A comprehensive and systemic review of ginseng‐based nanomaterials: Synthesis, targeted delivery, and biomedical applications

Among 17 Panax species identified across the world, Panax ginseng (Korean ginseng), Panax quinquefolius (American ginseng), and Panax notoginseng (Chinese ginseng) are highly recognized for the presence of bioactive compound, ginsenosides and their pharmacological effects. P. ginseng is widely used for synthesis of different types of nanoparticles compared to P. quinquefolius and P. notoginseng. The use of nano‐ginseng could increase the oral bioavailability, membrane permeability, and thus provide effective delivery of ginsenosides to the target sites through transport system. In this review, we explore the synthesis of ginseng nanoparticles using plant extracts from various organs, microbes, and polymers, as well as their biomedical applications. Furthermore, we highlight transporters involved in transport of ginsenoside nanoparticles to the target sites. Size, zeta potential, temperature, and pH are also discussed as the critical parameters affecting the quality of ginseng nanoparticles synthesis.

much energy for the synthesis of metal nanoparticles with broad diversity of sizes, shapes, composition, and physicochemical properties. 38 For the efficient drug delivery, polymers can be used broadly in wide range of applications in the field of biomedicine. However, use of synthetic polymers such as nylon, polyethylene, and polyester have several disadvantages including poor biocompatibility, lack of processing ability, poor drug loading ability, increased instability, and enhanced fragility. Therefore, the synthesis of biopolymers such as chitosan and biodegradable poly (lactic-co-glycolic acid) has gained promising role in drug delivery system due to their controlled delivery system, good biocompatibility, stability, nontoxic and nonimmunogenic, and enhanced therapeutic effects. 39 Microfluidics is also used to study the behavior of fluids at the microenvironments and therefore widely used in various field including biomedicine. 40 Compared to the polymer nanoparticles, microfluidic system possesses significant advantages including ease of fabrication, coherence, rapid, and cost effectiveness for synthesis of ginseng polysaccharide nanomaterials. 41 Furthermore, green approach of selfassembly such as Rb1 into ultra-micelles also showed promising therapeutic tool. 42 In the past decade, only few reviews are reported at systemic and local delivery of ginsenoside using specific polymeric nanoparticles and nanofibers, increasing bioavailability considering administration route, delivering behavior, biocompatibility, and biodegradability of ginseng nanoparticles. Furthermore, a short review emphasized on various types of ginseng nanoparticles and application toward specific cancer treatment. 24,43,44 This review provides a systemic and comprehensive overview on the standard methods for synthesis of ginseng nanoparticles.
To provide a quick glance on synthesis methods, we have also explained detailed process of various methods for synthesis of ginseng nanoparticle. This review highlights the role of different transporters involved in ginsenosides F I G U R E 2 Representative nanostructures used to deliver ginseng biomaterials. (A) Gold, 5 (B) silver, 5 (C) zinc, 6 (D) mesoporous silica, 7 (E) liposome based, 8 (F) polymer based, 9 and (G) microfluid based nanoparticles, 10 (H) nano-formulation. 29 This figure was created using biorender software https://biorender.com/. [Color figure can be viewed at wileyonlinelibrary.com] F I G U R E 3 Schematic representation of different types of ginseng nanoparticle synthesis. (A) Metals such as gold, silver, and zinc are widely used to synthesis metal nanoparticles using various ginseng extracts including leaf, root, and berry from different Panax species. 24,25 (B) Fe@Fe3O4 nanoparticles synthesis through the microfluidic process. 10,26 (C) Microbial synthesis of gold nanoparticles using ginsenoside compound K, gold (III) chloride trihydrate, and probiotic bacterial strains. 27,28 (D) Ginsenoside Rb1/protopanaxadiol (PPD) nanoparticles (Rb1/PPD NPs) fabrication using precipitation method. 29 (E) Self-assembly of diblock copolymers of PPS and PEG for Rg3 encapsulation and delivery. 9 APTMS, (3-aminopropyl) trimethoxysilane; CK, compound K; DSS, disuccinimidyl suberate; EDC, 1-ethyl-3-(3dimethylamino-propyl)carbodiimide hydrochloride; GNP, gold nanoparticles; MCH, 6-mercapto-1hexanol; MUA, mercaptoundecanoic acid; PVA, polyvinyl alcohol; Sulfo-NHS, N-hydroxysulfosuccinimide. This figure was created using biorender software https://biorender.com/. [Color figure can be viewed at wileyonlinelibrary.com] BOX 1 Experimental and mechanistic steps for producing ginseng nanoparticles from plants, iron oxide ginsenoside nanoparticles, microorganisms, and polymeric ginsenoside nanoparticles Metal nanoparticle synthesis-using ginseng extract For green synthesis, ginseng leaf, berry or root extracts were washed thoroughly, and boiled with relevant solvents or using aqueous solution. Depending on the type of metal nanoparticle synthesis, extract was mixed with (gold III) chloride trihydrate, silver nitrate or zinc sulfate heptahydrate and kept in magnetic stirrer at 70-80°C. The formation of nanoparticles with respect to metals were monitored by the color change in the medium. Later, the filtrates were purified by centrifugation and washed three times. Finally, the nanoparticles formed were collected in the form of pellet ( Figure 3A).

Nanoparticle synthesis-microbial synthesis
The probiotic bacteria such as, Gluconacetobacter liquefaciens kh-1 and Lactobalillus kimchicus DCY51 T were cultured in bacterial growth medium for 24 h. The well grown bacterial biomass were washed twice with 10 mM PBS buffer solution and re-suspended with sterilized dH 2 O, gold salt (gold III chloride trihydrate) and dose-dependent concentration of compound K/ginsenosides. The sample mixture along with bacterial strains were incubated at 37°C with shaker for 24 h. The color (dark purple) changes were observed visually in culturing tubes denoted gold nanoparticle synthesis. The culture mixture was centrifuged at 2500g to remove supernatant has excess unloaded drugs and cultured medium. Further, drug-loaded trapped bacterial strains were sonicated and centrifuged again at 2500g. Lastly, the bacterial strains loaded with nanoparticles were centrifuged further with high-speed centrifugation at 28,000g and washed the pellet with dH 2 O ( Figure 3C).

Nano-formulations-precipitation
A precipitation method to prepare Rb1 NPs and PPD-loaded nanoparticles includes PPD and Rb1 dissolved in dimethyl sulfoxide, is followed by injection into water, stirring for 10 min, and dialysis in normal saline ( Figure 3D).

Copolymer nanoparticles-emulsification
The critical parameters to be considered for synthesizing ginseng nanoparticle are provided in Table 1.
Methoxy polyethylene glycol (mPEG), methacryloyl chloride, and trimethylamine are added to dichloromethane, stirring for 24 h, precipitation using cold diethyl ether. The precipitate, 2, BALUSAMY ET AL. | 1379 transport across blood brain barrier to increase bioavailability. It also addresses biomedical applications of different types of reported ginseng and ginsenoside nanoparticles. We further emphasis on critical parameters, challenges, and prospects for synthesizing ginseng and ginsenosides based nanoparticles.

| Gold nanoparticles (AuNPs)
Gold nanoparticles are widely studied metal nanoparticles in the field of medicine to deliver the drugs to the target sites. The temperature and time dependent extracellular multifunctional gold nanoparticles were 2-azobisisobutyronitrile, and thioacetic acid are mixed and dissolved in tetrahydrofuran (THF), cooled, degassed, stirred at 60°C for 24 h, and precipitated using cold diethyl ether. Precipitate and sodium methylate dissolved in THF with stirring for 30 min at 25°C. Then, poly propylene sulfide (PPS) is added to the mixture, and the solution was maintained at 60°C overnight with constant stirring. The solvent was removed, and the viscous liquid was extracted twice with methanol. Rg3 is dissolved into saline/ethanol and PEG-b-PPS in chloroform. The solution is emulsified by sonication for 3 min, followed by slow addition of saline with polyvinyl alcohol, and emulsification by sonication for 60 s. Solvents of ethanol and chloroform are removed from the final emulsion at 40°C ( Figure 3E).
The different types of nanoparticles synthesis and critical parameters were summarized ( Figure 3 & Table 1).
T A B L E 1 Critical parameters to be considered during ginseng nanoparticle synthesis.

S. No.
Ginseng nanoparticles Critical parameters

1.
Metal nanoparticle synthesis-using ginseng extracts pH, time, temperature, metal concentration, surface charge and so forth.

Fe@Fe3O4 nanoparticle synthesis-microfluidic process
Thermodynamic and kinetic parameters of Fe@Fe3O4 need to be precisely controlled by microfluidic strategy.

3.
Nanoparticle synthesis-microbial Synthesis Bacterial growth condition, culture medium, pH of growth medium before and after addition of metal ion, type and concentration of plant extracts or secondary metabolites, toxic nature of metals and so forth.

4.
Nano-formulations-precipitation For self-assembly of nano-formulations, choosing same backbone structure ginsenosides and optimizing composition of selected ginsenosides are crucial for self-assembly of hydrophobic structures and controlling particle size.

5.
Copolymer nanoparticles-emulsification Selection of hydrophobic and hydrophilic polymers is crucial for efficient synthesis of amphiphilic block copolymer encapsulated ginsenoside. synthesized using aqueous extract of black P. ginseng root (BG-AuNPs) ( Figure 3A). The phytochemicals present in the extract act as a reducing and stabilizing agent for the synthesis of purple color nanoparticles. 45 An unprecedented one-step green synthesis of AuNPs were synthesized in 10 min using red ginseng root extract without any additional special reducing/capping agents. 46 The gold nanoparticles were synthesized within 1 h using the root extract of Korean red ginseng. The presence of phytochemicals and ginsenosides in the root aided in reducing and stabilizing the nanoparticle synthesis. 47 P. ginseng leaves was also used in synthesis of spherical shaped AuNPs within 3 min. 25 The nanoparticle formation is due to the reduction of Au 3+ to Au 0 causing the solution to turn dark purple in 3 min at 80°C. 48 Synthesis of AuNPs using berry extract was also obtained without using hazardous solvents and capping agents. 49 Beside AuNPs, utility of red ginseng was also reported as an efficient and environmentally friendly reducing agent in reduction of graphene oxide (GO) as compared with the reduction by hydrazine. 50 The single ginsenoside Rg3 was conjugated to spherical AuNPs using bifunctional linker of heptaethylene glycol. Hydroxyl group of Rg3 was bound with carboxylic acid end in heptaethylene glycol through the ester bond while the sulfhydryl group in heptaethylene glycol was bound with the gold nanoparticles. 51 The single ginsenosides of compound K (CK) and Rh2 were also utilized by one-pot green chemistry in the oil bath at 80°C to accommodate a synergistic chemical reduction of gold salts. 52

| Silver nanoparticles (AgNPs)
A simple, rapid, inexpensive, and eco-friendly ultra-sonication-assisted silver nanoparticles have been successfully synthesized from roots of P. ginseng. It showed consistent dispersal of nanoparticles in liquid medium, and it was effective in breaking aggregates and enhanced the reaction rates. Ultrasonication for 3 h after addition of silver nitrate to the aqueous ginseng root extract resulted in conversion of solution color from colorless to dark yellow indicating the nanoparticle formation. 53 The green and rapid synthesis of monodispersed silver nanoparticles (BG-AgNPs) synthesized using black ginseng root extract exhibited temperature and time dependent changes. Increase in temperature (80-90°C) showed major absorption peaks at 412 nm indicating that 90°C is the optimum temperature for BG-AgNPs synthesis. 45 The one-pot, simple, and rapid green synthesis of highly stable and colloidal AgNPs has been performed using P. ginseng root extract as a reducing and capping agent by taking different parameters under consideration including time, concentration of precursor, concentration of both reducing and capping agent, and pH. 33 A facile, monodispersed, and stable AgNPs were synthesized using leaves of P. ginseng. The presence of phenolic acids, flavonoids, ginsenosides, and polysaccharides influenced the formation of brown color after 45 min of incubation at 80°C, indicating the formation of AgNPs. 48 Polysaccharides and phenolic compounds present in the P. ginseng berry extract were suggested to be involved in stabilization and functionalization of nanoparticles using green synthesis method. 49

| Zinc nanoparticles (ZnNPs)
The utilization of plant extracts in the synthesis of zinc nanoparticles (ZnNPs) has gained much interest in the pharmaceutical and biomedical field because zinc is used as the preservative in various products including foods, pigments, plastics, and glasswares. 54,55 The extract of red ginseng roots can reduce zinc heptahydrate to produce ZnNPs. The heating temperature at 70°C is optimal to increase activation energy and reduce organic molecules for ZnNPs synthesis. 6 BALUSAMY ET AL.

| Silica nanoparticles (SiNPs)
Interesting structural properties of mesoporous silica nanoparticles (MSiNPs) for drug delivery include stability, large surface area, narrow particle size distribution, and surface silanol groups (Si-OH) for binding with drugs. 56 However, trapping drugs into the inner wall of the nanosized pores in MSiNPs protects the drugs from enzymatic degradation and premature release. 57 CK and Rh2 were loaded in 200 nm MSiNPs (4-nm pore size) with amide bond to enhance their efficacy. Under acidic condition, the amide bond could be hydrolyzed and thereby releasing CK and Rh2. In this method, addition of 3-(aminopropyl)triethyloxysilane (APTEOS) results in activated free amine groups on the surface and pores. 7
The activated NPs cross-linked with the pre-activated ginsenosides by APTMS formed the desired nanomedicine with an excellent coupling effect ( Figure 3B). 10,26 The direct conjugation of ginsenosides CK and Rg3 with superparamagnetic iron oxide nanoparticles (SPIONs) was also a simple, fast, low-cost, high yield, and eco-friendly method. The maximum percentage of ginsenosides attachment to the SPIONs was 5%. 58

| MICROBIAL SYNTHESIS OF GINSENG NANOPARTICLES
The microorganisms can interact with inorganic materials through direct or indirect approaches by various biochemical reactions. Predominantly, some bacterial strains such as, Lactobacillus sp. and Gluconacetobacter sp. can detoxify the metal by-products through oxidation or reduction process and convert metal ions into nanoparticles.
The hydrophobic and large-sized ginseng secondary metabolites such as, Rh2, compound CK, and other ginsenosides were transformed into gold or silver coated nanoparticles by using bacterial strains including Lactobacillus kimchicus DCY51 T and liquefaciens Kh-1 ( Figure 3C). 27,28 However, only few bacterial strains were reported the synthesis of microbial mediated ginseng nanoparticles using silver and gold. Extensive research should be carried out in this area for exploiting the use of microbes for ginseng nanoparticles synthesis and investigating their biomedical applications.
This viable green synthesis approach could be considered as an alternative approach to evade environmental pollution by physical or chemical synthesis. Recently, most of the microbes are abundantly engaged with different metal ions for nanomaterials synthesis including, gold, silver, iron, copper, pallidum, and titanium. 59,60 However, due to the toxicity of inorganic metals, most of them were not considered yet for biological synthesis of nanoparticles. The silver metal ions are well-known for its antibacterial activity, however some of the bacteria such as, into silver nanoparticles through the biochemical modification by their defensive mechanism. 61 Thus, the defense mechanism for their survival toward interaction with metal ions facilitates conversion of metals into nanomaterials through various biochemical reactions, such as oxidation or reduction to detoxify the metal ions into phosphate, carbonate, and sulfide forms. 62 3.1 | Extra-or intracellular mediated ginseng nanoparticle synthesis Microbial synthesis of ginseng nanoparticles is mainly involved microbial extra-or intracellular mediated surface modification through various biomolecules including, protein, enzymes, carbohydrates, and sugars of microbial population ( Figure 4). 28,63 The successful microbial ginseng nanoparticle synthesis was obtained through one-pot synthesis method using lactic acid producing bacterial strains such as, Lactobacillus and Gluconacetobacter strains. 28,63 The extracellular mediated nanoparticle synthesis of inorganic metals (gold or silver) is mostly performed on the bacterial cellular surface or membrane through various biological reduction process such as extracellular enzyme reductase, nitrate reductase or hydroquinone-mediated redox reaction. 59,64,65 Specifically, the extracellular synthesis of gold or silver nanoparticles is mediated through enzymatic reduction process that converts silver ions into nanosized silver materials. The exopolysaccharides on the cellular surface or cell membrane of Lactobacillus kimchicus DCY51 T contribute to a possible biochemical enzymatic process for the reduction of gold metal ions into CK coated gold nanoparticles. 27 However, smaller sized DCY51 T mediated ginseng gold nanoparticles are transported and internalized into the cells through the cytoplasmic membrane.
Meanwhile, microbial synthesized nanoparticles (peptide CopA3 surface conjugated CK loaded gold NPs) were internalized by membrane bounded cell organelles such as endosomes or lysosomes in inflammatory murine RAW 246.7 cells. 28 The intracellular mediated nanoparticle synthesis is influenced by electrostatic interaction of positively charged metal ions with negatively charged cellular surface of bacteria that promotes enzymatical surface modification and helps diffusion of nanoparticles through cell wall by various stepwise mechanisms including, trapping, reduction, capping, and stabilization. 66,67 The physicochemical properties of inorganic metal ions such as size and surface modification play crucial roles to interact with microbial environment for the nanoparticle synthesis through various biochemical reactions including, reduction, aggregation, electrostatic interaction, van der walls, and hydrophobic forces. The secondary metabolites of ginseng have poor permeability and low solubility due to large-sized hydrophobic characteristic resulting in decreased bioavailability and intestinal absorption. 22,68 Whereas microbial mediated synthesis of ginsenoside nanoparticles negotiated well with the cellular membrane. They are internalized into the cytoplasmic organelles that aids structural conversion due to gastric juice and digestive bacterial enzymes through reduction, aggregation, and electrostatic interaction. 69 Furthermore, the bacterial strains such as, Lactobacillus sp. and Pseudomonas sp. gene sequences of suggested bacteria were obtained from the GenBank database (http://blast.ncbi.nlm.nih.gov/ Blast.cgi). The multiple sequence alignments were performed by using the CLUSTAL_X program 74 and the phylogenetic tree were constructed based on 16S rRNA gene sequences using the neighbor-joining method 75 in the MEGA 7.0 program, 76 with bootstrap values based on 1000 replications.

| SYNTHESIS OF GINSENG NANO-FORMULATIONS
Ginseng derived nanoparticles in the form of nanoemulsion were synthesized using P. ginseng root extract that enhanced the efficacy and kinetics by improving their oral bioavailability. 77 The amphiphilic molecules spontaneously self-assembled in an aqueous environment is the core-shell structure nanoparticle that is used for drug delivery system. Ginsenoside Rb1/protopanaxadiol (PPD) nanoparticles (Rb1/PPD NPs) called as "nano-ginseng" were fabricated using precipitation method that is a scalable, simple, and green economy process ( Figure 3D). In this case, two PPD type ginsenosides of PPD and Rb1 self-assemble and form the inner hydrophobic core structure, while the sugar residues of Rb1 form the outer shell to enhance the dispersibility and stability. 29

| SYNTHESIS OF POLYMER-BASED GINSENG NANOPARTICLES
To enhance the drug efficacy and improve the bioavailability, the biodegradable polymers are extensively used.
Encapsulation of drugs with polymeric nanoparticles increases solubility, controls drug release, avoids effect of βglycoproteins, improves drug targeting, and often resulting in improved efficacy while protecting the drugs from premature degradation.

| Serum albumin
Albumin, the most abundant plasma protein (more than half of the human plasma proteins), is stable over a wide range of pH (4-9) and it is also thermally stable while heating up to 10 h at 60°C without deleterious effects. 80 Considering the highest binding rate of albumin proteins and ginsenoside Rh2, albumin nanoparticles are recognized as the prominent nanocarrier produced using desolvation method. 81,82 Several albumin-based drugs and imaging agents are produced and available in the market, while some are undergoing clinical trials for various applications. 83 Ginsenoside CK and Rh2 entraped within bovine serum albumin (BSA) to form BSA-CK/Rh2 nanoparticles using desolvation method, that enhanced their aqueous solubility and stability. 82

| Chitosan
Chitosan has received much attention due to several properties including nontoxicity, biocompatibility, biodegradability, low cost, and abundance. 85 However, due to poor solubility of chitosan, various chitosan derivatives are used extensively for the delivery of poor soluble drugs. The synthesis of ginsenoside CK loaded O-carboxymethyl chitosan nanoparticles (CK-NPs) enhanced solubility, stability, cytotoxicity, and cellular uptake of CK in prostate cancer cells. 86 Similarly, CK-NPs were prepared using amphipathic deoxycholic acid-O carboxymethyl chitosan carrier via combined self-assembly and sonication techniques. It improved the solubility of CK in water and promoted cellular uptake in vitro. 87 To enhance the stability and solubility of CK, an ionic cross-linking between Ca 2+ ions of CaCl 2 and carboxyl groups of O-carboxymethyl chitosan was also used to entrap ginsenoside CK within O-carboxymethyl chitosan nanoparticles. The principal parameters affecting the formation of the nanoparticles are the concentration of polymer and cross-linker. In vitro drug release from the nanoparticles was pH dependent and the drug release rate was consistently higher at lower pH than that of higher pH. 86 This can be due to the protonation of amino groups that causes O-carboxymethyl chitosan swelling in an acidic environment. 88 Furthermore, the hydrophobic ginsenoside CK covalently conjugated to the backbone of hydrophilic glycol chitosan (GC) through the ester bond is hydrolyzed under acidic conditions to enhance the targeted delivery and water solubility. For that purpose, succinic anhydride treatment is needed to prepare CK-COOH, after which the amino group of GC was covalently coupled to CK-COOH in the presence of N-hydroxyl succinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide·hydrochloride (EDC·HCl). 89

| Poly (ethylene glycol) (PEG)
FDA approved PEG as the Generally Regarded as Safe. PEG with nontoxic, hydrophilic, nonionic, and low polydispersity index characteristics is a widely used drug carrier. The succinic anhydride can prepare active carboxyl-terminus of PEG to be used for conjugation with PPD aglycone ginsenoside (aPPD). Thus, the hydrophilic PEG was covalently conjugated to the hydrophobic aPPD through a pH-sensitive ester linkage. It triggers release in acidic conditions (endosomes, lysosomes, and tumor tissues) while reducing cytotoxicity to nontargeted regions. 90 The micelles formed by amphiphilic block copolymers are also potential carriers for the delivery of hydrophobic drugs. 9 PEG with resistance to protein adsorption is usually used as the hydrophilic block. PPS with an extreme hydrophobicity is the hydrophobic block, oxidatively converted into a hydrophile in response to reactive oxygen species (ROS). Since a variety of injuries causes ROS accumulation, designing a ROSresponsive drug release system could improve the drug efficacy at the injury site. Thus, the ROS-responsive nanoparticles (PEG-b-PPS) was generated through the self-assembly of diblock copolymers of PPS and PEG for Rg3 encapsulation and delivery at the ROS-generating sites 9 ( Figure 3E). Similarly, PEG-b-PPS homologous, pluronics F127 could also encapsulate Rg3 to enhance the antioxidant effects and solubility in heart injury model induced by doxorubicin (DOX). 91 Moreover, the mPEG-b-P(Glu-co-Phe) copolymers were self-assembled in aqueous solution and then co-loaded with Rg3. Three components of the copolymer include, (1) PEG involved in nanoparticles protection from the enzymatic damage and prolonged circulation time, (2) glutamic acid units assist in the electrostatic interaction between the nanoparticles and Rg3, and thereby caused a pH sensitive drug delivery system within tumor tissues, (3) phenylalanine units increase the aromatic/hydrophobic interactions within the inner core of the nanoparticles and cellular uptake of nanoparticles. 92

| Hyaluronic acid (HA)
Amphiphilic HA derivative-based nanoparticles are fabricated for cancer therapy and diagnosis. Gelatin and HA nanoparticles were prepared and followed by addition of ginsenoside Rg3 in the electrostatic field preparation system. 93  Moreover, introducing PEGylated lipid (DSPE-PEG) could also form the hydrophilic PEG shell on the outer surface of nanocomplex. Rg3 with hydrophobic structure was encapsulated into the hydrophobic cavity in the nanocomplexes using a solvent evaporation method. 94 Ginsenoside-modified nanostructured lipid carrier loaded with insoluble bioactive curcumin was also prepared by melt emulsification technique, in which water added to the melted lipids was homogenized to provide a uniform suspension. 95 Furthermore, the oral absorption of 25-OCH3-PPD with poor lipophilicity and hydrophilicity could be improved by nanoemulsion loaded with 25-OCH3-PPD-phospholipid complex that was prepared by solvent evaporation. 96

| SYNTHESIS OF GINSENG-BASED MICELLES AND VESICLES
Plant derived extracellular vesicles are proven to have no toxicity to human and can be used to deliver chemoprotective agents and other biomaterials during cancer treatments. 97 A novel nanoparticle like extravesicular was isolated using P. ginseng root extract through density gradient centrifugation using ultrasonification method. 98 Micelles could be formed by ginsenoside Rb1 in aqueous solutions. Self-assembled Rb1 micelles with ultrasmall particle size (<8 nm) were utilized for the ocular diclofenac delivery system. The encapsulation of drug within Rb1 micelles using thin film hydration technique strengthened the drug therapeutic action and reduced the side effects. 42 Solutol HS15 is the nonionic surfactant and tocopherol polyethylene glycol succinate (TPGS) is an FDA certified surfactant of polymer materials. Solutol HS15 and TPGS forming a block copolymer micelle that were applied by thin film dispersion method to prepare self-assembled micelles loaded with ginsenoside Rh2. The prepared micelles enhanced antitumor efficacy and solubility of Rh2. 99 Liposomes are also considered as the useful carriers for the drug delivery. Methyl ether poly(ethylene glycol)poly(lactide-co-glycolide) (mPEG-PLGA) nanoparticles loaded with Panax notoginsenoside were prepared using a water-in-oil-in-water double emulsion solvent evaporation method. These notoginsenoside-loaded core-shell hybrid liposomal vesicles resolved the restricted bioavailability of Panax notoginsenoside and enhanced its protective effects upon oral administration. 8 Cholesterol, the crucial component of liposomes, could be replaced by ginsenosides. A ginsenoside Rh2-based  Figure 6. P. ginseng extracts such as root, leaf, and berry are used in the synthesis of gold, silver, and zinc nanoparticles that showed antioxidant, antibacterial, anticancer and anti-inflammation activity ( Table 2). Ginseng nano formulations are broadly applied in drug delivery, testicular apoptosis, and anticancer activity ( Table 2).
Polymer based ginseng nanoparticles are widely used in the field of antitumor, cytotoxicity, drug delivery, and cerebral function ( Table 2). Bone regeneration is also demonstrated by P. ginseng root extract within nanofibers and gelatin microspheres encapsulated with ginsenoside Rg1 (Table 2).

| Anti-inflammatory
The      using P. ginseng leaves prompted dose-dependent repression of nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW 264.7 cells 5 ( Figure 6).
Comparing the cytotoxicity of microbial synthesized ginseng nanoparticles (GNP-CK-CopA3) in different cell lines including inflammatory murine RAW 246.7 cells, human dermal fibroblast cell lines NHDF, and HaCaTs, showed less to no cytotoxicity at 50 µg/mL. 28 Similarly, the microbial synthesized ginseng nanoparticles DCY51 T -AuCK NPs showed less or no toxicity toward murine RAW 246.7 cells at different concentrations. 108 The inflammatory response of murine RAW 246.7 cells is elevated by ROS from intracellular organs caused by cellular modification or other external factors. However, the localization of synthesized ginseng nanoparticles in cytoplasmic organelles including lysosome or mitochondria reduced the effect of LPS mediated ROS elevation. 28,108 Thus, ROS reduction was proved in murine RAW 246.7 cells following treatment with microbial synthesized ginseng nanoparticles.
The production of ROS, inducible nitric oxide synthase, and nitric oxide were significantly reduced by SPIONs conjugated with ginsenosides CK and Rg3 in LPS-activated RAW 264.7 murine macrophage cells in a dosedependent manner. 58 Thus, these nanoparticles are non-cytotoxic to normal cells and can be used as a ginsenosides carrier for intracellular release in inflammatory diseases.
The higher anti-inflammatory efficacy of MSiNPs-CK and MSiNPs-Rh2 were shown in RAW264.7 cell lines as compared to ginsenosides CK and Rh2. Meanwhile, the superior biocompatibility in normal cell lines (HaCaT skin cells) and anticancer effect on A549 lung cancer, HepG2 liver carcinoma, and HT-29 colon cancer cell lines were shown at 10 μM concentration. 7 Improved stability and solubility of BSA-Rh2 nanoparticles significantly enhanced the therapeutic behavior of Rh2. 82 Therefore, BSA-Rh2 nanoparticles can be potentially used as a delivery vehicle for ginsenoside in inflammatory and cancer cell lines.

| Antitumor
The green synthesized AuNPs and AgNPs exhibited significant cytotoxicity to cancer cell lines including A549 lung cancer and B16B15 melanoma cell lines while no cytotoxicity to HaCaT skin and 3T3-L1 pre-adipocytes cells. 5  The Rb1/PPD nanoparticles exhibited high drug loading efficiency and capacity, appropriate size (~110 nm), long half-time in systemic circulation (nine-fold longer than free PPD), and higher accumulation at the tumor site.
Thus, better antitumor efficacy in vitro and in vivo and reduced damage to normal tissues were demonstrated. 29 Ginsenoside Rb1 self-assembled with anticancer drugs produced stable nanoparticles with tumor inhibition and fewer side effects than that of free drugs. 130 The encapsulation of ginsenoside Rg3 using PLGA also promoted its antitumor activity. 78 Ginsenoside GS25 encapsulated into PEG-PLGA nanoparticles for GS25 oral delivery improved anticancer efficacy, molecular targeting, and oral bioavailability. 79 Greater in vitro therapeutic efficacy was found by bovine serum albumin-CK (BSA-CK) nanoparticles in HaCaT skin, HT29 colon cancer, HepG2 liver carcinoma, and A549 lung cancer cell lines in comparison with CK. 109 HSA, BSA, and bovine serum were used to reduce the cytotoxicity of Rh2 in HepG2 cells. HSA was suggested to enhance Rh2 water solubility, and thus it can be used as nanoparticles in Rh2 delivery process. 81  with similar genotype to HT29 can be improved by this strategy. 95 Ginsenoside Rh2-mixed micelles also increased the solubility of ginsenoside Rh2 up to 150-folds compared to free Rh2, hence, improving the antitumor efficacy. 99 Rh2 liposome also prolonged the blood circulation and stabilized the structure of liposomes, while the paclitaxel-loaded Rh2 liposome realized the efficient tumor growth suppression. 118 Graphene based water-soluble nanosheets including graphene oxide (GO) sheets are efficient drug delivery system. 131 Beside water dispersibility, GO sheet shows good biocompatibility, and it is also capable of targeted drug release in the acidic tumor microenvironment. GO itself disruptes glutathione biosynthesis and induces ROS accumulation in human cells. However, GO conjugated with the antioxidant ginsenoside Rg3, prior to loading with chemotherapy drug DOX, significantly reduced the toxicity of the GO carrier by abolishing ROS production. 132

| Antimicrobial
Some of the known mechanisms involved in the antibacterial activity of nanomaterials are: (1) direct physical interaction of extremely sharp edges of nanomaterials such as graphene oxide nanowalls with cell membrane, 133,134 (2) ROS generation by nanomaterials for example by rGO under visible light, 135 and even by ZnO under dark condition, 136 (3) wrapping bacteria by nanomaterials such as graphene nanosheets, 137 (4) oxidative stress, 138 (5) reduction of nanomaterials such as graphene oxide to bactericidal graphene through the glycolysis process in the bacteria, 139 (6) DNA damage, 140 (7) ion release such as zinc from ZnO/GO composites, 141 and (8)  from the respiratory chain of the bacteria by rGO, and transferring the trapped electrons into the O2 nanobubbles for ROS generation. 142 The silver nanoparticles using 4-year-old fresh root extract of P. ginseng showed antimicrobial effect against pathogenic microorganisms 48 ( Figure 6). The red ginseng root extract synthesized AgNPs exhibited antimicrobial activity against pathogenic microorganisms including Vibrio parahaemolyticus, Staphylococcus aureus, Bacillus cereus, and Candida albicans, while they exhibited biofilm degrading activity against S. aureus and Pseudomonas aeruginosa. 48 BG-AgNPs showed significant antibacterial activity against Escherichia coli and S. aureus. 45 Quasi spherical AgNPs using aqueous extract from P. ginseng roots showed virucidal against the influenza A virus. 53 The main mechanisms behind the antibacterial effect of ginseng extract synthesized NPs are explained as the biofilm inhibition activity, 48 free radical-scavenging activity, 45 and loss of permeability due to the structural changes in the cell membrane. 143 Ginseng displayed antiviral effects by modulating both acquired and natural immunity. It is suggested as the potential therapeutic agent preventing SARS-CoV-2 infection along with the vaccine. 144 PEGylated nanoparticle albumin-bound-steroidal ginsenosides could treat symptoms such as coagulation, and cytokine storm that are associated with severe SARS-CoV-2 patients. 145

| Detection
A simple, rapid, and miniatured portable system for the detection of Hg 2+ in samples was designed using P. ginseng dried root powder. 33 The ginsenosides cross-linked with Fe@Fe3O4 nanoparticles are developed as the nanomedicine, enhanced magnetic resonance imaging, and auto-targeting in liver cancer therapy 10 ( Figure 6).
The PLGA-Rg3 nanoparticles were also offered as the theranostic material for encapsulating natural nutraceuticals for the treatment and detection of Alzheimer's disease. 18

| Reproductive function
Testicular toxicity of methotrexate (MTX) is a clinically important adverse effect. Ginseng and ginseng nanoparticles alleviate MTX-induced testicular toxicity in rats, possibly by the inhibition of MTX-induced testicular apoptosis. The protective effect of ginseng nanoparticles showed better effect compared to the ginseng extract treatment. 77 Ginsenosides could also increase the reproductive function on the hypothalamus pituitary-testis axis when the P.
ginseng formulated into the form of nanoparticles 112 ( Figure 6). The adjuvant ginsenoside-based nanoparticles (ginsomes) could also promote subunit vaccine to induce a strong immune response and protective effects. 117

| Cerebral function
Rg3-loaded PLGA nanoparticles showed improved biocompatibility, versatility, enhanced ability to cross bloodbrain barrier, decreased Aβ plaques, ROS generation, reduced mitochondrial dysfunction and thus alleviates Alzheimer disease progression 18 ( Figure 6). It was demonstrated that Rg1 nanoparticles could penetrate to brain tissue, stimulate neuronal recovery, and reduce the volume of cerebral infarction. 110,146 Furthermore, neural stem cells and their neural differentiation on graphene, and graphene-based neuronal tissue engineering can promisingly realize the regenerative therapy of various incurable neurological diseases/disorders and the fabrication of neuronal networks. 147

| Myocardial infarction
Cardiac function was improved by intramyocardial injection of Rg3-loaded PEG-b-PPS nanoparticles while reducing the infarct size ( Figure 6). Rg3 targets Forkhead box O3 (FoxO3a) protein that has anti-inflammatory, anti-fibrotic, and antioxidative functions. 9 Rg3 encapsulated pluronics F127 also enhanced the antioxidant effects in doxorubicin-induced heart injury model. 91 7.8 | Antioxidant activity P. ginseng leaves-mediated gold NPs exhibited antioxidant activity in a dose-dependent manner. 25 Ginseng berry AgNPs showed the highest free radical scavenging activity compared to ginseng berry AuNPs and ginseng berry extract. 49 The increased antioxidant activity of ginseng berry AgNPs could be correlated to the adsorption of bioactive compounds of extract over large surface area of spherical nanoparticles. Furthermore, the interaction of metal ions with plant metabolites within NP synthesis might result in improved free radical scavenging activity.
The increased free radical-scavenging activity of metal nanoparticles was attributed to the antioxidant activity of BG root extract. AuNPs and AgNPs biosynthesized using black ginseng root extract show promising prospect for the development of novel and biologically synthesized antioxidant agents.

| GINSENOSIDE BASED NANOPARTICLES DELIVERY
Most of the ginsenosides including Rh2, Rg3, CK, and Rg1 are the substrates for P-glycoprotein (P-gp) efflux transporters causing poor ginsenosides bioavailability. [17][18][19][20] Even, two ginsenoside isomers such as 20(S)-Rh2 and 20(R)-Rh2 exhibited significant difference in their cellular uptake due to the affinity and recognition difference between the two stereoisomers by efflux ABC transporters. 20 Nano-sized ginsenoside delivery system improved ginsenoside efficacy by penetrating to the cell to reach specific target sites and thereby increasing bioavailability.
The list of transporters and transport mechanism of ginsenoside and ginsenoside nanoparticles were summarized in Figure 7. The use of novel multifunctional liposome delivery system successfully delivered ginsenosides (Rg5, Rg3, Rh2) through GLUT carrier-mediated endocytosis into gastric tumor sites. Ginsenoside Rg5 liposomes were transported through GLUT5 or GLUT2, ginsenoside Rg3 liposomes were mainly transported through GLUT1 and SGLT1, and Rh2 liposomes entered the cell mainly through GLUT1 transporters. 100 Angiopep-2, kunitz domain-derived peptide from protease enzyme, is the specific ligand for low-density lipoprotein receptor-related protein-1 (LRP-1). Considering the overexpression of LRP-1 on glioblastoma or glioma cells and blood-brain barrier (BBB), angiopep-2 could be used for targeting brain. 148 Angiopep-2 eased the transport PLGA-Rg3 nanoparticles across the BBB from apical (blood/lumen) to basolateral side (brain) via a receptor mediated transcytosis. 18 Similarly, angiopep-2 functionalized ginsenoside Rg3 loaded nanoparticles crossed the BBB, enhanced the cellular uptake of nanoparticles, showed sustained drug release, and inhibited the C6 glioma cells proliferation dose-dependently. 113 On the other hand, development of poly-γ-glutamic acid-based nanoparticles loaded with Rg1 could successfully penetrate BBB via receptor mediated endocytosis and it could be a therapeutic agent for cerebral infarction treatment. 110   Abbreviations: mPEG-b-P(Glu-co-Phe), PEG-block-poly (L-glutamic acid-co-L-phenylalanine); NPs, nanoparticles; PEG, polyethylene glycol; PLGA, poly L-lactic-co-glycolic acid.
accumulation on tumor sites, targeted delivery in cancer cells and reduced side effect in healthy cells. 111 Overall, these ginseng nanoparticles are proposed as the promising carriers for drug delivery to the brain and tumor sites (Table 3).
9 | CRITICAL PARAMETERS 9.1 | Size and time Selection of organs for nanoparticle synthesis greatly affect the synthesis reaction time, nanoparticle size and it needs to be considered as one of the critical parameters when synthesizing ginseng nanoparticles using green methods. The size of various types of nanoparticles were summarized in Figure 8. So far, roots, leaves, and berries of P. ginseng have been extensively used for green synthesis. Fresh leaves mediated green synthesis of AgNPs is rapid, facile, stable, and costeffective. Furthermore, it enhanced the pharmacological effect of nanoparticles probably due to the smaller size (10-20 nm) of synthesized nanoparticles compared to the nanoparticles synthesized using P. ginseng root (100 nm). 48 Compared to the fresh root and red ginseng powder, P. ginseng fresh leaves initiated rapid synthesis (3 min) of AuNPs and AgNPs (45 min). 48 Like leaves, fresh berry extract of P. ginseng synthesized smaller sized AuNPs (5-10 nm) and AgNPs (10-20 nm). The AuNPs and AgNPs synthesis using fresh berry extract were completed in 270 min and 24 h, respectively indicating that fresh samples initiate rapid synthesis compared to dried or root samples. 49 Therefore, use of fresh berry and leaves contribute to rapid, smaller sized nanoparticle synthesis and improved biomedical applications.

| Temperature
Temperature is considered as one of the important critical parameters when synthesizing nanoparticles using P.
ginseng extracts. The synthesis of AuNPs and AgNPs using black P. ginseng root extract at 90°C significantly reduced the reaction time and the size of nanoparticles. 49 When the AuNPs and AgNPs were heated up to 700°C, there is complete degradation of organic compounds present in the P. ginseng leaves. 5 Certain ginsenosides are liable to changes in low pH and high temperature conditions. For instance, CK heated at 90°C for 3 h at pH 3 showed different bands of CK indicating the possibility of degradation at high temperature and low pH (3)(4)(5). 52,149 Therefore, optimum temperature for efficient synthesis of nanoparticles needs to be selected without affecting the pharmacological activity that is crucial for maintaining the bio-application.

| pH
The pH of nanoparticle is crucial to maintain stability, arrest aggregation of nanoparticles, and considered to be a significant factor when synthesizing metal nanoparticles. During the synthesis of silver and gold nanoparticles using P. ginseng fresh root, no shift in absorbance was observed even when a broad range of pH (3)(4)(5)(6)(7)(8)(9)(10)(11)(12) was used denoting the stability and possible application in drug delivery. 47 One step green synthesis of AuNPs synthesized using Korean red ginseng roots also showed sustainable stability at pH 2-10 indicating phytochemicals coated well onto the nanoparticle surface and thus prevents the aggregation of nanoparticles. 46 Similarly, the use of P. ginseng leaves did not alter the stability of AuNPs and AgNPs at pH 3-12 even after a month of storage denoting the leaf extract itself acts as the reducing and stabilizing agents. 48 Meanwhile, a green synthesis of AgNPs using dried roots of P.
ginseng showed pH dependent shift in nanoparticle formation. Alkaline pH (8)(9)(10)(11)(12) was more favorable than acidic pH (pH 6) in the formation of AgNPs using P. ginseng root. 33 Thus, use of fresh organs provided favorable environment to maintain stability of nanoparticles.

| Surface charge
The zeta potential of nanoparticles indicating their surface charge that is a critical factor determining the stability of nanoparticles dispersion. The different nanoparticles' zeta potential was summarized in Figure 8.
Nanoparticles are stable when the absolute value of zeta potential is larger. The zeta potential of the bovine F I G U R E 8 Size (nm) and surface charge (mV) of nanoparticles using different fabrication methods. Heatmap of size (nm) and surface charge (mV) of ginseng nanoparticles were constructed in Microsoft excel using conditional formatting option. serum albumin-compound K (BSA-CK) nanoparticles was −70.80 mV while it was highly stable. 109 The electrostatic repulsive force of the negatively charged nanoparticles prevents the nanoparticles from agglomeration and it could also give high stability to the colloidal solution. 58,109 It is also suggested that the interparticle interactions might partially contribute to the charge of Rb1/betulinic acid, Rb1/dihydroartemisinin, and Rb1/hydroxycamptothecine nanoparticles to be easily dispersed and resuspended after a period of sedimentation. 130 The zeta potential of AuNPs-HEG-Rg3 was -4.12 mV while AuNPs itself had zeta potential of -18.25 mV. 51 Thus, it is suggested the contribution of HEG linker to the surface charge of final product by formation of steric layers, and thereby reducing the opsonization and aggregation of particles. However, the zeta potential of chitosan nanoparticles loaded with ginsenoside CK was roughly same as chitosan nanoparticles, demonstrating no significant difference of surface charge by encapsulation. 87 Meanwhile, due to the presence of many COO-groups in Ocarboxymethyl chitosan, the zeta potential of the O-carboxymethyl chitosan nanoparticles was -14.6. After ginsenoside CK loading, nanoparticles showed stable dispersibility and the zeta potential was -29.6, that followed by a unimodal and concentric distribution. 86 The folic acid modification of Rg5-BSA nanoparticles also increased their negative surface charge from −14.9 to −22.5 mV, because after this modification the free amino groups are reduced. 111 The negatively charged nanoparticles are generally internalized by clustering, then through the nonspecific binding with plasma membrane cationic sites, and their subsequent endocytosis. 150 The leaf extract AuNPs and AgNPs showed negative surface charge of -16.0 and -19.3 mV resulting in relative colloidal stability. 5 However, due to the positive surface charges on peptide-capped gold nanoparticles, they showed more internalization to the cells and prolonged intracellular retention in comparison with citrate-capped gold nanoparticles. 151 It was shown that metal ions interacting with plant metabolites within formation of nanoparticles might improve the free radical scavenging activity of nanoparticles. Moreover, positively or neutrally charged AgNPs electrostatically attracting negatively charged phytochemicals act synergistically to improve the bioactivity of plants. 49 It was demonstrated that the negative charge of Rg3 nanoparticles (-28.5 ± 2.5 mV) and angiopep-2 functionalized ginsenoside Rg3 loaded nanoparticles (-14.6 ± 3.2 mV) could prevent the plasma protein dilution and prolong the circulation time. While it could provide a better interaction with the cell membrane in vivo through the electrostatic repulsion. 152 Similarly, Rb1 and Rb1/PPD nanoparticles have negative surfaces charges due to the attached sugar residues on C-3 and C-20 of the PPD aglycone. While it could positively affect their interaction with the cancer cells. 29 The zeta potential of nanoparticles increases when the nanoparticles enter an acidic tumor environment. It could affect the electrostatic interaction between the drugs such as Rg3 and Rg5 and nanoparticles, decreasing the stability of the nanoparticles, and thus resulting in drug release within tumor tissues. 92,111 Thus, the surface charge is a critical parameter affecting the stable dispersibility, circulation time, cellular internalization, and drug release of nanoparticles.

| PROSPECTS
In view of high demand for ginseng metabolites, and abundant distribution of Panax species around the world, further research should focus on other Panax species to unravel the exciting facts about the variation of age, organs, species specific nanoparticle synthesized for treating various diseases. This will further deepen the knowledge about the application of various types of Panax species in the nanomedicine era. Certain ginsenosides are heat unstable at high temperatures of synthesis and therefore it will be interesting to understand whether ginsenosides or other phytochemicals are responsible for the observed pharmacological effects. Further, it will be interesting to elucidate whether ginseng nanoparticle's cellular uptake reciprocate biomedical applications.
Compared to the dried extracts, fresh extracts provide efficient nanoparticle synthesis. The reason for the efficient green synthesis using fresh ginseng plant extracts should be addressed in the future. Even though metal nanoparticles synthesis using ginseng extracts is best, stable, and reliable method with least toxic effects in preliminary studies, there is a lack of sufficient evidence about their long-time exposure to the cells when treating health ailments. Therefore, more clinical studies are needed to investigate and optimize the efficiency of these nanoparticles in vivo. This can be addressed by exploring their toxicity, monitoring absorption, distribution, metabolism, and excretion, immunological response, and blood cell parameters of healthy subjects. Addition to that, bioavailability, and biocompatibility of ginsenoside nanoparticles are at the budding stage and therefore the involvement of respective ion channels and receptors for the delivery of ginsenosides to the target sites should be investigated in the future.
Moreover, inevitability and surplus successful microbial green synthesis approach yet to be discovered by utilizing various microbes as well as inorganic metal ions. Simultaneously, the precise mechanism of microbial synthesis nanoparticles is yet to be elucidated due to their distinct characteristic of microbial organism and physicochemical properties of metal ions including shape, size, surface modification and other environmental factors such as, temperature, pH, and pressure. Microbial mediated ginsenoside nanoparticle synthesis is emerging and therefore there is lack of sufficient reports on both intra-and extracellular synthesis of ginseng nanoparticles and their biomedical applications. It will be fascinating to explore the microbial mediated ginseng nanoparticle synthesis in the future.
Polymer-based ginsenoside nanocarriers have several beneficial characteristics including non-toxicity, biocompatibility, biodegradability, and cost-effective. Besides that, the possibility of engineering the polymerbased nanocarriers for targeting ability needs to be further investigated at in vitro and in vivo levels to achieve tissue-specific drug release, noninvasive systematic delivery, and ascertain noncytotoxicity toward any noncarcinoma cells. Furthermore, molecular docking studies can be used to elucidate the interaction between polymers and ginsenosides to ultimately elucidate their interaction sites and provide the optimal efficiency for ginsenoside nanocarriers.
Considering the facts about the high toxicity of anticancer drugs that seriously harm the organs, investigation of ginsenosides nanoparticles as an effective and biocompatible anticancer drugs or anticancer adjuvants holds good prospect. Selection of appropriate inorganic nanocarriers for the delivery of ginsenosides would be a remarkable step to deliver controlled release, high drug loading capacity, targeted delivery, photoimaging by incorporating fluorescence dye, and bio clearance of carrier thereby achieving enhanced efficacy of ginsenosides.
Some applications of ginseng nanoparticles are limited including dental application, corneal and reproductive function ( Figure 4, Table 2). Similarly, microbial based nanoparticle synthesis, nano emulsion, micelles, liposomes, nanotubes, microfluidic, and graphene-based ginseng nanoparticles are budding tools and need to be explored in depth to study their various biomedical applications.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

AUTHOR BIOGRAPHIES
Sri Renukadevi Balusamy, PhD is an Assistant Professor in Food Science and Biotechnology at the Sejong University, Korea. She is expertise in molecular biology, genetic engineering, nanomaterials synthesis as well as their application in plants and human cell lines. Currently, her research majorly focusses on identifying novel plant bioactive compounds and synthesizing novel nanomaterials to treat human chronic diseases including cancer, inflammation, and obesity using omics, and molecular biology.