regular intervals and the wet mass of the nanoparticles was determined. The swelling percentage (% Sw) was then calculated using the following formula:

$${\text{SW}}(\% ) = \frac{{{\text{Wet weight}}~ - {\text{Initial weight}}}}{{{\text{initial weight}}}}~ \times 100\%$$
(3)

Curcumin’s in vitro release profile from LNP-C was performed using the direct dispersion method [38]. Curcumin release was monitored separately for 156 h at two different pH, 1.2 and 7.4, respectively. A known amount of LNP-C (50 mg) was dissolved in 50 mL of PBS at pH 1.2 and 7.4. The tubes were incubated at 37 °C and 150 revolutions per minute. The samples were taken at regular intervals, and the absorbance at 425 nm was measured with a UV-Vis spectrophotometer to determine the amount of curcumin released.

Antioxidant Assay

DPPH assay was performed for the antioxidant properties of the free LNP and curcumin-loaded LNP (LNP-C) [39]. In the DPPH assay, 3 mL of curcumin-loaded LNP and Free LNP at various concentrations was added to 0.1 mM stock of DPPH kept in the dark for 30 min, and absorbance was measured at 517 nm to calculate the IC50 value and percentage of radical scavenging.

Haemolytic Assay

Using the in-vitro hemolysis assay, the impact of curcumin-loaded LNP (LNP-C) on the blood vascular system was examined and compared to that of free LNPs and curcumin. Blood was drawn from healthy volunteers with their consent. A 2% suspension of RBCs was treated with various concentrations of the samples (10–500 µg ml−1) and centrifuged at 3000 rpm for 5 min before being tested for hemoglobin at 541 nm. Positive and negative control tubes were made using 0.5% Triton-X100 and normal saline, respectively, and the percentage of hemolysis was calculated.

$$\% {\text{ relative hemolysis }} = {\text{ }}\left( {{\text{A}}_{{{\text{sample}}}} - {\text{A}}_{{{\text{negative control}}}} } \right){\text{ }}/{\text{ }}\left( {{\text{A}}_{{{\text{positve control}}}} - {\text{ A}}_{{{\text{negative control}}}} } \right){\text{ }} \times {\text{1}}00$$

was used to calculate the percentage of hemolysis [40].

Plant Biocompatibility Assay

The plant bioassay was performed to evaluate the early stages of seed development, plant growth, and biomass increase of young seedlings so that their use as bio-activators and nanocarriers in agriculture can be predictable. For this, the seeds of Vigna radiata were surface sterilized with 0.1% HgCl2 followed by 70% ethanol and distilled water. About ten seeds were placed in a Petri dish (15 cm) on a Whatman No. 1 filter paper moistened with 5 ml of different concentrations of LNPEG solutions in double distilled water and kept in a plant growth chamber at 22 °C under proper light and dark phase (14/10 h). As a control, double distilled water was used. The roots of V. radiata were measured using a thread and scale on the fifth day of exposure [41].

Result and Discussion

Characterization of Prepared Lignin Nanoparticles (LNP) from Kraft Lignin (KL)

Dynamic Light Scattering Study for Size Distribution

Following Dia et al. (2017) with some modifications, lignin nanoparticles (LNPs) were synthesized from KL as a source of biopolymer using five distinct organic solvents: methanol (LNPMet), THF (LNPTHF), Ethylene Glycol (LNPEG), DMSO (LNPDMSO), and 70% ethanol (LNPEth). The LNPs were then characterized. The yield of the lignin nanoparticles designated as LNPMet, LNPTHF, LNPEG, LNPDMSO, and LNPEth was found to be 59.26, 67, 63.12, 63.57, and 64.49%, respectively which appears close to earlier reports [42, 43]. . Dynamic Light Scattering analysis was performed for size distribution and shown in Fig. 1 indicating the relationship between the particle diameter and the percentage of total particles. The particle size distribution of all prepared LNPs is compared regarding the percentage of particles with their hydrodynamic diameters, such as D10 to D90 category and Daverage (Supplementary Table 1). Among all the five formulations, the LNPEG size is the smallest, with Daverage in the range of 189.32 nm, revealing that ethylene glycol is more favorable in maintaining the size. It is noticed that LNPTHF and LNPEG have more than 90% of the particle size below 255 nm. These results are close to the previous studies where using ethylene glycol, Richter et al. (2016) found the average size below 250 nm and Gupta et al. (2014) showed that 60% of the LNP below 250 nm [36, 44]. Lievonen et al. found that the average size can be between 320 and 360 nm using THF, while Mishra and Ekielski (2019) found the average size to be 150 nm using DMSO as the solvent [35, 44]. In brief, the diameters of lignin nanoparticles can be influenced when we use different solvents. The solubility of lignin and its interaction and affinity with solvent probably could have affected the size in this study as the solvent influences the self-assembly techniques of lignin nanoparticles [45, 46]. A recent study indicates that the solvent—lignin hydrophobic interaction and the solvent—lignin H-bonds contribute to the size of the particles [45]. Apart from this, for other polymer-based Nanoparticles, size is influenced by the affinity of the solvents towards the polymer [47].

Fig. 1
figure 1

Size distribution of lignin nanoparticles synthesized from kraft lignin through dynamic light scattering. Different colors represent the size distribution of different LNPs. Black (LNPMet), Red (LNPTHF) Blue (LNPEG), Green (LNPDMSO) and Purple (LNPEth)

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Zeta Potential Analysis

The Zeta Potentials of the LNPMet, LNPTHF, LNPEG, LNPDMSO, and LNPEth were about—16.3, − 26, − 18.5, − 12.2, and − 25.5 mV respectively at neutral pH. Similar values were reported by Lievonen et al. (2016) and Dia et al. (2017) [34, 44]. The prepared particles thus appear to have good stability as it is generally believed that a higher zeta potential value has sufficient repulsive force to achieve better physical colloidal stability [49]. It has been found that zeta potential depends on solvent polarity. It could be possible that the specific Solvent-lignin interactions affected the zeta potential of the synthesized nanoparticles [48].

Structural Morphology of Lignin Nanoparticles: FESEM Study

FESEM analysis depicted in Fig. 2 reveals that particles are quasi-spherical with smooth boundaries and in the nano-size range (below 200 nm). However, in some places, merged spheres were also noticed. This study further confirms that a higher surface area to volume ratio may enhance reactivity and efficiency in catalytic reactions and other applications [49]. LNPs will be more biocompatible due to their quasi-spherical structure, making them ideal for biomedical applications such as drug delivery and imaging studies. The high surface area to volume ratio of quasi-spherical nanoparticles enables surface labeling, functional imaging, and monitoring of cells, tissues, and organs, enhancing delivery efficiency. Quasi-spherical nanoparticles can specifically target and enhance the efficacy of specific cells or tissues. Enhanced biodistribution is attained by employing quasi-spherical nanoparticles within the size range of 10–200 nm, which exhibit prolonged circulation in the bloodstream [50,51,52]. Comparable LNP morphology and shape were also observed by other researchers [34, 35, 53].

Fig. 2
figure 2

Field-emission scanning electron microscopy (FESEM) images of lignin nanoparticles (LNPs). a FESEM of LNPMet, b FESEM of LNPTHF, c FESEM of LNPEG, d FESEM of LNPDMSO, e) FESEM of LNPEth

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EDAX

Energy-dispersive X-ray examination indicates significant structural remodeling of the lignin’s chemistry. EDAX analysis of lignin nanoparticles indicates that the elements sulfur and sodium are present in more significant proportions than carbon and oxygen, with the former arising primarily from the kraft pulping process (Table 1).

Table 1 Elemental analysis of five lignin nanoparticles formulated from kraft lignin
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XRD

Figure 3 depicts the XRD pattern of LNPs, revealing numerous significant reflection peaks at 2\({\uptheta }\) These prominent peaks of lignin nanoparticles are similar to the actual diffraction pattern detected in the lignin, as reported earlier [54]. The crystallite size and lattice strain are crucial indications of the drug release mechanism. The smaller the crystallite size, the more particles are released. Similarly, higher lattice strain in a plane (more significant lattice misfit) causes increased instability [55]. In Table 2, the crystallite size (nm) has been shown as calculated from the primary diffraction peaks data using the Debye–Scherrer Eq. 4 and for lattice microstrain (ε) using Eq. 5.

$${\text{Crystallite Size }} = k\lambda /\beta \text{Cos} \theta,$$
(4)
$${\text{lattice microstrain }}\left( \varepsilon \right) = \beta /4\tan \theta,$$
(5)

where \(k\) denotes the correction factor (0.94), \({\uplambda }\) denotes radiation wavelength, \({\upbeta }\) (FWHM) denotes line broadening Full Width at Half Maxima in any degree, \({\uptheta }\) denotes peak position in the XRD graph.

Table 2 Characteristics peak, crystallite size and lattice microstrain of five LNPs formulated from kraft lignin
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By using the Debye-Scherrer Equation, it was shown that the crystallite sizes of LNPMet, LNPTHF, LNPEG, LNPDMSO, and LNPEth were around 0.59, 0.65, 0.63, 0.62, and 0.63 nm, respectively. Gupta et al. (2014) found that the crystallite sizes of LNPs were around 5, 7, and 15 nm [48]. . The micro lattice strain values for LNPMet, LNPTHF, LNPEG, LNPDMSO, and LNPEth were 0.31, 0.29, 0.29, 0.30, and 0.29 nm, respectively. The synthesized LNPs have substantial stability, as evidenced by the crystallite size and lattice strain. Consequently, they hold the potential as an advantageous drug delivery mechanism.

Fig. 3
figure 3

XRD spectra of lignin nanoparticles synthesized from kraft lignin using five different solvents. Different colors represent different XRD peaks of LNPs. Black (LNPMet), Red (LNPTHF) Blue (LNPEG), Green (LNPDMSO), and Purple ( LNPEth)

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Investigation of Functional Group in LNP via UV/Visible and FTIR Study

The UV–Visible scan of the nanoparticles synthesized showed representative λmax as that of lignin, i.e., ∼ 280 nm [56, 57]. For the presence of a specific functional group in LNP, FT-IR spectra of LNPs along with the kraft lignin were analyzed and displayed in Fig. 4. The distinctive peaks and band positions are tabulated (Supplementary Table 2). It is noticed that all LNPs showed specific peaks at 3400–3440 cm−1(–OH stretching), 2930–2970 cm−1(–CH stretching), aromatic skeletal vibration, 1590–1620 cm−1(C=O stretching, adsorbed O–H), 1510–1520 cm−1(C=C–C aromatic ring stretching and vibration), 1420–1470 cm−1(C–H deformation in methyl and methylene), 1250–1275 cm−1 (C–O stretching of guaiacyl unit of lignin), 1100–1150 cm−1 ( C–O–C stretching), 1020–1040 cm−1 ( C–O stretching, aromatic C–H in-plane deformation of syringyl residue), and 795–815 cm−1(aromatic C–H out of plane bending) [58]. These specific peaks confirm the presence of sinapyl (S), guaiacyl (G) and p-hydroxy phenyl (H) moieties present in the lignin. Different solvents did not affect the peak position and intensity of LNP. Moreover, they were identical to that of functional groups of the conventional lignin [46, 59].

Fig. 4
figure 4

FT-IR spectra of five lignin nanoparticles synthesized from kraft lignin using different solvents are shown in different colors. Black for LNPMet, Red for LNPTHF, Blue for LNPEG, Green for LNPDMSO, Purple for LNPEth

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Thermal Stability of LNP: TG/DTA Analysis

In selecting the range of applications for LNPs, thermal stability is a crucial factor. Thermogravimetric (TG) and its derivatives (DTG) (Fig. 5) curves of LNPs showed that there is an initial minimal weight loss below 100 °C associated with the evaporation of loosely bound surface water and excess solvent. Till 300 °C, there is a progressive decrease [60].

Fig. 5
figure 5

TG/DTA analysis of lignin nanoparticles synthesized from kraft lignin for Thermal stability. a TGA Curve of lignin nanoparticles. b DTA Curve of lignin nanoparticles. Representing LNPMet (Black), LNPTHF (Red), LNPEG (Blue), LNPDMSO (Green), LNPEth (Purple)

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According to TGA analysis, 80% of the synthesized LNPs are stable up to 300 °C. The DTA curve shows a sharp peak (above 400 °C), indicating a phase change of powder from amorphous to anatase phase. Thus, it might be concluded that LNPs made from KL had a high degree of thermo stability. In the study by Košíková (2007), it was shown that lignin was thermally degraded between 220 and 800 ℃ depending upon its source [61]. The reason for this might be attributed to the presence of several aromatic rings in lignin, which possess various types of chemical linkages and functional groups.

Application of LNP as Drug Delivery System for Curcumin: Characterization of LNP-C

The hunt to utilize curcumin for its chemotherapeutic and chemo preventive potential in the clinical setting has motivated the development of drug delivery systems for this herbal drug. So, after comparing all five formulations based on size, shape, and physicochemical characterization, the LNPEG formulation was selected to examine its potential for a drug delivery system (Table 3). Ethylene glycol was selected as the solvent choice ahead of THF because lignin is more soluble in ethylene glycol [37]. Also by the Cramer guidelines, ethylene glycol is classified as low hazardous (class I) whereas THF is classified as a class II (Intermediate) compound [62]. As described in the method section, the most valuable herbal drug, curcumin of Indian origin, was the choice. The curcumin-loaded lignin nanoparticle (LNP-C) was prepared and characterized with the help of DLS, zeta potential, FESEM, EDAX, XRD, and FT-IR, as shown in Fig. 6. Its size distribution was analyzed by DLS [35]. The average particle diameter of LNP-C was 88 nm, and 90% of particles were in the range of less than 125 nm (D90). At neutral pH, the zeta potential − 41.67 mV of LNP-C represents that the particle is relatively stable. FESEM revealed LNP-C as quasi-spherical nanoparticles with nanoscale dimensions and clean borders. EDX of the particles uncovered that the LNP-C is integrated with 68% carbon, 0.8% nitrogen, 30.1% oxygen, and 1.1% silicon. According to an XRD analysis, the particle crystallite sizes and lattice strain for the peak of 49.17° were 0.017388 nm and 5.00191, respectively. In Fig. 6e, FT-IR spectra of LNP-C showed similar peaks to that of Free LNP with distinct peaks at 3434.6 (–OH stretching), 2963.7 (–CH stretching, aromatic skeletal vibration), 1599.8 ( C=O stretching, adsorbed O–H), 1513.2 (C=C–C aromatic ring stretching and vibration), 1261.6 ( C–O stretching of guaiacyl unit of lignin), 1095 ( C–O–C stretching), 1022.3 ( C–O stretching, aromatic C–H in-plane deformation of syringyl residue), and 799.9 (aromatic C–H out of plane bending) [58]. It is known that free curcumin does have similar peaks reflecting the stretching vibrations due to phenolic hydroxyl groups at 3200–3500 cm−1, stretching vibration at 1490 cm−1 associated with the aromatic C=C bond, a bending vibration at 1246 cm−1 attributed to the phenolic C–O group and 720–750 cm−1 attributed to stretching of the plane [63].

Table 3 Comparative study of size, morphology and physico–chemical nature of LNPs
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Fig. 6
figure 6

Physico–Chemical characterization of curcumin-loaded lignin nanoparticles (LNP-C): a Size distribution, b FESEM image, c EDAX, d XRD spectra, e FT-IR spectra

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Drug Loading and In-Vitro Drug Release of Curcumin Loaded LNP

The therapeutic efficacy of any drug delivery system is significantly influenced by the amount of drug encapsulated in the nanoparticles. LNP–C showed a loading capacity of 16.65 ± 0.127 (% LC) and encapsulation efficiency of 83.70% ± 1 (% EE) for the selected drug curcumin-loaded lignin nanoparticles. The polymer swelling is crucial for detangling and relaxing the polymer chains and promoting mucus membrane penetration [64]. The LNP-C had shown a swelling index of 35.7% (pH 7.4) and 28.6% (pH 1.2), respectively, shown in Fig. 7a. The primary reason for the better swelling of these nanoparticles at pH 7.4 may be due to the lignin’s swelling behavior at an alkaline pH. At this pH, the swelling force is more significant due to the electrostatic repulsion between the increasingly ionized groups, which results in a more significant release and diffusion of water molecules toward the centre of the nanoparticle [65].

Fig. 7
figure 7

Invitro Drug release profile of LNP-C with a for the Swelling profile of LNP-C, b drug release profile in two different stimulated fluids

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To test the in vitro drug release, the simulated gastric (SGF) and intestinal fluids (SIF) were used and tabulated in Supplementary Table 3. Orally administered nanoparticles come into contact with SGF and SIF before being absorbed. After 4 h, a 13.8% curcumin was released at pH 1.2 (SGF). While at pH 7.4 (SIF), it was 26.79% and more persistent (Fig. 7b). The results showed that the release rate of curcumin from the nanoparticles was significantly affected by the pH as the release rate was low at pH 7.4, which is closer to blood pH than pH 1.2.

Under acidic conditions, the LNPs release curcumin slowly and steadily, reaching only about 28% after 8 h under intestinal conditions. It appears that the LNP delayed the release of the encapsulated drug curcumin in the stomach’s highly acidic environment in contrast to potential rapid erosion of the particles at high pH. Curcumin is released and diffused more readily at higher pH levels due to a more significant swelling force brought on by the electrostatic repulsion between the progressively ionized groups. This is essential for delivering drugs or macromolecules that are easily broken down in acidic media. According to our findings, the minimum burst releases for SGF and SIF are 13.5% and 25%, respectively. The duration of curcumin release into aqueous media continued up to 6 days is a significant observation as it indicates the sustained release of the curcumin which can protect the decomposition of curcumin in the alkaline condition of the intestine. It thus reflects that lignin-based nanoparticles may be effective in the sustained release of the drug [66]. However, the in vitro release profile of curcumin from chitosan nanoparticles evaluated by Gao et al. (2019) found that the release was sustained over 24 h [67].

LNP as an Antioxidant Agent

Both Free LNP and drug-loaded LNPs were checked for their antioxidant properties. The DPPH assay identified curcumin-loaded LNP as an effective antioxidant agent and demonstrated a concentration-dependent activity with an IC50 value of 12.2803 ± 1.397 µg/ml. The IC50values were 40.04 ± 0.7669 µg/ml and 37.931 ± 1.153 µg/ml when tested for free nanoparticle (LNPEG) and free curcumin, respectively. When encapsulated in the current study, the improved antioxidant activity indicates their potential to act as a drug delivery system (see Fig. 8).

Fig. 8
figure 8

Antioxidant assay of curcumin-loaded lignin nanoparticles using DPPH assay

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Compatibility Study of LNP-C: Hemolytic Assay

The compatibility of any drug or drug delivery system with the vascular system is provided by the in-vitro hemolytic assay. Various studies have demonstrated that the hemolytic effect can be utilized to assess carrier-caused membrane damage. Our study shown in Fig. 9 revealed that in comparison to curcumin (10–100 µg/ml), LNP and LNPC (10–500 µg/ml) showed a deficient hemoglobin release (5%) which suggests that lignin-based NPs are less likely to cause membrane damage. Thus, non-hemolytic curcumin with less bioavailability can be delivered using lignin-based nanoparticles as vehicles (LNP-C) for humans as it could be safe [68, 69].

Fig. 9
figure 9

Relative hemolysis % in the presence of Different concentrations of LNP-C (orange), LNPEG (Grey) and Free Curcumin (yellow) (Color figure online)

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LNP as Plant Biostimulants

LNP from Kraft lignin could be a valuable and significant compound with various applications. The awareness of studying LNP and their biological outcome on plants is still less explored. However, it could have a beneficial effect because other nanomaterials and nanoparticles exhibit interesting biological properties [70, 71]. Few experimental results on the usefulness of lignin nanoparticles as a plant biostimulant have recently been highlighted; this impact may be connected to their size and morphological characteristics [32]. LNP must be free from any detrimental consequences to be used in applications involving biological systems. Vigna radiata, a model plant for the preliminary assessment of the toxicity of natural bioactive and other substances, was used. As far as our knowledge goes, this is the first observation on the role of lignin nanoparticles as a growth stimulator, specifically for Vigna radiata. As represented in Table 4, the germination profile of V. radiata in response to LNP shows enhancement of root length after treatment with LNPEG in a concentration-dependent manner (up to ∼ 246%). The probable reason for this enhancement could be due to the potentiality of nanoparticles to increase the availability of water and minerals or due to their highly antioxidant property. The antioxidants can neutralize the ROS generated in plant systems during stress. It is studied that the defense mechanism in the plant system may be more triggered with the help of enhanced antioxidant availability in the plant system and thus may affect the growth in response to the application of LNPs in this study [41].

Table 4 Effect of LNPEG on the germination of Vigna radiata
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Conclusion

Here kraft lignin (KL) a byproduct produced by the paper mill industry was used, to synthesize lignin nanoparticles using five different solvents. The size, shape, and physicochemical characterization of the LNPs formed are observed to be somewhat unaffected by the solvent. It was found thermally stable. The effectiveness of selected LNPs for enhancement of the bioavailability of curcumin was found effective with a limited release in a stomach environment and showed encouraging entrapment efficiency. The particle’s antioxidant potential indicated it would be more beneficial than other biopolymer-based drug delivery systems. A more comprehensive range of applications could be possible by these lignin nanoparticles due to their non-hemolytic nature. Apart from these, it is a good plant growth stimulator. This work demonstrated that KL, the industrial byproduct can be explored in an interesting and useful manner as it may be used as a drug delivery system as well as a plant growth stimulator, Further research on mechanisms for positive results observed on plant growth will help explore in agricultural sector. The positive effects on plant growth also indicate eco-friendly nature indirectly.