2.1.1. Nanoparticles (NPs)

Numerous studies have explored the pharmaceutical significance and therapeutic viability of the polymeric NPs for the delivery of a wide range of therapeutic agents and in the treatment of various skin inflammatory diseases [54], [55], [121].The success of polymeric NPs-based delivery system is attributed to their ultra-small size, high encapsulation efficiency, adequate surface potential, biodegradability and biocompatibility [126]. In addition, these delivery systems can prevent premature degradation of encapsulated drugs, provide controlled delivery, achieve target-oriented delivery of the drugs [54], [55], and prevent systemic toxicity. These delivery systems have also been proposed to augment percutaneous delivery of the drugs without permanent damage to the stratum corneum [54], [55]. Shao et al. [121] have recently written an excellent review article after critical evaluation of the literature and suggested that nanoencapsulation of a wide range of pharmacological moieties significantly improved their therapeutic feasibility in treating various skin inflammatory disorders.

Recently, Krausz et al. [6] evaluated the pharmaceutical significance, antioxidant and antimicrobial efficacies of CUR-encapsulated polymeric NPs. After the successful fabrication of CUR-loaded polymeric NPs, they tested these NPs for the mean particle size, zeta potential, in vitro release characteristics, cellular cytotoxicity, zebrafish cytotoxicity, and antibacterial activity against MRSA and P. aeruginosa [6]. Due to susceptibility of MRSA and P. aeruginosainfections in burn wounds, the activity of CUR-NPs against these species was evaluated in vitro. Results showed that CUR-NPs exhibit strong antibacterial activity against MRSA and P. aeruginosa. After 6 h incubation with CUR-NPs, MRSA displayed edema and damage with subsequent lysis and extrusion of contents after 24 h, in contrast to untreated MRSA with intact cellular structures, uniform cytoplasmic density, and well-defined cell wall [6]. The quantification of CFU showed that CUR-loaded NPs caused approx. 97% reduction in MRSA growth and around 60% reduction of P. aeruginosa growth which was significantly greater compared to both untreated control and control np (p ≤ 0.0001).

To further investigate the mode of action of antimicrobial activity of CUR-NPs, MRSA incubated with CUR-NPs as well as with control NPs were studied over time using TEM (Fig. 2). Untreated MRSA (Fig. 2A) showed intact cellular architecture with uniform cytoplasmic density and highly contrasting cross wall. After 24 h, MRSA incubated with control NPs did not exhibit changes in cellular architecture compared to untreated control despite visible interaction with nanoparticles (Fig. 2B). In contrast, after 6 h treatment with CUR-NPs (Fig. 2C), the cells of MRSA which were in contact with particles displayed edema and distortion, with subsequent lysis and extrusion of contents after 24 h (Fig. 2D).

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Fig. 2. CUR-NPs induced cellular damage to MRSA. High-power transmission electron microscopy demonstrated interaction of nanoparticles (arrows) with MRSA cells. (A) Untreated MRSA showed uniform cytoplasmic density and central cross wall surrounding a highly contrasting splitting system. (B) After 24 h, cells incubated with control NPs (5 mg/mL) did not exhibit changes in cellular morphology compared to untreated control. (C) After 6 h, cells incubated with CUR-NPs (5 mg/mL) exhibited distortion of cellular architecture and edema, followed by lysis and extrusion of cytoplasmic contents after 24 h (D). All scale bars = 500 nm [6].

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The delivery of CUR-loaded PLGA NPs has also shown promising potential in improving in vitro performance of CUR [73]. In this study, authors prepared CUR-loaded PLGA NPs by emulsification-solvent evaporation method [13], [138]. Briefly, 50 mg of PLGA was dissolved in 2 mL dichloromethane and stirred to obtain a uniform PLGA solution. After a homogenous solution of PLGA was obtained, 5 mg of CUR was added and sonicated (at 70 W) for 30 s. The solution was then emulsified with 10 mL of 1% (w/v) PVA solution and again sonicated for 1 min to generate the o/w emulsion. The CUR-loaded NP suspension was then washed twice in distilled water using centrifugation techniques. In order to evaluate successful fabrication, PLGA-CC NP were characterized for particle size, zeta potential, encapsulation efficiency, morphology, and in vitro release studies [73]. In addition, the tissue-associated MPO assay was performed to quantify the degree of inflammatory infiltration in the wounds. MPO is an enzyme that is found predominantly in the azurophilic granules of neutrophils and can be used as a quantitative index of inflammatory infiltration.

The findings demonstrated that the prepared PLGA-CC NP showed optimal physicochemical characteristics including smaller particle size (176.5 ± 7.0 nm), narrow distribution index (0.105 ± 0.025), acceptable zeta potential (− 23.2 ± 3.8 mV), and greater encapsulation efficiency (89.2 ± 2.5%). On the other hand, in vitro release studies suggested biphasic release pattern with the initial burst release (approx. 40% of encapsulated CUR) within the first 24 h, followed by a sustained and gradual (approx. 75%) over a period of 8 days [138]. The assessment of anti-inflammatory activity of CUR revealed that on day 5 there was a significant increase in the activity of MPO in wound tissue of untreated and PLGA NP-treated mice due to inflammatory response. In contrast, CC and PLGA-CC NP-treated group showed significantly lower MPO activity. The MPO inhibition was much evident on day 10 in the case of PLGA-CC NP-treated mice as shown in Fig. 3 [73]. Furthermore, the authors also evaluated the significant downregulation of anti-oxidative enzymes like glutathione peroxidase (GPx) and NFκB which is a well-known transcription factor involved in cellular inflammatory responses. The results revealed that encapsulation of CUR in PLGA nanoparticles improve their anti-inflammatory efficacy.

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Fig. 3. Inhibition of MPO by CC, PLGA NP and PLGA-CC NP (mean ± SD, n = 3) [73].

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2.1.2. Nanovesicles

Liposomes, spherical vesicles having at least one lipid bilayer, have widespread recognition as carriers or vehicle system for the administration of nutrients, pharmaceuticals and gene delivery [137]. Liposomes are versatile carriers having bilayer arrangement of hydrophilic core surrounded by hydrophobic membrane and thus are capable of carrying hydrophilic and hydrophobic drug cargo simultaneously. They are mainly composed of phospholipids, especially phosphatidylcholine, but may also include other lipids, such as egg phosphatidylethanolamine [137].

Liposomal systems have been numerously used to encapsulate a wide range of pharmaceutical moieties such as NSAIDs [39], immunoglobulins [41], growth factors [147], opioid analgesics [15], hyaluronic acid [93], superoxide dismutase [142], quercetin [21] and CUR [91], aiming to improve their antioxidant, antimicrobial and anti-inflammatory activities.

Recently, encapsulation of CUR in sodium hyaluronate immobilized vesicles (hyalurosomes) has been investigated and compared with conventional liposomes for in vitro and in vivo performance [91]. In this work, Manca and co-workers developed highly biocompatible nanovesicles by direct addition of polyanion sodium hyaluronate to the polyphenol CUR to fabricate polymer immobilized vesicles, so called hyalurosomes. CUR-loaded hyalurosomes were then characterized for particle size, zeta potential, encapsulation efficiency, stability, rheology, in vitro skin delivery, antioxidant activity, cellular toxicity, and anti-inflammatory activity [91].

The antioxidant activity of both curcumin ethanolic solution and vesicles was tested by measuring their ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH). Each sample was added (1:50) to DPPH methanolic solution (25 μM), stored at room temperature for 30 min in dark, and absorbance was measured at 517 nm against blank. The percent antioxidant activity was calculated according to the following formula:$\left(\right)\left[\left({}_{}{}_{}\right){}_{}\right]\mathrm{}$

In vitro cryogenic TEM and small-angle X-ray scattering analyses showed that CUR-loaded hyalurosomes were nanosized (112–220 nm) with spherical morphology. Skin drug delivery studies suggested that CUR-loaded hyalurosomes significantly enhanced drug permeation across the intact skin and showed approx. 57% of drug accumulated in the SC, 18% in the epidermis, and approx. 16% in the dermis only after 4 h. These findings indicated excellent ability of CUR-loaded hyalurosomes to promote delivery of CUR throughout the skin and thus can be utilized for various dermatological disorders. Cellular toxicity studies suggested that CUR-loaded hyalurosomes were biocompatible, non-toxic to human keratinocytes, could protect keratinocytes from oxidative stress damages, and were able to promote tissue remodeling by increasing cellular proliferation and differentiation.

To evaluate in vivo anti-inflammatory activity, female CD-1 mice were shaved and applied with TPA topically on the shaved back. TPA induces oxidative stress, inflammatory reactions, edema, and infiltration of inflammatory cells and loss of epidermis or ulceration. The results demonstrated that animals treated with CUR-loaded hyalurosomes showed minimal signs of inflammation, edema, and redness. The authors proposed that these results were due to greater ability of encapsulated CUR as hyalurosomes to diminish initiation of inflammatory reactions, in comparison to other treatment groups. These results were also in agreement with previous studies by Castangia et al. [21] and El-Refaie et al. [33]who have suggested a greater impact of nanoencapsulation of CUR to enhance its antioxidant, antimicrobial and anti-inflammatory efficacies.

Castangia and colleagues attempted for the nanodelivery of CUR and quercetin (QUE) by encapsulating these drugs in nanovesicles (e.g., liposomes and PEVs). In vitro TEM, cryogenic-TEM, and small-angle X-ray scattering analyses showed that CUR-loaded- and QUE-loaded nanovesicles were ultra-small in size (approx. 112–220 nm) with spherical morphology. The nanovesicular encapsulation of CUR and QUE was also evaluated for in vitro drug permeation across the new born pig skin using Franz-diffusional vertical cells and calculation of percentage of drug accumulated in various skin layers (epidermis and dermis). The findings demonstrated that as compared to other formulations, nanovesicles of CUR and QUE namely Oramix®CG110-mediated surface modified nanovesicles, showed noticeably greater permeation across the skin and remarkably higher accumulation of the encapsulated drugs into various skin layers. According to the experiment, highest accumulation of CUR and QUE was found in the SC, followed by in the epidermis and then in dermis, respectively [21].

The therapeutic significance of the surface modified nanovesicles was also evaluated in vivo using female CD-1® mice (5–6 weeks old, approximately 25–35 g in weight) by investigating edema and MPO activity (which is used to estimate severity of inflammation). These studies revealed that the mice group dressed with surface modified nanovesicles of CUR or QUE demonstrated remarkable control over skin ulceration, curst formation, loss of epidermal integrity, and pathological damage to the epidermis, dermis and the subcutaneous tissues, in comparison to the control groups. Results from MPO activity suggested that PEG-modified nanovesicles and liposomes remarkably suppressed MPO activity by approx. 68% and 59% respectively, in comparison to the control groups. Histology results also showed significantly higher re-epithelization and collagen formation in mice group treated with nanovesicles system as compared to other treatment groups [21].

2.2.1. Nanocomposite hydrogel

Hydrogel-based delivery systems have also been used for improving pharmaceutical significance and therapeutic applicability of a wide range of pharmacological moieties including growth factors [44], nerolidol [106], stem cells [30], sodium fusidate [72], peptides [42], antibiotics [24], [78], hyaluronic acid [20], and CUR [85], [148], [149].

Recently, a novel nanocomposite hydrogel composed of nano-CUR, CS and oxidized alginate has shown promising ability to accelerate dermal wound repair [85], [148], [149]. In this study, they evaluated in vitro and in vivo efficacy of CUR-encapsulated nanocomposite hydrogel which was previously developed by thin-film evaporation method using MPEG-PCL as a carrier/vehicle [83]followed by incorporating into the CS-OA hydrogel. The developed nanocomposite hydrogel was evaluated for in vitro stability, release profile, antioxidant activity and in vivo wound healing efficacy.

In vitro stability studies demonstrated that the stability of nano-CUR in phosphate buffered saline (PBS, pH 7.4) was significantly increased when encapsulated in nanocomposite hydrogel compared to the unmodified CUR. In case of nano-CUR, 100% of CUR remained intact after 5 h incubation, in comparison to the unmodified CUR that underwent significant rapid degradation (only about 50% of curcumin remained intact after 5 h of incubation) in PBS. The encapsulation of CUR into nanocomposite hydrogel was also evaluated for antioxidant activity and the results showed that there was a slight improvement in antioxidant activity. The release study revealed that the encapsulated nano-CUR was slowly released from CCS-OA hydrogel in a diffusion-controllable manner during initial phase followed by release in corrosion manner from hydrogel during terminal phase.

ROS are generally produced during normal physiologic conditions and can easily initiate the peroxidation of membrane lipids, resulting in the accumulation of lipid peroxides. ROS are also capable of damaging crucial biomolecules such as lipids, nucleic acids, proteins and carbohydrates and may cause DNA damage that can lead to mutations [7]. However, if ROS are not effectively scavenged by cellular constituents, the disease conditions might progress. In this study, Li et al. [85], [148], [149] demonstrated that the antioxidant activity of nano-CUR was about 99.22%, which was not significantly different than that of unmodified curcumin (approx. 98.73%). This result suggested that incorporated curcumin into polymer to form nano-CUR could not influence the intrinsic antioxidant activity of CUR.

2.2.2. Nanofibers

A great degree of improvement in antioxidant and antimicrobial efficacies of CUR was reported when loaded onto and administered using poly(ε-caprolactone) (PCL)/gum tragacanth (GT) electrospun nanofibers [95]. They developed PCL/GT/CUR-loaded nanofibers by electrospinning technique. Briefly, GT solution (7% w/v) blended vigorously with PCL solution (20% v/v prepared in acetic acid) in 2:1 (PCL/GT mass ratio). After making a homogenous solution, CUR (3% w/w based on solid contents in polymer blend) was introduced into the polymer blend and stirred for 30 min. Afterward, PCL/GT/CUR nanofibers were synthesized by electrospinning technique [95]. The in vitro performance of fabricated nanofibers was assessed through in vitro release study, antibacterial activity against MRSA and ESBL by broth dilution method [95].

In vitro studies revealed that PCL/GT/CUR nanofibers demonstrated biphasic release profile with approx. 60% of drug released in first hour with subsequent slow release until 20 h. The results of antibacterial activity suggested that the nanofibers demonstrated remarkable antibacterial activity against MRSA (99.9%) and ESBL (85.14%). The results of antibacterial activity of the nanofibers are shown in Fig. 4. These results suggested that CUR-loaded nanofibers are promising effective scaffold for antibacterial applications [95].

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Fig. 4. Antibacterial activity against MRSA and ESBL for samples containing Cur (average CFU/mL) [95].

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2.2.3. Foams (porous silk fibroin scaffold)

Substantial improvements in therapeutic efficacies of the CUR were also suggested by Kasoju and Bora [68] when CUR was fabricated as silk fibroin scaffold. They reported the fabrication of CUR-releasing porous silk fibroin scaffold by a simple mixing of fibroin solution (aqueous) with CUR solution (organic) followed by freeze–thaw of the mixture. The fabrication process is simple, reproducible, and does not require any sophisticated instruments or toxic crosslinking agents. The in vitro characterization of the scaffold demonstrated a uniform pore distribution with an average pore size of approx. 115 μm and a degree of swelling of 2.42% and water uptake capacity of 70.81%. Fibroin showed thermal stability up to approx. 280 °C, whereas the encapsulated CUR was disintegrated at around 180 °C. Fourier transform infrared, powder X-ray diffraction, and nuclear magnetic resonance studies together with UV–visible and fluorescence spectroscopy investigations revealed that the solvent (which was used to dissolve CUR) induced conformational transition of fibroin from silk-I to silk-II that led to the formation of water-stable structure. Fluorescence spectroscopy data also suggested the presence of hydrophobic domains in fibroin and encapsulation of CUR in such domains through hydrophobic interactions. Release kinetics and mathematical modeling studies indicated a slow and sustained release profile with diffusion as the predominant mode of release. Further, in vitro anticancer, antioxidant, and antimicrobial assays suggested that the biological activity of encapsulated CUR improved significantly [68]. It was anticipated that the CUR-loaded fibroin scaffold could be used in soft tissue replacements including localized postsurgical chemotherapy against tumors, dressing material for quick healing of wounds and burns, and other related applications.

2.3.1. Nanohybrid scaffolds

A significant improvement in in vitro performance of CUR was suggested by Karri et al. [140] after encapsulating CUR in nanohybrid scaffold. The authors attempted the synthesis of a novel nanohybrid scaffold system by encapsulating CUR in CSNPs to improve aqueous solubility and stability of CUR, followed by impregnation of CUR-loaded CSNPs into collagen scaffold (nanohybrid scaffold). The authors prepared CUR-loaded CSNPs by ionic-crosslinking of CS with ionic cross-linker, TPP [140]. The resulting NPs were examined for particle size, zeta potential, morphology, biocompatibility, biodegradability, and in vitro release. In vitro studies demonstrated that encapsulation of CUR in nanohybrid system improved aqueous solubility, stability, biocompatibility, biodegradability and water uptake of CUR. The release studies demonstrated biphasic release pattern of CUR with burst release in 24 h followed by slow and sustained release. The data obtained suggested that nanohybrid system of CUR demonstrated better sustained release pattern compared to CUR-loaded CSNPs that can diminish inflammatory cascades over extended time period and reduce dressing frequency.