2.2.1. Polymeric nanoparticles

Polymeric nanoparticles (PNPs, Fig. 3) are the most widespread delivery systems. These include nanospheres and nanocapsules. Nanospheres are matrix systems in which the drug is homogeneously distributed, while in nanocapsules, the drug is contained in a core, which is walled in by an outer shell. Polymers used for the preparation of PNPs are classified as synthetic or natural (i.e., biopolymers), and are commonly classified as biodegradable or non-biodegradable as well. Biodegradation of a polymer involves the cleavage of hydrolytic or enzymatic bonds in the polymer, leading to polymer erosion. Most naturally occurring polymers undergo enzymatic degradation and are thus the first biodegradable biomaterials used in pharmaceutical technology. Natural polymers include proteins (e.g., albumin, gelatin, soy protein hydrolysate, and casein) and polysaccharides (pectin, cellulose, starch, gum arabic, carrageenan, alginate, xanthan gum, gellan gum, and chitosan) [15].

Roy et al. [16] developed PLGA PNPs loaded with AG and coated with chitosan for antitumor therapy. The PNPs were nontoxic and increased the anticancer activity by threefold in the MCF-7 cell line and in vivo studies using Ehrlich ascites carcinoma (EAC) cells, in comparison with unformulated AG. The anticancer activity of AG loaded in other PNPs was tested for specific targets. Kim et al. [17] formulated water-soluble PNPs for systemic administration, using a polymer obtained from phenylboronic acid. This nanocarrier displayed exceptional targeting properties both in vitro and in vivo, and resulted in a significant decrease of in vivo tumor growth. Roy et al. [18] prepared AG loaded in PLGA PNPs (50:50), which were stabilized with polyvinyl alcohol (PVA). The study focused on AG delivery into macrophage cells infected with leishmanial parasite. The PNP average size was 173 nm, with a surface charge of −34.8 mV. Activity on the infested macrophages was significant in the case of PNPs containing 4% PVA (IC50 of 34 μmol·L−1), which is a one-quarter dose of pure AG (IC50 of 160 μmol·L−1). The same authors recently prepared PNPs with hepatoprotective activity for use in hepatotoxic conditions. Cationic modified PLGA PNPs loaded with AG were developed, and showed superior dissolution behavior in comparison with pure AG and favorable cytokine regulation in the hepatic tissues, leading to a rapid recovery of mouse liver [19]. Moreover, in 2017, Das et al. [20] explored the effectiveness of AG encapsulated in PLGA PNPs against liver damage in mice induced by arsenic. The PNPs had an average diameter of 65.8 nm and the EE was 64%. The NPs increased the level of reduced glutathione and antioxidant enzymes, including superoxide dismutase and catalase. The protective efficiency of AG loaded in PNPs was about five times greater in comparison with unformulated AG. NP administration improved the liver tissue architecture, suggesting a beneficial effect against arsenic-induced liver toxicity. AG has also been reported to ameliorate neurodegenerative disorders. To overcome its low brain distribution, Guccione et al. [10] loaded AG into albumin NPs (ANPs) and ethylcyanoacrylate NPs (ENPs). The ability of both NPs to permeate the BBB was investigated using an in vitro BBB model, hCMEC/D3. Although AG was unable to cross the BBB model to any significant degree, the ANPs improved the permeation of AG by twofold. In addition, the integrity of the cell layer was maintained. In contrast, while ENPs increased the cross properties of AG, the BBB was temporarily disordered. Another nanovector that was tested involved pH-sensitive PNPs based on a cationic poly methacrylate copolymer (Eudragit® EPO). The optimized formulation consisted of Pluronic® F-68 (0.6%, w/v) and Eudragit® EPO (0.45%, w/v). It had a very high EE (93.8% ± 0.67%), homogeneous particle size ((255 ± 9) nm), and good superficial charge values ((29.3 ± 3.4) mV). The in vivo absorption of AG and of AG loaded in the NPs was studied at a dose of 10 mg·kg−1 in male Wistar albinorats. The AG loaded in PNPs caused a tremendous increase in area under the curve (AUC0–∞) (ca. 2.2 fold) and the maximum concentration (Cmax) (3.2 fold) in comparison with unformulated AG. In addition, the relative bioavailability increased by 121.53% (P < 0.05) in comparison with pure AG. Other parameters were favorably affected by the loading of AG in NPs: A smaller amount of time that AG is present at the maximum concentration in serum (Tmax) (4.0 fold) and a reduction in Cl/F (2.2 fold) were observed [21].

2.2.2. Polymeric micelles

Polymeric micelles (Fig. 3) are self-assembling colloids of amphiphilic polymers, which can spontaneously aggregate in a particular solvent (generally water). AG was entrapped in a micellar formulation based on an amphiphilic triblock copolymer of D,L-lactic acid, glycolic acid, and ethylene glycol(poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PLGA-PEG-PLGA)), and then evaluated for bioavailability in vivo and for in vitrocytotoxicity. Cellular uptake and cytotoxicity, including cell cycle arrest, proliferation inhibition, and pro-apoptosis effects were tested against human breast cancer MAD-MD-231 cells. The loading efficiency of the micelles was about 92%, and the particle size was (124.3 ± 6.4) nm. The micelles exhibited a higher inhibition of proliferation when compared with the unformulated AG. The highest effectiveness of cellular uptake and intracellular transport, pro-apoptotic properties, and cell cycle arrest at the G2/M phase were found in MAD-MD-231 cells using the micelles. Pharmacokinetics studies were carried out in rats, and both the mean residence time and plasma AUC0–∞ were found to increase by almost threefold in comparison with unformulated AG [22].

In a further study, a series of copolymers obtained from methoxy poly(ethylene glycol)-poly(D,L-lactic acid) (mPEG-PLA) with various ratios of hydrophilic to hydrophobic portions was synthetized to encapsulate AG. The micelles had a size of (92.84 ± 5.63) nm, a high EE of 91.00% ± 11.53%, and a loading capacity of 32.14% ± 3.02% (w/w). mPEG-PLA loaded with AG was found to have good stability against salt dissociation, protein adsorption, and anion substitution. The solubility of AG and a derivative of AG (14-deoxy-11,12-didehydroandrographolide) in micelles increased by 4.51 times and 2.12 times in water in comparison with unformulated AG. mPEG-PLA loaded with AG showed the same release profile in a different dissolution medium. Cytotoxicity testing in vitro demonstrated that AG loaded in mPEG-PLA exhibited higher cell viability inhibition in mouse breast cancer 4T1 than free AG [23].

2.2.3. Vesicles

Vesicles (Fig. 3) are colloidal vectors formed by bilayers, which can load hydrophilic and hydrophobic compounds. Liposomes are principally constituted of natural phospholipids and cholesterol, while niosomes are nonionic surfactant-based vesicles [24].

A very recent study by Kang et al. [25] reported on liposomal co-delivery of AG and doxorubicin to inhibit breast cancer growth and metastasis. The liposome was prepared with a cell-penetrating peptide, which was able to inhibit the in vitro proliferation of 4T1 cells. Two distinctive tests were done: a wound-healing assay and a trans well invasion assay. The liposome was found to enhance AG accumulation in tumors and to result in high intra-tumor penetration in a tumor mouse model of breast. A synergistic effect of doxorubicin and AG was found. In another study, AG-loaded niosomes were prepared. The niosomes improved the tissue distribution and bioavailability of AG in mice. In particular, AG-loaded niosomes accumulated in the liver much more than the free drug. In vitro studies on anti-hepatocellular carcinoma (HCC) efficacy in HepG2 cells disclosed no significant differences between the free drug and AG-loaded niosomes [26]. In a further study, loading AG in a natural soya-phosphatidylcholine mixture was found to enhance the absorption and hepatoprotective activity of AG in comparison with unformulated AG. The study proved the hepatoprotective potential of AG using a rat model of hepatotoxicityinduced by carbon tetrachloride. The results showed significantly increased absorption, bioavailability, and hepatoprotective potential of AG loaded in vesicles when compared with the unformulated drug. The effects of AG-loaded vesicles were comparable to those of silymarin, which is used as a standard drug [27]. Maiti et al. [28] obtained similar results in a study using the same formulation in rats. The formulation had an improved bioavailability and a better hepatoprotective activity than unformulated AG. The formulation was very helpful in solving the problem of rapid clearance and low elimination caused by the short half-life of AG. In an additional study, Sinha et al. [29]reported the capacity of AG encapsulated in liposome and decorated with mannosyl or fucosyl (as active targeting for macrophages) to reduce hepatic and renal toxicity. Furthermore, a decrease was observed in the parasitic burden of experimental leishmaniosis in a hamster model and in the splenic tissue histological architecture when compared with treatment with unformulated AG or conventional AG-loaded liposomes. In a further study, Li et al. [30] developed AG-loaded liposomal dry powder inhalers (LDPIs) for pulmonary delivery for the treatment of pneumonia induced by Staphylococcus (S.) aureus. The AG-loaded liposomes were freeze-dried to formulate LDPIs, and were found to be suitable for pulmonary delivery. A stronger in vivo anti-S. aureus pneumonic effect of the formulation was found at a tenfold dose, in comparison with unformulated AG or penicillin. The LDPIs significantly reduced the tumor necrosis factor α (TNF-α) and interleukin (IL)-1 pro-inflammatory cytokines. Phosphorylation of IκB-α in the nuclear factor-κB pathway was also inhibited to an extraordinary degree.

Piazzini et al. [31] developed liposomes for the central nervous system (CNS) delivery of AG by adding Tween 80 alone or in combination with didecyldimethylammonium bromide in order to modify the surface of the vesicles. The ability of liposomes to increase the permeability of AG was evaluated by a parallel artificial membrane permeability assay (PAMPA) and hCMEC/D3 cells. The size of the liposomes ranged from (96.4 ± 9.5) to (82.1 ± 9.3) nm, and the EE ranged from 44.7% ± 3.2% to 47.5% ± 3.3%. The liposomes showed excellent stability as suspensions or freeze-dried products. PAMPA and hCMEC/D3 transport studies revealed that all the developed liposomes increased the permeability of AG, in comparison with free AG. No alterations in cell viability were found. The main uptake mechanism was caveolae-mediated endocytosis, and the increased cellular internalization of the formulations was related to the positive charge [31].

2.2.4. Solid lipid nanoparticles

SLNs (Fig. 3) are formulated with physiological lipids, which are dispersed in water or in an aqueous surfactant solution. SLNs are typically spherical, with an average diameter between 10 and 1000 nm. The solid lipid core is stabilized by surfactants (emulsifiers) and formulated using fatty acids, monoglycerides, diglycerides, triglycerides, steroids, and waxes. All classes of emulsifiers can be used to stabilize the lipid dispersion, thus preventing particle agglomeration more efficiently [15].

AG encapsulated into SLNs was found to enhance antitumor activity in Balb/c mice because of an enhancement of bioavailability due to the improvement of the AUC0–∞ and Cmax of AG in comparison with unformulated AG. A sustained-release pattern of AG-loaded SLNs was indicated by an increased value of Tmax[32].

AG-loaded SLNs with an average diameter of 286.1 nm and a zeta potential of −20.8 mV were developed to improve AG bioavailability. The AG EE was 91.00%, while the drug loading was 3.49%. Both the bioavailability and anti-hyperlipidemic efficacy of AG-loaded SLNs were improved by increasing the stability and solubility of AG in the gastro-enteric tract. The bioavailability of AG loaded in SLN was increased to 241% in comparison with unformulated AG [33].

AG-loaded SLNs were formulated using Compritol 888 ATO and Brij 78. The EE was 92%. The SLNs showed exceptional physical and chemical stability at 4 and 25 °C after storage for one month. The SLNs were also stable when dispersed in human serum albumin and plasma. The in vitro release of AG-loaded SLNs at physiological pH was prolonged and sustained. The SLNs’ ability to cross the BBB was evaluated in vitro by the PAMPA test and hCMEC/D3 cells in order to predict the passive transcellular permeability. The SLNs improved the permeability of AG in comparison with free AG, and the data from the two tests were comparable. Intravenously administered fluorescent SLNs were detected in brain parenchyma outside of the vessel, thus establishing their ability to cross the BBB [34].

2.2.5. Nanoemulsions and microemulsions

Both NEs and MEs (Fig. 3) are nanoscale emulsions characterized by high stability and transparency. However, the terms “ME” and “NE” are not interchangeable; an ME is an isotropic liquid mixture that forms spontaneously and is thermodynamically stable, whereas an NE is a nanoscale dispersion that is obtained by means of mechanical force and is only kinetically stable. The two phases of NEs and MEs may be water-continuous or oil-continuous. In addition, MEs can present bicontinuous systems (also called sponge-like systems) [15], [35]. An AG-loaded NE was formulated in order to improve oral bioavailability and protective action against inflammatory bowel disease. The AG-loaded NE was prepared with water, ethanol, α-tocopherol, and Cremophor EL. The optimized AG-loaded NE was stable at 4 and 25 °C for three months, and had a droplet size of (122 ± 11) nm and a viscosity of 28 centipoise. An ex vivo test using an everted rat gut sac indicated that the jejunum was the optimal site for AG loaded in the NE. AG activity was 8.21 and 1.40 times higher than the values obtained with AG suspension and AG ethanol solution, respectively. The pharmacokinetic results showed a relative bioavailability of 594.3% when the AG-loaded NE was compared with an AG suspension. In addition, the ulcer index and histological damage score of indomethacin-induced intestinal lesions in mice were significantly reduced by pretreatment with AG-loaded NE [36].

An oil-in-water (O/W) ME loaded with AG was developed using isopropyl myristate, Tween 80, ethanol, and water. The mean droplet size was 15.9 nm, and the solubility of AG was 8.02 mg·mL−1. The formulation was stable, with a higher anti-inflammatory effect and bioavailability than AG tablets; it displayed no acute oral toxicity [37].

A special NE loaded with AG was prepared using layer-by-layer technology via electrostatic deposition of chitosan over alginate-encrusted O/W NE by means of ultra-sonication. The best stability was obtained after 20 min of sonication. The particle size of the multi-layered NE was measured to be within the range of 90.8–167.8 nm, with a zeta potential between 22.90 and 31.01 mV. The NE showed a strategic release pattern when assessed in vitro in various simulated biological fluids. It showed significant modulation in a liver function test(alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate transaminase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), and liver glycogen) and serum cytokines (IL-β, TNF-α, IL-10, and IL-6) when assessed in vivo in galactosamine-lipopolysaccharide-intoxicated mice, thus exhibiting significantly improved hepatoprotection [38].

2.2.6. Gold nanoparticles

Functionalized gold NPs (Fig. 3) are smart vectors for biomedical applicationsthat can control geometrical and optical properties. Their large surface can be coated with diverse molecules (i.e., targeting agents, drugs, and anti-fouling polymers), making them versatile carriers. In particular, the targeted delivery of drugs is one of the most encouraging medicinal uses of gold NPs [39].

Engineered gold NPs loaded with AG, with a spherical geometry of 14 nm and a polydispersion index (PDI) value of 0.137, were developed. They exhibited resilient anti-leishmanial activity, against both wild-type (IC50 of (19 ± 1.7) µmol·L−1) and sodium stibogluconate (IC50 of (55 ± 7.3) µmol·L−1)/paromomycin (IC50 of (41 ± 6.0) µmol·L−1)-resistant strains. Complete macrophage uptake of AG gold NPs occurred within 2 h of exposure, and the cytotoxicity was significantly lower than that of amphotericin-B [40].

2.2.7. Nanocrystals and nanosuspensions

Ma et al. [41] designed a fast-dissolving nanocrystal-based solid dispersion to enhance the dissolution of AG. The nanodispersion was formulated using various ratios of hydroxypropyl methylcellulose (HPMC) and three super disintegrant excipients: sodium carboxymethyl starch, mannitol, and lactose. The dispersion with the highest concentration of HPMC exhibited the most rapid AG dissolution properties, probably due to the enhanced wettability. The performance of the super-disintegrants at a level of 20%, in combination with 25% HPMC, was tested. The formulation developed with 15% sodium carboxymethyl starch, 15% HPMC, and 10% lactose improved the dissolution (drug loading was up to 67.83% ± 1.26%). The in vivo pharmacokinetics studies revealed a significantly enhanced bioavailability of AG in comparison with unformulated AG. A nanodispersion was prepared and its performance tested. The dissolution rate (85.87%), Cmax ((299.32 ± 78.54) ng·mL−1), and AUC0–∞((4440.55 ± 764.13) mg·(h·L)−1) of the AG nanodispersion were significantly higher (P < 0.05) in comparison with crude AG. The AUC0–∞ was three times higher [42]. AG nanosuspensions containing glycyrrhizin had a mean particle size of 487 nm and exhibited excellent performance in comparison with those obtained with trehalose. The dissolution percentage of these AG nanosuspensions (99.87%) was significantly increased in comparison with that of unformulated AG (42.35%) [43].